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WHO Expert Consultation on Rabies: second report

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982 WHO Expert Consultation on Rabies

W H O

Te c h n i c a l

R e p o r t

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982 Although there is debate about the estimated health burden of rabies, the estimates of direct mortality and the DALYs due to rabies are among the highest of the neglected tropical diseases. Poor surveillance, underreporting in many developing countries, frequent misdiagnosis of rabies, and an absence of coordination among all the sectors involved are likely to lead to underestimation of the scale of the disease It is clear, however, that rabies disproportionately affects poor rural communities, and particularly children. Most of the expenditure for postexposure prophylaxis is borne by those who can least afford it. As a result of growing dog and human populations, the burden of human deaths from rabies and the economic costs will continue to escalate in the absence of concerted efforts and investment for control. Since the first WHO Expert Consultation on Rabies in 2004, WHO and its network of collaborating centres on rabies, specialized national institutions, members of the WHO Expert Advisory Panel on Rabies and partners such as the Gates Foundation, the Global Alliance for Rabies Control and the Partnership for Rabies Prevention, have been advocating the feasibility of rabies elimination regionally and globally and promoting research into sustainable cost-effective strategies. Those joint efforts have begun to break the cycle of rabies neglect, and rabies is becoming recognized as a priority for investment. This Consultation concluded that human dog-transmitted rabies is readily amenable to control, regional elimination in the medium term and even global elimination in the long term. A resolution on major neglected tropical diseases, including rabies, prepared for submission to the World Health Assembly in May 2013 aims at securing Member States’ commitment to the control, elimination or eradication of these diseases. Endorsement of the resolution would open the door for exciting advances in rabies prevention and control.

WHO Expert Consultation on Rabies Second report

WHO Technical Report Series

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SELECTED WHO PUBLICATIONS OF RELATED INTEREST

WHO Position Paper on Rabies Vaccines Weekly Epidemiological Record, 2010, 85: 309-320 WHO Expert Consultation on Rabies. First report. Geneva, World Health Organization, 2005 WHO Technical Report Series, No. 931 WHO Expert Committee on Rabies. Eighth report. Geneva, World Health Organization, 1992 WHO Technical Report Series, No. 824 Laboratory Techniques in Rabies. Fourth edition. Geneva, World Health Organization, 1996

Further information on these and other WHO publications can be obtained from WHO Press, World Health Organization ■ 1211 Geneva 27, Switzerland ■ www.who.int/bookorders tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: bookorders@who.int

W H O

Te c h n i c a l R e p o r t 9 8 2

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WHO Expert Consultation on Rabies Second report

This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the World Health Organization

WHO Library Cataloguing-in-Publication Data WHO Expert Consultation on Rabies: second report. (WHO technical report series ; no. 982) 1.Rabies – prevention and control. 2.Rabies – diagnosis. 3.Rabies – epidemiology. 4.Rabies vaccines. 5.Rabies virus. 6.National health programs. I.World Health Organization. II.Series. ISBN 978 92 4 120982 3 ISBN 978 92 4 069094 3 (PDF) ISSN 0512-3054 (NLM classification: WC 550)

©World Health Organization 2013 All rights reserved. Publications of the World Health Organization are available on the WHO web site (www.who.int) or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: bookorders@who.int). Requests for permission to reproduce or translate WHO publications –whether for sale or for noncommercial distribution– should be addressed to WHO Press through the WHO web site (www. who.int/about/licensing/copyright_form/en/index.html). The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use. This publication contains the collective views of an international group of experts and does not necessarily represent the decisions or the policies of the World Health Organization. Design: WHP (Sophie Guetaneh Aguettant)

Contents Introduction 1. The burden of rabies 1.1 1.2 1.3 1.4 Methods for estimating the burden of rabies Estimated burden of rabies in the world Global summary References

1 2 2 3 8 10 13 13 13 14

2. Classification of lyssaviruses 2.1 Distinguishing features of lyssaviruses 2.2 Criteria for differentiating between lyssaviruses 2.3 Present structure of the Lyssavirus genus 2.4 References

15 19 23

3. Pathogenesis 4. Diagnosis 4.1 Standard case definitions for rabies 4.2 Clinical diagnosis 4.3 Biosafety, sampling and specimen transport for laboratory diagnosis 4.4 Laboratory techniques for post-mortem diagnosis of rabies 4.5 Techniques for intra-vitam diagnosis of rabies in humans 4.6 Virus identification with molecular techniques: epidemiological considerations 4.7 References

23 24 25 27 28 30 31

5. Management of patients before and after death 5.1 Rabies survivors and treatment protocols 5.2 Clinical management of rabies patients 5.3 Transmission via organ transplantation 5.4 Recommendations for health care personnel and patients’ family members 5.5 Management of the bodies of patients who have died of rabies 5.6 References

34 34 35 35 36 36 36

6. Vaccines and rabies immunoglobulin for humans 6.1 Vaccine types 6.2 WHO prequalification of human rabies vaccines 6.3 Requirements for human rabies vaccines 6.4 Routes of vaccine administration 6.5 Adverse events after active immunization 6.6 Duration of immunity 6.7 Rabies vaccine and full post-exposure prophylaxis failures 6.8 Rabies immunoglobulins 6.9 References

37 37 39 40 41 42 42 42 43

iii

7. Vaccines for animals 7.1 Vaccine types 7.2 Potency requirements for animal rabies vaccines 7.3 Safety of animal vaccines 7.4 Parenteral rabies vaccination 7.5 References

47 48 49 50 51 51

8. Prevention of human rabies 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 General considerations Pre-exposure prophylaxis Post-exposure prophylaxis Requirements for periodic booster injections Vaccination of immunocompromised individuals Rabies immunoglobulin for passive immunization Contraindications and precautions Travellers to and residents of rabies-affected countries and areas, and indications for pre-exposure prophylaxis 8.9 References

54 54 54 55 59 59 60 60 61 62

9. National programmes for dog rabies control 9.1 Canine mass parenteral vaccination campaigns 9.2 Strategic planning and management of vaccination campaigns 9.3 Implementing and monitoring dog vaccination campaigns 9.4 Increasing access to dogs for vaccination 9.5 Supplementary measure: humane dog population management 9.6 Main components of a dog rabies control programme 9.7 Operational research for dog rabies control 9.8 References

63 64 66 67 69 70 70 72 74

10. Prevention and control of rabies in wild animals 10.1 Epidemiology and ecology of rabies in carnivore species 10.2 Epidemiology and ecology of rabies in bats 10.3 Rabies in rodents 10.4 Wildlife species of special concern 10.5 Elimination of rabies in wild carnivores 10.6 Bat rabies control 10.7 Other public health measures 10.8 References

77 77 80 82 83 83 88 88 88

11. Rabies surveillance 12. Rabies-free countries or areas 13. International movement of animals 13.1 International transport of dogs, cats and ferrets from rabies-infected countries or areas 13.2 International transport of livestock and animals for zoos, research, shows and other activities from rabies-infected countries or areas iv

92 94 97 97 97

13.3 Special exemption of guide dogs for people with disabilities and of other service dogs 13.4 References

98 98

14. Global and regional activities on rabies 14.1 WHO global and regional activities 14.2 Examples of activities by partners 14.3 References

98 99 102 107

15. Research 15.1 Diagnostics 15.2 Epidemiology 15.3 Molecular, genetic and epidemiological characterization of new viral isolates 15.4 Biological medical products 15.5 Human rabies prophylaxis 15.6 Pathobiology 15.7 Host ecology 15.8 References

110 110 110 111 112 113 113 114 114

Concluding remarks Acknowledgements Annex 1 List of participants

117 118 119 125

Annex 2 Record form for cases of possible exposure to rabies Annex 3 Four steps for replacing nervous tissue vaccine by modern rabies vaccines produced on cell culture or embryonated eggs Annex 4 Technique for intradermal administration of rabies vaccine and precautions to be taken Annex 5 Recommended post-exposure prophylaxis according to type of exposure Annex 6 Suggested rabies vaccination certificates for humans Annex 7 International rabies vaccination certificate for dogs, cats and ferrets Annex 8 WHO collaborating centres on rabies, neurovirology, viral zoonoses and zoonoses control

127

128 130 131 134

138 v

Introduction

Introduction The World Health Organization (WHO) Expert Consultation on Rabies met in Geneva, Switzerland, on 18–20 September 2012. Dr Denis Daumerie, Project Manager, welcomed the participants on behalf of Dr Lorenzo Savioli, Director, Department of Control of Neglected Tropical Diseases, and the Director-General. He pointed out that rabies, like the tropical diseases covered by the Department, affected mainly people whose deaths are not accounted for. The disease continues to occur mainly in poor communities, where measures that could prevent it in humans by controlling dog rabies are not implemented, even though a resolution adopted by the Third World Health Assembly in 1950 already mentioned the need for prevention of rabies in humans and its control in dogs. Advances have been made in the field of rabies, particularly in the production and use of human and animal biologicals, but the disease is still neglected, and no new WHO resolutions on rabies have been proposed to address human rabies transmitted by dogs. Dr Daumerie described the successful collaboration between the Department of Control of Neglected Tropical Diseases and major drug manufacturers for the control and elimination of tropical diseases such as leprosy, lymphatic filariasis and human African trypanosomiasis, and advised the consultation to explore the benefits of such partnerships for rabies prevention and control. Dr François-Xavier Meslin, Neglected Zoonotic Diseases, recalled that WHO had been denouncing and combating the ‘cycle of neglect’ with regard to rabies for more than a decade. Since the first WHO Expert Consultation on Rabies, in 2004, WHO and its network of collaborating centres on rabies, specialized national institutions, members of the WHO Expert Advisory Panel on Rabies and partners such as the Bill & Melinda Gates Foundation, the Global Alliance for Rabies Control and the Partnership for Rabies Prevention, have been advocating the feasibility of rabies elimination regionally and globally, and promoting research into strategies. Those joint efforts have begun to break the cycle of rabies neglect, and rabies is becoming recognized as a priority for investment. Dr Louis Nel was appointed Chairperson and Dr Naseem Salahuddin was appointed Rapporteur of the Consultation. The list of participants is given in Annex 1. The information in this report should be considered the most current data on rabies prevention and control, and supersedes that of the report of the first WHO Expert Consultation on Rabies, published in 2005 (1).

WHO Expert Consultation on Rabies Second report

The burden of rabies

Information on disease burden is widely used to set public health priorities, allocate limited resources for disease prevention and control and assess the impacts and cost–effectiveness of interventions (1). Standardized metrics, such as the disability-adjusted life year (DALY), have been widely adopted to evaluate the burden of disease at regional and global levels and have become an essential tool for decision-making by policy-makers (2). The major burden of rabies is attributable to dog-mediated transmission, and therefore this chapter focuses on dog-mediated rabies and only briefly addresses the burden attributable to other host species (3). Estimates of disease burden can be contentious when the underlying data are of poor quality; nevertheless, the resulting information is a useful starting point for more accurate estimates as better data become available.

1.1

Methods for estimating the burden of rabies

Several factors contribute to significant underreporting of human deaths from rabies in many parts of the world. Methods have therefore been developed to estimate the mortality attributable to rabies, which account for the quality of reporting in countries with endemic canine rabies. In particular, a predictive approach based on a probability decision-tree has been devised to determine the likelihood of the onset of clinical rabies in humans after a bite by a dog suspected of being rabid. This method, initially used to estimate human deaths from rabies in the United Republic of Tanzania (4), has resulted in a revised estimate of the burden of rabies in Africa and Asia (5). More recently, the approach has been tailored for estimating mortality due to rabies in specific countries in Asia (e.g. Bhutan (6) and Cambodia (7)). Empirical studies to both parameterize and validate such estimates include community surveys (8), large-scale verbal autopsy surveys (9) and active surveillance and contact tracing (10). DALYs incorporate both premature mortality and disability (2). The most critical element in calculating DALYs for rabies is premature death (5); because of the short duration of the disease, disability accounts for a relatively small part of the burden of rabies. Disability can, however, occur after administration of nerve tissue vaccine, which is still in use in a few countries. These vaccines have severe side-effects lasting from 4 to 7 months, depending on the type of vaccine used, in an estimated 0.3–0.8 cases out of 1000 (5). The economic burden of disease is typically calculated from a combination of direct and indirect costs. For rabies, the direct costs of post-exposure prophylaxis depend on the vaccine, regimen and route of administration as well as the type of rabies immunoglobulin used; the indirect costs include those for visiting a clinic (or accompanying a bite victim to a clinic) and associated income

WHO Technical Report Series No. 982, 2013 2

The burden of rabies

loss. The scale of such costs is particularly important for rabies, as lack of postexposure prophylaxis translates directly into human deaths. A further economic component is productivity loss, calculated by weighting the discounted years of life lost by the country’s gross domestic product and using a 3% discounting rate. Up to now, productivity losses have not been considered in studies of the burden of rabies. The cost of rabies prevention, control and elimination (including surveillance) in animal reservoirs and losses in the animal production sector should also be taken into account. An additional component of the burden of rabies is its emotional and psychological impact, particularly the trauma and the long periods of uncertainty after a bite by a rabid animal when post-exposure prophylaxis is either unreliable or unavailable. A working group was convened by the Partners for Rabies Prevention to collate and review the most recent data and use the probability decision-tree approach to assess the global burden of canine rabies. The Institute for Health Metrics and Evaluation, as part of the study, also generated estimates of the global burden of rabies using a ‘cause of death ensemble’ model (11,12). The preliminary results of these studies are discussed here; however, both indicated that their estimates are highly uncertain, owing to lack of accurate data. Field data to validate these estimates are therefore needed to address this enduring problem.

1.2 Estimated burden of rabies in the world In the following section, information on the rabies burden in various countries is grouped according to epidemiological similarity and geographical proximity. For each region, results of local studies that have provided the most accurate data are given, as well as less certain regional estimates based on extrapolations.

1.2.1 Countries that are free of canine rabies Canine rabies has been eliminated from western Europe, Canada, the United States of America (USA), Japan, Malaysia and a few Latin American countries; while Australia is free from carnivore rabies, and many Pacific island nations have always been free from rabies and related viruses. In these areas, human deaths from rabies are restricted to people exposed while living or travelling in areas endemic for canine rabies. About two deaths per year due to imported human rabies have been reported in Europe, North America and Japan (13,14). One third of the imported cases in 1990–2010 originated in South and South-East Asia (predominantly India and the Philippines), another third in Africa, almost 20% in Latin America and the Caribbean and over 10% in eastern Europe and Central Asia. The costs of post-exposure prophylaxis for travellers returning from overseas and for pre-exposure prophylaxis are often substantial. The cost of post3

WHO Expert Consultation on Rabies Second report

exposure prophylaxis in otherwise rabies-free areas escalates after incidents of imported rabid animals and is higher in places where illegal entry from endemic countries is common, putting a considerable burden on the health services (15). In countries bordering areas endemic for canine rabies, border campaigns and intensified surveillance are required to maintain rabies-free status. Quarantine procedures and legislation are needed in all rabies-free countries. The costs of prevention in many countries where wildlife rabies or bat rabies viruses circulate must also be taken into account. Millions of dollars have been spent annually to eliminate wildlife rabies by administering oral rabies vaccine, and the cost varies substantially according to the setting and tactics (16). For instance, one to eight human rabies deaths occur annually in the USA as a result of wildlife rabies (17), and, according to the Centers for Disease Control and Prevention, an estimated US$ 300 million are spent per annum for rabies prevention. Several states are attempting to eliminate raccoon rabies to reduce the demand for post-exposure prophylaxis. Since fox rabies was eliminated from western Europe, the costs for oral vaccination have been substantially reduced (Table 1), but other European countries now striving to eliminate fox rabies are incurring high costs. Recent incursions into Italy, although now under control, required substantial financial commitments, and costs may escalate elsewhere, given the threat of emergence in rabies-free countries such as Greece. The cost of setting up a cordon sanitaire along the entire eastern border of the European Union to prevent such incursions is estimated to exceed US$ 6.5 million per year (21). Table 1 Examples of costs associated with rabies and its elimination from Europe Country (reference) France (18) Germany (19) Estonia (20) Period of programme 1988–1993 1983–2008 2005–2010 Costs included in programme Cost of programme (million US$)

WHO Technical Report Series No. 982, 2013

Post-exposure prophylaxis, preventive 261 vaccination of cattle, dogs and cats, oral rabies vaccination Oral rabies vaccination Oral rabies vaccination and surveillance 122 15.5

The burden of rabies

1.2.2 Countries in which canine rabies is endemic Latin America and the Caribbean

Canine rabies control programmes during the past two decades have had substantial success in this region. Official reports of cases of human rabies transmitted by dogs decreased from about 250 in 1990 to fewer than 10 in 2010, with concomitant declines in dog rabies (22). In foci where canine rabies continues to circulate, however, official reports probably underestimate the scale of the problem, particularly in the Plurinational State of Bolivia, Cuba, the Dominican Republic, El Salvador, Guatemala, Haiti, Honduras and parts of Brazil, Mexico and Peru. In these countries, human deaths from rabies are either still occurring or are at risk of occurring. Preliminary estimates with the probabilistic decisiontree model suggest that the number of human deaths due to canine rabies in the Americas is more likely to be of the order of 200 cases per annum, most occurring in Haiti. Although progress has been made in phasing out nerve tissue vaccines in the Americas, their use is still widespread in Argentina, the Plurinational State of Bolivia, Honduras, Peru and the Bolivarian Republic of Venezuela, and therefore adverse events and the resulting disabilities are still a problem. The annual public health burden of rabies in this region probably exceeds 15 000 DALYs, about 100 of which are probably attributable to adverse events from nerve tissue vaccines; however, appropriate systems for reporting adverse events are required to accurately quantify the number. The Pan American Health Organization has set a target to eliminate canine rabies from the Americas by 2015. To reach this target, an estimated total budget of more than US$ 20 million per year is required (23); however, there is currently an annual budget shortfall of around US$ 4 million (24). Almost 75% of this estimated total annual budget is allocated to dog vaccination, and 5–10% is associated with post-exposure prophylaxis. The costs incurred by people seeking post-exposure prophylaxis (including time lost, income loss and side-effects) were not included in these estimates, nor were the costs of bat-related rabies in humans or livestock. Asia

More human deaths from rabies occur in Asia than anywhere else in the world, with estimates of human mortality due to endemic canine rabies exceeding 30 000 per annum (95% confidence interval [CI], 8100–61 400) in 2003 (5). Since 2003, the epidemiological situation in many parts of the region has changed, with improvements in rabies control and prevention in many areas, particularly in delivery of post-exposure prophylaxis. There have, however, been emergences elsewhere. 5

WHO Expert Consultation on Rabies Second report

Nerve tissue vaccines have been almost completely phased out in the region; only Mongolia, Myanmar and Pakistan still use these vaccines. The DALYs attributable to adverse events from the vaccines are estimated to have decreased from over 40 000 (5) to around 10 000 in 2010. Bangladesh phased out nerve tissue vaccines in late 2011, and plans are under way to discontinue their production and use in both Myanmar and Pakistan. Wider availability of post-exposure prophylaxis might have reduced the death toll in many areas, including India, but its increased use has been costly, as dog rabies control programmes have not been given the same priority, and exposure to the risk of contracting rabies remains and may be increasing. The costs associated with postexposure prophylaxis are higher in Asia than anywhere else, estimated at around US$ 1.5 billion. Extreme examples include Sri Lanka and Thailand, where the annual direct costs of post-exposure prophylaxis in both countries exceed US$ 10 million (25). Estimates suggest that in 2010 between 15 900 (‘cause of death ensemble’ model approach) and 34 500 (probability decision-tree approach) human rabies deaths occurred in Asia, excluding Central Asia,with about 1.2 million DALYs lost in the region. Both estimates are uncertain, with overlapping confidence intervals, and field data are required to validate the model results. In excellent examples of such studies, the incidence of human deaths from rabies was estimated to be 1.1–1.8 deaths/100 000 in rural Bangladesh (8), 2.5–7.5 deaths/100 0000 in populations at risk in Bhutan (6) and 2.8–11.5 deaths/100 000 in Cambodia (7). India is reported to have the highest incidence of rabies globally. A multicentre study in 2003 showed that 20 565 human deaths occur annually (26), and a large-scale verbal autopsy study in 2005 put the figure conservatively at 12 700, without adjustment for atypical cases not captured by this latter method (9). Most cases were reported in rural communities (9, 26) where no large-scale dog vaccination programmes have been conducted and where the incidence of dog rabies presumably remains high. While the availability of post-exposure prophylaxis has improved, it is not clear how much rural communities have benefited; furthermore, most deaths occur among people who do not seek medical care. The number of deaths due to rabies in India therefore remains uncertain. Estimates of the burden of rabies in China are also uncertain. Surveillance records indicate that the incidence has decreased since 2007, when over 3300 suspect (clinically diagnosed) rabies deaths were recorded officially (27). These records may, however, underreport the incidence of the disease (27), and field investigations are therefore urgently needed. Despite the uncertainty of these estimates, rabies is clearly a major problem in Asia, mainly affecting the rural poor. In many countries, official records substantially underestimate the scale of the problem (6,7,9), and reassessments are therefore encouraged. This is already planned for India.

WHO Technical Report Series No. 982, 2013

The burden of rabies

Africa

The number of deaths from endemic canine rabies in Africa was estimated in 2003 to be about 23 700 (95% CI, 6900–45 900) (5). Estimates of the burden of rabies in Africa have always been uncertain, however, because of the lack of good data. Few large-scale dog vaccination programmes were implemented in the region during the past decade, and the disease continues to circulate largely unregulated. Recent surveys have also indicated extremely limited availability of post-exposure prophylaxis in most of sub-Saharan Africa. In-depth studies show that official reports may underestimate the incidence of rabies by more than 100-fold, because most deaths occur in communities rather than in hospitals (4,10), and those that occur in hospitals are frequently misdiagnosed as forms of encephalitis (29). The revised 2010 estimate of the rabies burden in Africa by the probability decision-tree approach of about 23 800 deaths (95% CI, 21 000–28 000) and 609 000 DALYs (95% CI, 522 000–707 000) is consistent with the earlier estimate (5). In the study of the Institute for Health Metrics and Evaluation, 9500 rabies deaths were estimated to occur in 2010 (11), although the number of DALYs was similar (750 000; 95% CI, 169 000–2 733 000). These figures should be interpreted with caution, as there are few data for validation, and they should be the subject of further investigation in the region. Use of nerve tissue vaccine remains widespread in Ethiopia, contributing about 1000 DALYs per annum. Algeria still produces nerve tissue vaccines, but the situation in some other countries in North Africa and in the horn of Africa is unknown. Central Asia and the Middle East

Little information is available on rabies in the Middle East or Central Asia, and the scale of the rabies burden in these regions has not been investigated previously. On the basis of the literature and population data in the probability decision-tree model, initial estimates can be made of 350 deaths (95% CI, 270–450) and 13 100 DALYs (95% CI, 11 100–15 900) in the Middle East and 1900 deaths (95% CI, 1600–2350) and 55 200 DALYs (95% CI, 47 500–66 600) in Central Asia.

1.2.3 Vampire bat rabies In Latin America and the Caribbean, cases due to vampire bat rabies virus are largely underreported. In 1985, it was estimated that the death toll among cattle was of the order of 100 000 per year, at an annual estimated cost of US$ 30 million. Evidence suggests, however, that the incidence of bat rabies has increased, probably resulting in more human cases and livestock losses (30). 7

WHO Expert Consultation on Rabies Second report

1.3 Global summary The annual number of human rabies deaths globally is estimated in 2010 to be from 26 400 (95% CI 15 200–45 200) (‘cause of death ensemble’ model approach) to 61 000 (95% CI 37 000–86 000) (probability decision-tree approach) (Table 2). The vast majority of deaths (84%) occur in rural areas. These estimates represent about 1.9 million (95% CI, 1.3–2.6 million) DALYs. About 12 600 DALYs are due to morbidity following adverse events due to nerve tissue vaccine. The estimated annual cost of rabies is US$ 6 billion (95% CI, 4.6–7.3 billion), with almost US$ 2 billion (~40%) due to lost productivity after premature deaths and a further US$ 1.6 billion spent directly on post-exposure prophylaxis. Although there is considerable debate about the estimated burden of neglected tropical diseases, the estimates of direct mortality due to rabies are among the highest (possibly the highest), and the DALYs due to rabies are also high (31). The cost of life-saving prophylaxis is a major burden both to national economies and to poor families as more data on increasing numbers of postexposure prophylaxis provided annually are becoming available from countries such as China (e.g. reports of 10 million post-exposure prophylaxis treatments delivered in 2010) and India since 2004, suggesting higher exposure to the risk of contracting rabies even if a large proportion of these are not from rabid animals. The psychological impact of fear and trauma after a suspected rabid dog bite is difficult to translate into a monetary value but was estimated to account for about 32 000 DALYs in Africa and 140 000 DALYs in Asia (32). These effects are heightened by uncertainty about the availability, quality and affordability of postexposure prophylaxis in many countries endemic for canine rabies. Poor surveillance, underreporting in many developing countries, frequent misdiagnosis of rabies (29) and an absence of coordination among all the sectors involved are likely to lead to underestimation of the scale of the disease burden. Both country-specific burden studies and improved surveillance (see section 11) should be encouraged in order to obtain more reliable global estimates of the burden of rabies. Nonetheless, it is clear that rabies disproportionately affects poor rural communities, and particularly children. Most of the expenditure for postexposure prophylaxis is borne by those who can least afford it. For example, in India, patients pay nearly half the financial burden of rabies. Previous estimates indicated that a full course of post-exposure prophylaxis represents as much as 3.87% of the gross national income for a person in Asia and 5.80% for a person in Africa (equivalent to 51 days’ wages for an average African, and 31 days’ wages for an average Asian). A recent field study in the United Republic of Tanzania suggests, however, that these are still considerable underestimates of the true cost for high-risk populations. As a result, many patients do not complete their treatment courses and often use regimens that are not recommended. The

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Table 2 Estimated numbers of deaths from rabies (with 95% confidence intervals) in various areas of the world Africa China India Other Asian countries All Asia All Asia and World Africa

Year of Reference Methods estimate /source 20 565 (16 931– 24 198) 12 700 (10 000– 15 000) 23 700 (6900– 45 900) 9500** 2213 23 800 (21 000– 28 000) 7450 (2000– 13 000) 16 450 (6000– 27 000) 10 550 * (6000– 14 000) 34 500 * (14 000– 54 000) 58 300 (35 000– 82 000) 61 000 (37 000–86 000) 16 000 2336 (565– 5049) 19 713 (4192– 39 733) 9489 (2281– 19 503) 30–000 (8100– 61 400) 55 270 (23 910– 93 057) 25 500 26 400 (15 181–45 184)

2003

(26)

Multi-centre study (community surveys and hospital records)

2005

(9)

Verbal autopsies

2003

(5)

Probability decision-tree approach

2010

(11,12)

‘Cause-of-death ensemble’ model

2010

(27)

National surveillance data

2010

PRP

Probability decision-tree approach

PRP, Partners for Rabies Prevention

*Excluding Central Asia

The burden of rabies

**Excluding North Africa

WHO Expert Consultation on Rabies Second report

annual cost of livestock losses due to rabies is also substantial: approximately US$ 12.3 million (90% CI, 11–13.7 million) (5), disproportionately affecting the rural poor who depend upon livestock for subsistence. As a result of growing dog and human populations, the burden of human deaths from rabies and the economic costs will continue to escalate in the absence of concerted efforts and investment for control. Rabies is entirely preventable. As countries strive to reduce the number of human deaths and improve the availability of post-exposure prophylaxis, the costs will rise; however, if dog rabies control and ultimately elimination are achieved by mass dog vaccination, both the demand for post-exposure prophylaxis and the costs should decline. National vaccination programmes will require consistent, sustained commitment but will have widespread health benefits, particularly for the poorest communities in the world.

1.4 References 1. Murray CJL et al. Summary measures of population health: concepts, ethics, measurements, and applications. Geneva, World Health Organization, 2002. 2. Stein C et al. The global burden of disease assessments—who is responsible? PLoS Neglected Tropical Diseases, 2007, 1(3):e161. 3. Essential rabies maps. Geneva, World Health Organization (http://www. who.int/rabies/rabies_maps/en/; accessed March 2013).

4. Cleaveland S et al. Estimating human rabies mortality in the United Republic of Tanzania from dog bite injuries. Bulletin of the World Health Organization, 2002, 80(4):304–310. WHO Technical Report Series No. 982, 2013

5. Knobel DL et al. Re-evaluating the burden of rabies in Africa and Asia. Bulletin of the World Health Organization, 2005, 83(5):360–368. 6. Tenzin et al. Dog bites in humans and estimating human rabies mortality in rabies endemic areas of Bhutan. PLoS Neglected Tropical Diseases, 2011, 5(11):e1391. 7. Ly S et al. Rabies situation in Cambodia. PLoS Neglected Tropical Diseases, 2009, 3(9):e511.

8. Hossain M et al. Human rabies in rural Bangladesh. Epidemiology and Infection, 2012, 140(11):1964–1971. 9. Suraweera W et al. Deaths from symptomatically identifiable furious rabies in India: a nationally representative mortality survey. PLoS Neglected Tropical Diseases, 2012, 6(10):e1847.

The burden of rabies

10. Hampson K et al. Rabies exposures, post-exposure prophylaxis and deaths in a region of endemic canine rabies. PLoS Neglected Tropical Diseases, 2008, 2(11):e339. 11. Lozano R et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet, 2012, 380(9859):2095–2128. 12. Murray CJL et al. Disability-adjusted life years (DALYs) for 291 diseases and injuries in 21 regions, 1990–2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet, 380(9859):2197–2223. 13. Gautret P, Parola P. Rabies vaccination for international travelers. Vaccine, 2012, 30(2):126–133.

14. Malerczyk C, DeTora L, Gniel D. Imported human rabies cases in Europe, the United States, and Japan, 1990 to 2010. Journal of Travel Medicine, 2011, 18:402–407. 15. Lardon Zl et al. Imported episodic rabies increases patient demand for and physician delivery of antirabies prophylaxis. PLoS Neglected Tropical Diseases, 2010, 4(6):e723. 16. Sterner RT et al. Tactics and economics of wildlife oral rabies vaccination, Canada and the United States. Emerging Infectious Diseases, 2009, 15(8):1176–1184. 17. Blanton JD et al. Rabies surveillance in the United States during 2010. Journal of the American Veterinary Medicine Association, 2011, 239(6):773–783. 18. Aubert MF. Costs and benefits of rabies control in wildlife in France. Revue Scientifique et Technique (International Office of Epizootics), 1999, 18(2):533–543. 19. Müller T et al. Elimination of terrestrial rabies in Germany using oral vaccination of foxes. Berliner und Munchener tierarztliche Wochenschrift, 2012, 125(5–6):178–190.

20. Cliquet F et al. Eliminating rabies in Estonia. PLoS Neglected Tropical Diseases, 2012, 6(2):e1535. 21. Demetriou P, Moynagh J. The European Union strategy for external cooperation with neighbouring countries on rabies control. Rabies Bulletin Europe, 2011, 35(1):5–7. 11

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22. Sistema de Información Epidemiológica. Washington DC, Pan American Health Organization and World Health Organization. (http://siepi. panaftosa.org.br; accessed March 2013). 23. Elimination of neglected diseases and other poverty-related infections. Pan American Health Organization and World Health Organization. 49th Directing Council. 61st session of the Regional Committee. Washington DC, 2009 [resolution CD49.R19]. (http://new.paho.org/hq/ dmdocuments/2009/CD49.R19%20(Eng.).pdf; accessed March 2013).

24. Interagency meeting on planning the prevention and control of neglected zoonotic diseases, Geneva, 5–6 July 2011. Geneva, World Health Organization, 2011 (WHO/HTM/NTD/NZD/2011; also available at whqlibdoc.who.int/publications/2011/9789241502931_eng.pdf; accessed March 2013). 25. Strategic framework for elimination of human rabies transmitted by dogs in the South-East Asia Region. New Delhi, WHO Regional Office for South-East Asia, 2012 (http://www.searo.who.int/topics/rabies/en/; accessed March 2013). 26. Sudarshan MK et al. Assessing the burden of human rabies in India: results of a national multi-center epidemiological survey. International Journal of Infectious Diseases, 2007, 11(1):29–35. 27. Yu J et al. The spatial and temporal dynamics of rabies in China. PLoS Neglected Tropical Diseases, 2012, 6(5):e1640.

28. Yin C-P. Analysis on factors related to rabies epidemic in China from 2007–2011. Virologica Sinica, 2012, 27(2):132–143. WHO Technical Report Series No. 982, 2013

29. Mallewa M et al. Rabies encephalitis in malaria-endemic area, Malawi, Africa. Emerging Infectious Diseases, 2007, 13(1):136–139. 30. Streicker DG et al. Ecological and anthropogenic drivers of rabies exposure in vampire bats: implications for transmission and control. Proceedings of the Royal Society B. Biological Sciences, 2012, 279(1742):3384–3392. 31. Mathers CD, Ezzati M, Lopez AD. Measuring the burden of neglected tropical diseases: the Global Burden of Disease Framework. PLoS Neglected Tropical Diseases, 2007, 1(2):e114. 32. WHO Expert Consultation on Rabies. First report. Geneva, World Health Organization, 2005 (WHO Technical Report Series, No. 931).

Classification of lyssaviruses

2. 2.1

Classification of lyssaviruses Distinguishing features of lyssaviruses

Rabies is an acute encephalitis or meningoencephalitis due to a lyssavirus infection. The etiological agents of rabies encephalitis belong to the Mononegavirales order, the Rhabdoviridae family and the Lyssavirus genus. Lyssaviruses have a 12-kb nonsegmented RNA genome of negative polarity that encodes five viral proteins (3´ to 5´): a nucleoprotein (N), a phosphoprotein (P), a matrix protein (M), a glycoprotein (G) and an RNA-dependent RNA polymerase (or large protein, L). The lyssavirus particle is shaped like a bullet, 100–300 nm long and 75 nm in diameter. It is composed of two structural and functional units: an internal helical nucleocapsid and an external envelope. The nucleocapsid consists of a ribonucleoprotein complex comprising the genomic RNA and tightly bound N protein together with the L and P proteins. The nucleocapsid is active for transcription and replication: the N-RNA template is processed by the L protein, which contains most of the RNA polymerase activities, and its cofactor, the P protein. The lipid envelope is derived from the host cytoplasmic membrane during budding. Knobbed glycoprotein spikes (5–10 nm long and about 3 nm in diameter) consisting of three glycosylated ectodomains, which binds the virions to host cell receptors, protrude through the virion membrane. The M protein forms oligomers that bind to the outside of the nucleocapsid, giving rigidity to the virion structure and providing a binding platform for the viral glycoprotein and the envelope membrane (1,2).

2.2 Criteria for differentiating between lyssaviruses Until the 1950s, the rabies virus was considered to be unique. Identification of serologically related viruses in Nigeria—Lagos bat virus from a pteropodid bat (3) and Mokola virus from a shrew (4)—showed that the structure of this virus group was more complex, and the terms ‘rabies-related viruses’ and ‘rabies serogroup’ were introduced (4). Another serologically related virus, Duvenhage virus, was isolated from a man who died of rabies after a bite of an insectivorous bat in 1970 in South Africa (5), representing a fourth serotype. The viruses regularly isolated from bats in Europe since the 1950s were related serologically to Duvenhage virus and were initially included in the Duvenhage serotype (6,7). Later, use of monoclonal antibodies made it possible to refine the classification of the ‘rabies serogroup’ (8). European bat lyssaviruses were not only distinguished from the African Duvenhage virus (9) but also separated into two distinct serotypes (10), temporally termed ‘biotypes’ (11). This differentiation was later supported by gene sequencing and phylogenetic analysis (12,13). Extensive phylogenetic studies of the diversity of rabies-related

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viruses led to the creation of the operational term ‘genotype’, which has since been used broadly in the scientific literature (12). New genotypes were identified, and quantitative criteria for their differentiation were proposed (12,14–18). To accommodate the growing variety of ‘rabies-related’ viruses, the genus Lyssavirus was established under the auspices of the International Committee on the Taxonomy of Viruses. The name of the genus was derived from Greek mythology: Lyssa (Λυσσα) was a goddess or spirit of rage, fury, raging madness and frenzy. The existing ‘genotypes’ served as a basis for the taxonomy of lyssavirus but were refined to satisfy the official rules of the International Committee, which apply to more complex entities such as viral species. The demarcation criteria for lyssavirus species include (19): ■■ Genetic distance, with a threshold of 80–82% nucleotide identity for the complete N gene, which provides better quantitative resolution than other genes, or 80–81% nucleotide identity for concatenated coding regions of the N+P+M+G+L genes. In general, all isolates belonging to the same species have higher identity values than the threshold, except the viruses currently included in the Lagos bat virus species. For that reason, some authors have suggested that Lagos bat virus be subdivided into several genotypes (20,21). In the absence of other sufficient demarcation characters, however, Lagos bat virus has not been separated into several species, as these representatives segregate into a monophyletic cluster in most phylogenetic reconstructions. ■■ Topology and consistency of phylogenetic trees obtained with various evolutionary models WHO Technical Report Series No. 982, 2013

■■ Antigenic patterns in reactions with nucleocapsid monoclonal antibodies (preceded by serological cross-reactivity and definition of lyssavirus serotypes with polyclonal antisera) ■■ When available, additional characteristics, such as ecological properties, host, geographical range and pathological features.

2.3

Present structure of the Lyssavirus genus

Currently, the International Committee on the Taxonomy of Viruses recognizes 12 Lyssavirus species (Table 3). On the basis of genetic distances and serological cross-reactivity, the genus has been subdivided into two phylogroups: ■■ Phylogroup I contains the species rabies virus, European bat lyssaviruses type 1 and type 2, Duvenhage virus, Australian bat lyssavirus, Aravan virus, Khujand virus and Irkut virus.

Classification of lyssaviruses

■■ Phylogroup II contains Lagos bat virus, Mokola virus and Shimoni bat virus. The remaining species of the genus, West Caucasian bat virus, cannot be included in either of these phylogroups and is suggested to be considered a representative of an independent phylogroup III. A further potential extension of the genus, a novel Bokeloh bat lyssavirus, was recently isolated from an insectivorous bat (Myotis nattereri) in France and Germany. This virus is related phylogenetically to European bat lyssavirus type 2 and Khujand virus (17,22). Another divergent lyssavirus, related phylogenetically to West Caucasian bat virus (therefore potentially a member of the proposed phylogroup III) and tentatively named Ikoma lyssavirus, was detected in an African civet (Civettictis civetta) in the United Republic of Tanzania (18). Bats are the reservoirs and vectors of lyssaviruses for 12 of the 14 recognized and proposed species, while the reservoirs of Mokola virus and Ikoma lyssavirus remain to be determined. Lyssaviruses show broad antigenic cross-reactivity at the nucleocapsid level, mainly because of sequence conservation of the N protein. Therefore, similar reagents can be used for diagnosis by immunofluorescence. The ectodomain of the G protein (which carries the main antigenic sites) is more variable, and there is cross-neutralization among lyssaviruses of the same phylogroup (amino acid identity in the ectodomain, >74%) but not between phylogroups (amino acid identity in the ectodomain, <62%). Experimental evidence indicates that the available vaccine strains, which all belong to rabies virus species in phylogroup I, are ineffective against infection with lyssaviruses in phylogroup II and West Caucasian bat virus. A similar lack of protection is likely for Ikoma lyssavirus.

2.4 References 1. Graham SC et al. Rhabdovirus matrix protein structures reveal a novel mode of self-association. PLoS Pathogens, 2008, 4:e1000251. 2. Ge P et al. Cryo-EM model of the bullet-shaped vesicular stomatitis virus. Science, 2010, 327:689–693.

3. Boulger LR, Porterfield JS. Isolation of a virus from Nigerian fruit bats. Transactions of the Royal Society of Tropical Medicine and Hygiene, 1958, 52:421–424.

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Table 3 Viruses currently included in the genus Lyssavirus Recognized and proposed species (abbreviation) Rabies virus (RABV) Primary host Geographical range Comments

Carnivora and bats (Chiroptera)

Terrestrial 1 mammals worldwide except in Australia, Antarctica and several islands; bats in the New World only Australia (and perhaps several nearby islands) Most of Europe, from Spain to the Ukraine North-western Europe Central Asia Central Asia France, Germany Eastern Asia Sub-Saharan Africa Sub-Saharan Africa Sub-Saharan Africa Kenya 2 3 4 5 6 7 8 9 10 11 12

Australian bat lyssavirus (ABLV) European bat lyssavirus, type 1 (EBL1) European bat lyssavirus, type 2 (EBL2) Khujand virus (KHUV) Aravan virus (ARAV) Bokeloh bat lyssavirus (BBLV) WHO Technical Report Series No. 982, 2013 Irkut virus (IRKV) Duvenhage virus (DUVV) Lagos bat virus (LBV) Mokola virus (MOKV) Shimoni bat virus (SHIBV) West Caucasian bat virus (WCBV)

Pteropodid bats (at least four species of Pteropus genus) and insectivorous bats (Saccolaimus albiventris) Insectivorous bats (predominantly Eptesicus serotinus) Insectivorous bats (predominantly Myotis daubentonii and M. dasycneme) Insectivorous bat Myotis mystacinus Insectivorous bat Myotis blythi Insectivorous bat Myotis nattereri Insectivorous bat Murina leucogaster Insectivorous bats Pteropodid bats of several genera (e.g. Eidolon helvum, Rousettus aegyptiacus, Epomophorus spp.) Unknown Insectivorous bat Hipposideros commersoni Insectivorous bats from genus Miniopterus

South-eastern Europe United Republic of Tanzania

13 14

Ikoma lyssavirus Not known (IKOV)

Classification of lyssaviruses

1. 2. 3.

Responsible for the vast majority of human rabies cases in the world. All currently available human and veterinary vaccine strains originate from this species. Given limited surveillance, the host range among insectivorous bats may be greater. Two human cases have been documented. Given the limited surveillance in eastern Europe and Asia, may be distributed more broadly, along the reservoir species range. Spillover infections in wild and companion animals and a very small number of human cases have been documented. Two human cases have been documented. Known from a single isolate. Given the limited surveillance in eastern Europe and Asia, may be distributed more broadly. No human cases have been documented. Known from two isolates. Given the limited surveillance in eastern Europe and Asia, may be distributed more broadly. No human cases have been documented. Known from a single isolate. Does not have species status and is not listed in current International Committee on the Taxonomy of Viruses documents. No human cases have been documented. Known from two isolates, from a bat and from a human. Known from four isolates, three of which came from humans bitten by bats and one from a bat, presumably of the Miniopterus species.

4. 5. 6. 7. 8. 9.

10. Constitutes several lineages with long genetic distances. In the future, may be subdivided into two or three separate species. Spillover infections reported in wild and companion animals. No human cases documented to date. 11. Twice isolated from shrews, once from a rodent. Most other isolates were obtained from companion animals, such as cats, as the result of spillover infection. Two human cases have been reported. 12. Known from a single isolate. Serological surveys suggest that H. commersoni is the probable reservoir. No human cases have been documented. 13. Known from a single isolate; however, serological surveys suggest that West Caucasian bat virus (or another serologically related virus) is present in Miniopterus bats in Africa (Kenya). No human cases have been documented. 14. Known from a single isolate from an African civet (Civettictis civetta). The natural host is unknown. Given the phylogenetic relatedness to the West Caucasian bat virus, the index case in an African civet may have resulted from a spillover infection of bat origin. No human cases have been documented.

WHO Expert Consultation on Rabies Second report

4. Shope RE et al. Two African viruses serologically and morphologically related to rabies virus. Journal of Virology, 1970, 6:690–692.

5. Meredith CD, Rossouw AP, van Praag Koch H. An unusual case of human rabies thought to be of chiropteran origin. South African Medical Journal, 1971, 45:767–769. 6. Schneider LG. Antigenic variants of rabies virus. Comparative Immunology, Microbiology and Infectious Diseases, 1982, 5:101–107. 7. Schneider LG, Barnard BJH, Schneider HP. Application of monoclonal antibodies for epidemiological investigations and oral vaccination studies: I. African viruses. In: Kuwert E et al., eds, Rabies in the tropics. Berlin, Springer-Verlag, 1985:49–53.

8. Wiktor TJ, Koprowski H. Monoclonal antibodies against rabies virus produced by somatic cell hybridization: detection of antigenic variants. Proceedings of the National Academy of Sciences of the United States of America, 1978, 75:3938–3942. 9. Dietzschold B et al. Antigenic diversity of the glycoprotein and nucleocapsid proteins of rabies and rabies-related viruses: implications for epidemiology and control of rabies. Reviews of Infectious Diseases, 1988, 10(S4):785–798 10. Bourhy H et al. Antigenic and molecular characterization of bat rabies virus in Europe. Journal of Clinical Microbiology, 1992, 30:2419–2426.

11. King A, Davis P, Lawrie A. The rabies viruses of bats. Veterinary Microbiology, 1990, 23:165–174. WHO Technical Report Series No. 982, 2013

12. Bourhy H, Kissi B, Tordo N. Molecular diversity of the Lyssavirus genus. Virology, 1993, 194:70–81.

13. Davis PL et al. Phylogeography, population dynamics, and molecular evolution of European bat lyssaviruses. Journal of Virology, 2005, 79:10487–10497. 14. Fraser GC et al. Encephalitis caused by a lyssavirus in fruit bats in Australia. Emerging Infectious Diseases, 1996, 2:327–331. 15. Kuzmin IV et al. Bat lyssaviruses (Aravan and Khujand) from Central Asia: phylogenetic relationships according to N, P and G gene sequences. Virus Research, 2003, 97:65–79.

Pathogenesis

16. Kuzmin IV et al. Phylogenetic relationships of Irkut and West Caucasian bat viruses within the Lyssavirus genus and suggested quantitative criteria based on the N gene sequence for lyssavirus genotype definition. Virus Research, 2005, 111:28–43. 17. Freuling C et al. Novel lyssavirus in a Natterer’s bat (Myotis nattereri), Germany. Emerging Infectious Diseases, 2011, 17:1519–1522. 18. Marston DA et al. Ikoma lyssavirus: identification of a highly divergent novel lyssavirus in an African civet (Civettictis civetta). Emerging Infectious Diseases, 2012, 18:664–667.

19. Dietzgen RG et al. Family Rhabdoviridae. In: King AMQ et al., eds. Virus taxonomy: ninth report of the International Committee on Taxonomy of Viruses. Oxford, Elsevier, 2011:686–714. 20. Delmas O et al. Genomic diversity and evolution of the lyssaviruses. PLoS One, 2008, 3:e2057.

21. Markotter W et al. Phylogeny of Lagos bat virus: challenges for lyssavirus taxonomy. Virus Research, 2008, 135:10–21. 22. Picard-Meyer E et al. Découverte d’une chauve-souris de Natterer infectée par un lyssavirus Bokeloh en Moselle en 2012. Bulletin Epidémiologique Santé animale, Alimentation, 2012, 55:25 (http://www. anses.fr/bulletin-epidemiologique/).

3. Pathogenesis Rabies virus enters the body through wounds or by direct contact with mucosal surfaces. It cannot cross intact skin. Rabies virus replicates in the bitten muscle and gains access to motor endplates and motor axons to reach the central nervous system (1–5). Virions are carried in transport vesicles (6) and travel to the central nervous system exclusively by fast retrograde transport along motor axons, with no uptake by sensory or sympathetic endings (1–3,5). Viruses can also enter motor axons in peripheral nerves directly during a penetrating injury (1,3,4). In some bat variants, viral propagation may also occur via sensory nerves due to skin tropism (3,7,8). The incubation period varies from 5 days to several years (usually 2–3 months; rarely more than 1 year), depending on the amount of virus in the inoculum, the density of motor endplates at the wound site and the proximity of virus entry to the central nervous system (3–5). Muscle-specific micro-RNA may 19

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contribute to this eclipse phase by suppressing viral transcription and replication in the muscle (9,10). The estimated speed of virus migration depends on whether it moves by centripetal retrograde axonal transport or centrifugal spread. In centripetal retrograde axonal transport, migration is fast, with speeds of 5–100 mm/day or even faster, because neuronal populations of the same synaptic order located at various distances, e.g. 10 µm to 2 cm, are infected simultaneously (1,5). Conversely, centrifugal spread is slow, probably mediated by passive diffusion rather than active transport (1–3,5). The first rapid centripetal phase leads to wide transneuronal transfer within the central nervous system and to infection of dorsal root ganglia via their central connections with the initially infected motor neurons and spinal interneurons (1–3,5). The virus then moves centrifugally from the central nervous system via slow anterograde axoplasmic flow in motor axons to the ventral roots and nerves and in peripheral sensory axons of the infected dorsal root ganglia, leading to infection of muscle spindles, skin, hair follicles and other non-nervous tissues, such as salivary glands, heart muscle, lung and abdominal visceral organs via their sensory innervation (3–5). By the time of clinical onset, the virus is widely disseminated throughout the central nervous system and probably to extra-neural organs (11). The first specific clinical symptom is neuropathic pain at the site of the bite. This is caused by virus replication in dorsal root ganglia and inflammation induced by cellular immunity (12). Human rabies can manifest as furious or paralytic forms, which cannot be correlated with a specific anatomical localization of rabies virus in the central nervous system (12–14). The major clinical signs are probably due to different site-specific responses (14). Functional neuronal impairment also explains coma. Electrophysiological studies with pathological correlates show that peripheral nerve axonopathy or myelinopathy is responsible for weakness in paralytic rabies (7,12). Preferential entry via the motor route explains why subclinical anterior horn cell dysfunction precedes sensory loss in furious rabies and is initially localized at body segments corresponding to the site of the bite, progressively spreading to other locations (3,5,12). The same considerations apply to prodromal symptoms and signs in paralysed patients (3–5). It is likely that less virus is present in the brain in paralytic rabies (when consciousness is preserved) than in furious rabies. Diffusion tensor imaging in canine paralytic rabies showed that neural tract integrity is compromised at brainstem level, limiting viral propagation to the forebrain (5,15,16). A viral immune evasive strategy with blood–brain barrier integrity prevents eradication of the virus in the central nervous system (4,16–21). There is no evidence of immune suppression or accelerated death in rabies-infected patients (15,16). Rabies with atypical clinical and/or neuroimaging features is increasingly recognized (4,22–26). Whether this is due to atypical virus variants, a host immune

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Pathogenesis

response or large doses of virus inoculum (as in the case of organ transplantation from rabies-infected donors) is unknown. Without intensive care, death occurs within 2 weeks after the appearance of clinical symptoms (5,7).

3.1 References 1. Ugolini G. Use of rabies virus as a transneuronal tracer of neuronal connections: implications for the understanding of rabies pathogenesis. Developments in Biologicals (Basel), 2008, 131:493–506. 2. Ugolini G. Advances in viral transneuronal tracing. Journal of Neuroscience Methods, 2010, 194:2–20. 3. Ugolini G. Rabies virus as a transneuronal tracer of neuronal connections. Advances in Virus Research, 2011, 79:165–202. 4. Hemachudha T, Laothamatas J, Rupprecht CE. Human rabies: a disease of complex neuropathogenetic mechanisms and diagnostic challenges. Lancet Neurology, 2002, 1(2):101–109.

5. Hemachudha T et al. Human rabies: neuropathogenesis, diagnosis and management. Lancet Neurology, 2013, 12(5):498–513. 6. Klingen Y, Conzelmann KK, Finke S. Double-labeled rabies virus: live tracking of enveloped virus transport. Journal of Virology, 2008, 82(1):237–245. 7. Hemachudha T et al. Pathophysiology of human paralytic rabies. Journal of Neurovirology, 2005, 11(1):93–100.

11. Hemachudha T et al. Rabies. Current Neurology and Neuroscience Reports, 2006, 6(6):460–468. 12. Mitrabhakdi E et al. Difference in neuropathogenetic mechanisms in human furious and paralytic rabies. Journal of Neurological Science, 2005, 238(1–2):3–10.

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13. Dumrongphol H et al. Alteration of muscarinic acetylcholine receptors in rabies viral-infected dog brains. Journal of Neurological Science, 1996, 137(1):1–6. 14. Thanomsridetchai N et al. Comprehensive proteome analysis of hippocampus, brainstem, and spinal cord from paralytic and furious dogs naturally infected with rabies. Journal of Proteome Research, 2011, 10(11):4911–4924. 15. Laothamatas J et al. Furious and paralytic rabies of canine origin: neuroimaging with virological and cytokine studies. Journal of Neurovirology, 2008, 14(2):119–129. 16. Laothamatas J, Sungkarat W, Hemachudha T. Neuroimaging in rabies. Advances in Virus Research, 2011, 79:309–327.

17. Lafon M. Evasive strategies in rabies virus infection. Advances in Virus Research, 2011, 79:33–53. 18. Laothamatas J et al. MR imaging in human rabies. American Journal of Neuroradiology, 2003, 24(6):1102–1109. 19. Roy A et al. Failure to open the blood–brain barrier and deliver immune effectors to central nervous system tissues leads to the lethal outcome of silver-haired bat rabies virus infection. Journal of Virology, 2007, 81(3):1110–1118. WHO Technical Report Series No. 982, 2013

20. Roy A, Hooper DC. Immune evasion by rabies viruses through the maintenance of blood–brain barrier integrity. Journal of Neurovirology, 2008, 14(5):401–411.

21. Kasempimolporn S et al. Human immune response to rabies nucleocapsid and glycoprotein antigens. Clinical and Experimental Immunology, 1991, 84(2):195–199. 22. Hemachudha T, Phuapradit P. Rabies. Current Opinions in Neurology, 1997, 10(3):260–267. 23. Burton EC et al. Rabies encephalomyelitis: clinical, neuroradiological, and pathological findings in 4 transplant recipients. Archives of Neurology, 2005, 62(6):873–882. 24. Maier T et al. Management and outcomes after multiple corneal and solid organ transplantations from a donor infected with rabies virus. Clinical Infectious Diseases, 2010, 50(8):1112–1119.

Diagnosis

25. Shantavasinkul P et al. Failure of rabies postexposure prophylaxis in patients presenting with unusual manifestations. Clinical Infectious Diseases, 2010, 50(1):77–79. 26. Human rabies—Minnesota, 2007. Morbidity and Mortality Weekly Report, 2008, 57(17):460–462.

4. Diagnosis Rabies is an acute, progressive encephalitis caused by a lyssavirus. Clinical diagnosis of encephalitis can be challenging, and all suspected and probable clinical cases of rabies should be confirmed by laboratory methods when possible. During the past decade, significant progress has been made in laboratory diagnostic methods for clinical case confirmation. Each country should have a national reference laboratory with the capacity for basic rabies diagnosis and case confirmation by suggested modern techniques (1–7). Where such expertise is lacking, training and reference diagnostic capability can be obtained from WHO collaborating centres (8) (Annex 8) and from reference centres of the World Organisation for Animal Health (OIE) for animal rabies (9).

4.1 Standard case definitions for rabies All countries should use standard case definitions for rabies supported by laboratory-based surveillance of suspected cases in humans and animals. According to the WHO recommended standards and strategies for surveillance, prevention and control of communicable diseases, a clinical case of rabies is defined as: a subject presenting with an acute neurological syndrome (i.e. encephalitis) dominated by forms of hyperactivity (i.e. furious rabies) or paralytic syndromes (i.e. dumb rabies) progressing towards coma and death, usually by cardiac or respiratory failure, typically within 7–10 days after the first sign, if no intensive care is instituted.

One or more of the following laboratory criteria should be used to confirm a clinical case: ■■ presence of viral antigens; ■■ isolation of virus in cell culture or in laboratory animals;

■■ presence of viral-specific antibodies in the cerebrospinal fluid or the serum of an unvaccinated person; or 23

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■■ presence of viral nucleic acids detected by molecular methods in samples (e.g. brain biopsy, skin, saliva, concentrated urine) collected post mortem or intra vitam. Cases of rabies are basically classified as follows: ■■ suspected: a case that is compatible with a clinical case definition ■■ probable: a suspected case plus a reliable history of contact with a suspected rabid animal ■■ confirmed: a suspected or probable case that is laboratory-confirmed. In some situations, a clinical suspicion of encephalitis or a history of animal exposure may be lacking; however, a case would still be considered confirmed by appropriate laboratory diagnostic testing. A record form for possible exposure to rabies is given in Annex 2.

4.2 Clinical diagnosis A presumptive diagnosis of rabies, an acute, progressive encephalomyelitis, with the highest case fatality rate of any infectious disease, is simple in a person presenting with a compatible illness after documented exposure to a laboratoryconfirmed rabid animal. Specific clinical signs of hydro- or aerophobia in humans provide a strong suspicion of rabies, if they are well documented. In the absence of a history of exposure or paramount signs, however, the diagnosis of rabies on clinical grounds alone is difficult and often unreliable. For example, some patients can present with a paralytic or Guillain-Barré-like syndrome or other atypical features (10). Atypical or non-classical rabies is increasingly recognized and may be responsible for underreporting of cases. Detailed clinical information on patients with atypical rabies, especially cases associated with exposure to bats or other wildlife, has been reported (11,12). Human case reports can be found in a variety of peer-reviewed publications, national and international reports and electronic sources, such as the website of the United States Centers for Disease Control and Prevention. Classical signs of brain involvement include spasms in response to tactile, auditory, visual or olfactory stimuli (e.g. aerophobia and hydrophobia) alternating with periods of lucidity, agitation, confusion and signs of autonomic dysfunction (10). Spasms may occur in rabid patients in whom excitation is prominent. Spontaneous inspiratory spasms can occur continuously until death, and their presence may facilitate a clinical diagnosis. Excitation is less evident in paralytic rabies, and phobic spasms may appear in only 50% of such patients. During the early stages of paralytic rabies, notable signs may include myoedema at percussion sites, usually in the region of the chest, deltoid muscle and thigh, piloerection and fasciculations.

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Diagnosis

Magnetic resonance imaging, performed with adequate precautions for potentially infectious patients, can be helpful (10,13). Abnormal, illdefined, mildly hypersignal T2 images involving the brain-stem, hippocampus, hypothalamus, deep and subcortical white matter and deep and cortical grey matter indicate a diagnosis of rabies, regardless of clinical type. Gadolinium enhancement may appear clearly only in later stages, when patients lapse into a coma. Such patterns can help differentiate rabies from other viral encephalitides, not in terms of location, but in the T2 image appearance and in the pattern of contrast enhancement, when compared to consciousness status. Computerized tomography of the brain is of little diagnostic value. Rabies should be included in the differential diagnosis of all patients who present with unexplained, acute, progressive viral encephalitis, even in areas where the disease is rare, as it can occur locally in wildlife, such as bats, can be acquired during travel to enzootic areas and because imported cases of human and animal rabies continue to occur (2,12). In addition, rabies may be misdiagnosed and death ascribed to another cause (e.g. cerebral malaria), without adequate epidemiological scrutiny and laboratory confirmation (2,4,14). As transmission of rabies virus to recipients of solid organ transplants has been described, all potential organ donors who present with a compatible encephalitis should be screened and tested to determine whether they present an infectious risk, by examining suitable ante- or post-mortem specimens by sensitive, specific laboratory methods (2,4,6).

4.3

Biosafety, sampling and specimen transport for laboratory diagnosis

4.3.1 Biosafety Rabies has the highest case fatality rate of any currently recognized infectious disease. Safety is therefore of paramount importance when working with lyssaviruses. In general, biosafety level 2 safety practices are adequate for routine laboratory activities such as handling animals, necropsy, collection preparation and processing samples (5–7). The basic facility design should be adequate, and precautions should include personal protective equipment (e.g. clothing, gloves, eye protection) and vaccination. Certain activities may require a biosafety level 3 classification, such as production of large quantities of concentrated virus, procedures that may generate aerosols (e.g. homogenization of tissue suspensions) and working with newly isolated lyssaviruses for which the effectiveness of current prophylaxis is not known. All national safety guidelines for working with infectious agents should be followed. 25

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4.3.2 Sampling for intra-vitam diagnosis in humans Secretions, biological fluids (e.g. saliva, spinal fluid, tears) and tissues (skin biopsy samples and hair follicles at the nape of the neck) can be used to diagnose rabies during life (1,2,5,6,15,16). Three saliva samples taken at intervals of 3–6 h, skin and hair follicules are the most sensitive samples. Ideally, samples should be stored at –20 °C or less. Serum should be collected from blood samples before freezing and stored at –20 °C or less.

4.3.3 Sampling for post-mortem diagnosis in humans and animals Brain tissue is the preferred specimen for post-mortem diagnosis in both humans and other animals (4,5,7). If a brain biopsy cannot be performed, such as in field studies, tissue samples can be collected via the trans-orbital or trans-foramen magnum route (1). Preservation in glycerine (at +4 °C or –20 °C) or drying smears of brain tissue on filter paper containing proper inactivating chemicals (at +30 °C) allows safe, stable transport of infected material, but safe, effective viral inactivation must be ensured before shipment (1,17). Other specimens, such as skin and hair follicles taken at the nape of the neck, are also highly sensitive for post-mortem diagnosis (5,6,18).

4.3.4 Transport of specimens Specimens for a diagnosis of rabies should be shipped according to national and international regulations to avoid exposure. Information on the appropriate International Air Transport Association shipment classification can be found on the Association’s website (19), and packing instructions are given in the WHO recommendations on transport of infectious substances (20). Diagnostic specimens should be frozen or refrigerated; if they are shipped at ambient temperature, they should be preserved in 50% glycerine–saline solution. The source of specimens for diagnosis and the storage conditions clearly affect the results of any laboratory procedure. Rabies can be diagnosed in fresh (unfixed) specimens from several different tissue sources, but they are preferably refrigerated or frozen. If samples are stored in 50% glycerol–saline before testing, they must be washed thoroughly; freezing and long-term storage are not recommended. Unlike the processing of fresh or frozen tissues, acetone fixation is not recommended before direct fluorescent antibody testing of samples stored in glycerol saline. The choice of specimens and handling depend on the test to be performed and the stage of the disease (1,6). Examination of chemically fixed specimens for viral antigens can be both sensitive and specific if appropriate tissues and tests are used (21). Formalin fixation of brain tissue is not, however, a suitable method for routine diagnosis, because it delays the test results. If specimens are received in formalin, the

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Diagnosis

duration of fixation should be approximately 7–14 days before embedding in paraffin. Wet tissue specimens should be transferred from formalin to absolute ethanol for subsequent molecular diagnosis and antigen detection. Typical intracytoplasmic inclusions in fixed brain tissue can be detected in neurons by validated immunohistochemical methods (22).

4.4 Laboratory techniques for post-mortem diagnosis of rabies A definitive diagnosis of rabies can be made only with the appropriate laboratory methods. The basic techniques are described in the WHO publication Laboratory techniques in rabies (5) and the OIE Manual of diagnostic tests and vaccines for terrestrial animals (7). Participation in routine quality management is strongly recommended when using any of the laboratory techniques described (6).

4.4.1 Viral antigen detection The direct fluorescent antibody technique is a rapid, sensitive, specific method for diagnosing rabies in animals and humans (5–7,23,24) and is the gold standard for rabies diagnosis. The accuracy of the test depends, however, on variables such as the expertise of the examiner, the quality of the anti-rabies conjugate and basic equipment, including the fluorescence microscope. The test is based on microscopic examination of impressions or smears of brain tissue after incubation with anti-rabies polyclonal globulin or broadly cross-reactive monoclonal antibodies conjugated with fluorescein isothiocyanate. The diagnostic conjugate should be of high quality, and the appropriate working dilution for optimal performance and detection of virus-specific antigens must be determined. Impressions (or smears) of samples from the brain-stem and cerebellum are recommended for high sensitivity of the test (6). The hippocampi (Ammon horns) may be included but are not necessary for a definitive diagnosis. Other methods for the detection of lyssavirus antigens, such as enzymelinked immunosorbent assays (ELISAs) and direct rapid immunohistochemistry tests, have provided consistently reproducible results in several laboratories (6,25–28). Extensive evaluation of direct rapid immunohistochemistry tests has shown that their sensitivity and specificity are at least comparable to those of the direct fluorescent antibody test, the traditional standard in rabies diagnosis. This test allows rapid onsite testing by light microscopy and should facilitate decentralized epidemiological surveys if the reagents become commercially available. The Consultation recommends further development of direct rapid immunohistochemistry tests as an alternative to the direct fluorescent antibody test for improved decentralized laboratory-based surveillance. Lateral flow tests for rapid detection of rabies virus antigen under field conditions have been developed (29–31); however, the procedures for the 27

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commercially available assays have not been standardized or harmonized for proper use and adequate validation according to international standards (5).

4.4.2 Virus isolation Virus might have to be isolated to confirm the results of antigen detection tests and for further amplification or characterization of an isolate (5). Viruses can be isolated in cell cultures, such as neuroblastoma cells, or by intracranial inoculation into mice. Virus isolation in animals should be replaced by alternative methods, whenever possible. Murine neuroblastoma cells (e.g. NA C1300) are more susceptible to field isolates of lyssavirus than other cell lines tested (5,6). Virus isolation in neuroblastoma cell culture is at least as efficient as animal inoculation, especially for small quantities of virus. Cell culture isolation also reduces the time required for diagnosis, from 10–21 days with the mouse inoculation test to only 1–2 days. If the conditions are not optimal, however, such as decomposed brain, false-negative results may be obtained. When cell culture facilities or molecular methods are not available, animal inoculation can be used. If a rapid answer is required, suckling mice (<3 days old) are preferred to weanling or adult mice, because they are more susceptible than older animals. The observation period may be shortened by fluorescent antibody examination of brains of inoculated mice euthanized 14–21 days (or more) after inoculation or when clinical signs appear.

4.4.3 Viral RNA detection Molecular methods, such as the reverse transcription polymerase chain reaction (RT-PCR) and other amplification techniques, are playing an increasingly important role in many countries but are not recommended currently for routine post-mortem diagnosis of rabies if brain tissue is available, when the direct fluorescent antibody test should be used (5). Molecular techniques can be used, however, for epidemiological surveys in laboratories with strict quality control procedures and with experience and expertise in using such techniques; they can also be used for ante-mortem diagnosis in humans. The use of robust positive controls or in-process controls is strongly recommended.

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4.5 Techniques for intra-vitam diagnosis of rabies in humans Many laboratory methods can be used to confirm a clinical case of rabies while the patient is still alive (2,32). Use of intra-vitam techniques for the diagnosis of rabies in animals is, however, strongly discouraged. The sensitivity of a technique for diagnosing rabies varies widely according to the stage of the

Diagnosis

disease, immunological status, intermittent viral excretion and the training of the technical staff. While a positive validated result is indicative of rabies, a negative result does not necessarily rule out the infection. It is not recommended that a brain biopsy sample be taken solely for the diagnosis of rabies, but it can be useful when obtained (6,10). A diagnosis of rabies in a patient suspected of having the disease is valuable for multiple reasons, including: specific characterization of the causative agent and of the potential source of infection, especially when a history of exposure to an animal is lacking; identification of other people who may have been exposed to the same animal during the public health investigation; application of appropriate measures for infection control to prevent exposure from contact with the patient; administration of post-exposure prophylaxis to people exposed to the patient’s infectious secretions; case closure and grief counselling with family members; consideration of experimental therapeutic options; monitoring of viral loads and patient response if treatment is undertaken; less invasive techniques for documenting the human burden of disease, given the infrequency of autopsies; and indication of another infectious agent if the tests are negative.

4.5.1 Viral antigen detection Viral antigens can be detected with the direct fluorescent antibody test in skin biopsy samples or hair follices from patients with clinical rabies (33). The results are independent of the antibody status of the patient, and specimens may be positive during the early phase of the disease. Skin samples are usually taken from the nuchal area of the neck, with hair follicles containing peripheral nerves. Examination of several sections may be required to detect viral antigens around the base of hair follicles. The quality of the samples is of paramount importance, as the absence of follicles decreases the sensitivity of the test. This technique may not be practicable in all settings, because a cryostat is required to prepare frozen sections of skin; it should be replaced by detection of viral RNA (6,12,18,34). Fluorescent antibody testing of corneal impressions is rarely reliable in most clinical settings, and it is not recommended as a routine test because of the risk of corneal scarification, particularly in patients with encephalitis and not rabies. Immunochromatographic methods have been developed to detect rabies antigen directly in saliva or in brain tissue from animals (29–31) but still require standardization and stringent quality control.

4.5.2 Viral antibody detection Neutralizing antibodies in the serum of unvaccinated patients or in cerebrospinal fluid can be measured with a virus neutralization test, including the rapid fluorescent focus inhibition test and the fluorescent antibody virus neutralization test (27,35–37). If virus-neutralizing antibodies are present in serum, they tend 29

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to appear on average 7–8 days after clinical symptoms. Viral antibodies are infrequently found in cerebrospinal fluid, depending in part on the clinical stage of the disease. Antibody titres against rabies glycoprotein measured by ELISA correlate well with those measured by virus neutralization, and ELISA is easier to perform routinely (27,35). Rapid detection of antibodies (immunoglobulins G and M) to other viral antigens, (e.g. nucleoprotein) may also be useful, as they may appear before neutralizing antibodies (12).

4.5.3 Viral RNA detection Molecular detection methods are highly sensitive for diagnosis (1–3,5,10,11,14– 18,24,32,38–42), although, like all laboratory methods, they require standardization and stringent quality control. Lyssavirus RNA can be detected and amplified not only from brain tissue but also from other biological fluids and tissue samples (e.g. saliva, cerebrospinal fluid, tears, skin, concentrated urine and hair follicles). Serial samples of, for example, saliva and urine should be tested, as the virus is excreted intermittently.

4.5.4 Virus isolation Virus is preferably isolated from brain or saliva or other biological samples in which it is highly likely to be detected (1–7). The success rate depends in part on the immunological status of the patient (more positive results are obtained in those without antibodies), the intermittence of viral excretion and the number of consecutive passages in cell culture. Liquid specimens or swabs should be frozen after collection, the content of the swab having been expelled into the collection medium. Under no circumstances should preservatives be added to the collection medium. Specimens may contain no infectious virus even during the late stage of the disease.

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4.6 Virus identification with molecular techniques: epidemiological considerations Thousands of lyssavirus isolates from humans, domestic animals and wildlife have been compared with molecular techniques, leading to basic identification and classification of lyssaviruses and the demonstration that virus isolates from a given geographical area or species have unique genetic sequences. In most cases, these differences can be used to identify the principal animal hosts (e.g. bat, dog, fox) and to infer the source of infection when a definitive history of exposure is lacking (1–3,5,10–12,14–17,29,32,33,38–42).

Diagnosis

4.7 References 1. Barrat J et al. Rabies diagnosis. Developments in Biologics (Basel), 2006, 125:71–77. 2. Dacheux L et al. More accurate insight into the incidence of human rabies in developing countries through validated laboratory techniques. PLoS Neglected Tropical Diseases, 2010, 4:e765. 3. Dürr S et al. Rabies diagnosis for developing countries. PLoS Neglected Tropical Diseases, 2008, 2(3):e206.

4. Fooks AR et al. Emerging technologies for the detection of rabies virus: challenges and hopes in the 21st century. PLoS Neglected Tropical Diseases, 2009, 3(9):e530. 5. Meslin FX et al., eds. Laboratory techniques in rabies, 4th ed. Geneva, World Health Organization, 1996.

6. Orciari LA, Rupprecht CE. Rabies. In: Versalovic J et al., eds. Manual of clinical microbiology, 10th ed. Washington DC, ASM Press, 2011:1470– 1478. 7. Manual of diagnostic tests and vaccines for terrestrial animals, 6th ed. Paris, World Organisation for Animal Health, 2011 (http://www.oie. int/international-standard-setting/terrestrial-manual). 8. WHO collaborating centres database and portal. Geneva, World Health Organization (http://apps.who.int/whocc/).

9. Reference experts and laboratories. Paris, World Organisation for Animal Health (http://www.oie.int/our-scientific-expertise/references laboratories/list-of-laboratories/). 10. Rupprecht CE, Hemachudha T. Rabies. In: Scheld M, Whitley RJ, Marra C, eds. Infections of the central nervous system. Philadelphia, Lippincott, Williams & Wilkins, 2004:243–259. 11. Feder HM et al. Rabies: still a uniformly fatal disease? Historical occurrence, epidemiological trends, and paradigm shifts. Current Infectious Disease Reports, 2012, 14:408–422. 12. Petersen BW, Rupprecht CE. Human rabies epidemiology and diagnosis. In: Tkachev S, ed. Non-flavivirus encephalitis. Rijeka, InTech, 2011. 31

13. Laothamatas J et al. MR imaging in human rabies. American Journal of Neuroradiology, 2003, 24:1102–1109.

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14. Mallawa M et al. Rabies encephalitis in a malaria-endemic area of Malawi, Africa. Emerging Infectious Diseases, 2007, 13:136–139. 15. Madhusudana SN, Sukumaran SM. Antemortem diagnosis and prevention of human rabies. Annals of Indian Academy of Neurology, 2008, 11(1):3–12. 16. Wacharapluesadee S, Hemachudha T. Ante- and post-mortem diagnosis of rabies using nucleic acid-amplification tests. Expert Review of Molecular Diagnosis, 2010, 10(2):207–218. 17. Picard-Meyer E et al. Use of filter paper (FTA) technology for sampling, recovery and molecular characterisation of rabies viruses. Journal of Virological Methods, 2007, 140(1–2):174–182.

18. Dacheux L et al. A reliable diagnosis of human rabies based on analysis of skin biopsy specimens. Clinical Infectious Diseases, 2008, 47(11):1410– 1417. 19. Shipping guidelines for hazardous goods. Montreal, Quebec, International Air Transport Association (www.iata.org/whatwedo/cargo/dangerous_ goods/pages/infectious_substances.aspx). 20. Guidance on the regulations for transport of infectious substances 2007– 2008. Geneva, World Health Organization. (www.who.int/csr/resources/ publications/biosafety/WHO_CDS_EPR_2007_2cc.pdf). 21. Coertse J et al. A case study of rabies diagnosis from formalin-fixed brain material. Journal of the South African Veterinary Association, 2011, 82(4):250–253. WHO Technical Report Series No. 982, 2013

22. Stein LT et al. Immunohistochemical study of rabies virus within the central nervous system of domestic and wildlife species. Veterinary Pathology, 2010, 47(4):630–633. 23. Robardet E et al. International interlaboratory trials on rabies diagnosis: an overview of results and variation in reference diagnosis techniques (fluorescent antibody test, rabies tissue culture infection test, mouse inoculation test) and molecular biology techniques. Journal of Virological Methods, 2011, 177:15–25. 24. Rudd RJ et al. A need for standardized rabies-virus diagnostic procedures: effect of cover-glass mountant on the reliability of antigen detection by the fluorescent antibody test. Virus Research, 2005, 111(1):83–88.

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25. Lembo T et al. Evaluation of a direct, rapid immunohistochemical test for rabies diagnosis. Emerging Infectious Diseases, 2006, 12(2):310–313. 26. Madhusudana SN et al. Evaluation of a direct rapid immunohistochemical test (dRIT) for rapid diagnosis of rabies in animals and humans. Virologica Sinica, 2012, 27(5):299–302.

27. Welch RJ et al. An evaluation of two commercially available ELISAs and one in-house reference laboratory ELISA for the determination of human anti-rabies virus antibodies. Journal of Medical Microbiology, 2009, 58(6):806–810. 28. Xu G et al. WELYSSA: a simple tool using mouse monoclonal antibodies for the detection of lyssavirus nucleocapsid in rabies suspected specimens. Developments in Biologics (Basel), 2008, 131:555–561. 29. Kasempimolporn S et al. Evaluation of a rapid immunochromatographic test strip for detection of rabies virus in dog saliva samples. Journal of Veterinary Diagnostic Investigation, 2011, 23(6):1197–1201. 30. Markotter W et al. Evaluation of a rapid immunodiagnostic test kit for detection of African lyssaviruses from brain material. Onderstepoort Journal of Veterinary Research, 2009, 76(2):257–262. 31. Servat A et al Evaluation of a rapid immunochromatographic diagnostic test for the detection of rabies from brain material of European mammals. Biologicals, 2012, 40(1):61–66.

32. Hemachudha T, Wacharapluesadee S. Ante-mortem diagnosis of human rabies. Clinical Infectious Diseases, 2004, 39:1085–1086. 33. Crepin P et al. Intravitam diagnosis of human rabies by PCR using saliva and cerebrospinal fluid. Journal of Clinical Microbiology, 1998, 36(4):1117–1121. 34. Macedo CI et al. Diagnosis of human rabies cases by polymerase chain reaction of neck-skin samples. Brazilian Journal of Infectious Diseases, 2006, 10(5):341–345.

35. Feyssaguet M et al. Multicenter comparative study of a new ELISA, Platelia Rabies II, for the detection and titration of anti-rabies glycoprotein antibodies and comparison with the rapid fluorescent focus inhibition test (RFFIT) on human samples from vaccinated and non-vaccinated people. Vaccine, 2007, 25(12):2244–2251. 33

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36. Nishizono A et al. Evaluation of an improved rapid neutralizing antibody detection test (RAPINA) for qualitative and semiquantitative detection of rabies neutralizing antibody in humans and dogs. Vaccine, 2012, 30(26):3891–3896. 37. Wright E et al. A robust lentiviral pseudotype neutralisation assay for in-field serosurveillance of rabies and lyssaviruses in Africa. Vaccine, 2009, 27(51):7178–7186. 38. Hughes GJ et al. Evaluation of a TaqMan PCR assay to detect rabies virus RNA: influence of sequence variation and application to quantification of viral loads. Journal of Clinical Microbiology, 2004, 42:299–306. 39. Wacharapluesadee S et al. Development of a TaqMan real-time RTPCR assay for the detection of rabies virus. Journal of Virological Methods, 2008, 151:317–320.

40. Wacharapluesadee S et al. Comparative detection of rabies RNA by NASBA, real-time PCR and conventional PCR. Journal of Virological Methods, 2011, 175(2):278–282. 41. Wacharapluesadee S et al. Detection of rabies viral RNA by TaqMan realtime RT-PCR using non-neural specimens from dogs infected with rabies virus. Journal of Virological Methods, 2012, 184(1–2):109–112.

42. Wakeley PR et al. Development of a real-time, TaqMan reverse transcription-PCR assay for detection and differentiation of lyssavirus genotypes 1, 5, and 6. Journal of Clinical Microbiology, 2005, 43:2786– 2792. WHO Technical Report Series No. 982, 2013

Management of patients before and after death

5.1 Rabies survivors and treatment protocols Although rabies is considered a fatal disease, survival has been documented during the past few decades, particularly in cases associated with bat variants (1). After successful treatment in 2004 of an adolescent in the USA with the Milwaukee protocol (2), attempts were made in the USA and in some countries of South America to treat rabies patients, albeit with little success (3). In considering a possible treatment modality for rabies patients, the following should be kept in mind (1).

Management of patients before and after death

■■ Rabies is not invariably fatal in animals, but a very small number of humans have recovered. ■■ At present, it is not possible to predict which patients are likely to recover. ■■ All survivors, with or without treatment, had a vigorous, early immune response. ■■ Studies to identify management protocols, procedures for immunomodulation and new medications, including antiviral drugs, are encouraged. ■■ Human treatment must be proven to be safe and not further harm the patient.

5.2 Clinical management of rabies patients Patients remain conscious, are often aware of the nature of their illness and are usually extremely agitated, particularly when excitation is predominant. Furthermore, they are often isolated because of the perceived risk of transmission of the virus through contact. Patients with confirmed rabies should receive adequate sedation and care in an appropriate medical facility, preferably in a private room, with suitable emotional and physical support. Repeated intravenous morphine or benzodiazepines is effective in relieving the severe agitation, anxiety and phobic spasms that afflict patients with furious rabies (1). Once furious rabies has been diagnosed, invasive procedures should be avoided, and the patient should be cared for in a private, quiet, draft-free area. In view of the inevitability of death in most cases, treatment should focus on comfort, with heavy sedation (barbiturates, morphine) and avoidance of intubation or life-support measures once the diagnosis is certain (1).

5.3 Transmission via organ transplantation Rabies virus is present in many tissues in the terminal stages of disease. Caution should be exercised before transplanting organs from people who have died with neurological symptoms and signs, as several cases of rabies due to organ and tissue transplantation have been documented (4,5). Testing for common or highly fatal infections should be balanced against the urgency of transplanting a viable organ. Rare diseases will not be identified until the techniques become available. Corneal transplantation, which is common in developing countries, should be performed with caution.

WHO Expert Consultation on Rabies Second report

5.4 Recommendations for health care personnel and patients’ family members The care of people in whom rabies is diagnosed may create anxiety among medical and nursing staff and in the media and the public. Human rabies does not pose any greater risk to health care staff than most bacterial or viral infections if routine precautions are used, especially during intubation and suctioning. Post-exposure prophylaxis should be provided for health care personnel considered to be at risk after careful assessment, and they should be reminded of the importance of adhering to barrier nursing, as recommended for all infectious diseases. Hospitals that are likely to receive rabies patients may consider preexposure vaccination for health care staff who may be involved in their management. It may sometimes be necessary to immunize the partners of patients, as close contact and sexual intercourse in the early stages of the disease carry a risk for transmission.

5.5 Management of the bodies of patients who have died of rabies The body of a patient suspected to have died of rabies should be labelled as infectious. The risk for transmission to others is, however, small if normal precautions are taken. Blood does not contain the virus, but it is present in many tissues and fluids, such as those of the central nervous system and salivary glands (1). If embalming or autopsy is performed, it should be undertaken carefully, with appropriate precautions and personal protective equipment. Tissues and body fluids should be disposed of in the same manner as for other infectious diseases. The body of the deceased should be buried or cremated, depending on their religious practice.

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5.6 References 1. Hemachudha T et al. Human rabies: neuropathogenesis, diagnosis and management. Lancet Neurology, 2013, 12(5):498–513.

2. Willoughby RE et al. Survival after treatment of rabies with induction of coma. New England Journal of Medicine, 2005, 352:2508–2514. 3. Jackson AC. Therapy of human rabies. Advances in Virus Research, 2011, 79:365–375. 4. Srinivasan A et al. Transmission of rabies from an organ donor to four transplant recipients. New England Journal of Medicine, 2005, 352:1103– 1111.

Vaccines and rabies immunoglobulin for humans

5. Maier T et al. Management and outcomes after multiple corneal and solid organ transplantation from a donor infected with rabies virus. Clinical Infectious Diseases, 2010, 50(8):1112–1119.

Vaccines and rabies immunoglobulin for humans

Since their development more than four decades ago, concentrated, purified cell culture and embryonated egg-based rabies vaccines (jointly referred to as CCEEVs) have proved to be safe and effective in preventing rabies. These vaccines are intended for both pre- and post-exposure prophylaxis and have been administered to millions of people worldwide (1). Prompt administration of CCEEVs after exposure combined with proper wound management and simultaneous administration of rabies immunoglobulins is almost invariably effective in preventing rabies, even after high-risk exposure (1) (see also section 8).

6.1 Vaccine types 6.1.1 Cell culture and embryonated egg-based rabies vaccines CCEEVs contain rabies virus that has been propagated in cell substrates such as human diploid cells, Vero cells, primary chick embryo cells or embryonated duck eggs. Recently developed vaccines based on chick embryo and Vero cells are as safe and effective as human diploid cell vaccines and are less expensive. After growth in cell culture (or embryonic egg), the viral harvest is concentrated, purified, inactivated and lyophilized. In some CCEEVs, human albumin or processed gelatine is used as a stabilizer. Rabies vaccines are not supplied in multidose vials for intramuscular injection, and those prequalified by WHO do not contain preservatives such as thiomersal. The shelf-life of these vaccines is ≥3 years, provided they are stored at 2–8 °C and protected from sunlight. After reconstitution with sterile diluent, the vaccines should be used immediately or within 6 h if kept at the correct temperature (1), as partially used vials of rabies vaccine may become contaminated. Rabies vaccines for humans should meet WHO recommendations for characterization, production and control, as set out by the WHO Expert Committee on Biological Standardization (2). Presently, WHO recommendations apply only to inactivated rabies vaccines produced in cell culture or embryonated eggs.

6.1.2 Nerve tissue vaccines Nerve tissue vaccines induce more severe adverse reactions and are less immunogenic than CCEEVs. Since 1984, WHO has recommended discontinuation 37

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of the production and use of nerve tissue vaccines and their replacement by CCEEVs. Many developing countries have followed this recommendation (see below list of countries and dates at which discontinuation took place) and meet their requirements for rabies biologicals by either importing vaccine, developing or acquiring technology for producing CCEEVs. In a few countries, mainly in Asia and Latin America, populations at high risk for rabies still depend on vaccines derived from animal nerve tissues for post-exposure prophylaxis. Ecuador and Peru in Latin America and Myanmar and Pakistan in Asia are investigating affordable, sustainable alternatives to the use of nerve tissue vaccines. This Consultation again strongly recommends that production and administration of vaccines based on animal central nervous systems, including suckling mouse brain, be discontinued and replaced by CCEEVs. A four-step strategy to replace nervous tissue vaccine by modern rabies vaccines produced on cell culture or embryonated eggs has been developed (3) and is attached as Annex 3 to this report. Region, country and date of discontinuation of nerve tissue vaccines South East Asia (1987–2011) ■■ Bangladesh (2011) ■■ Bhutan (1995) ■■ India (2004) ■■ Indonesia (1992) ■■ Nepal (2006) ■■ Sri Lanka (1995) WHO Technical Report Series No. 982, 2013

■■ Thailand (1987) Western Pacific Region (1997–2007) ■■ Cambodia (2005) ■■ China (1990) ■■ Lao People’s Democratic Republic (2005) ■■ Philippines (1997) ■■ Viet Nam (2007) Region of the Americas (2002–2009) ■■ Brazil (2002)

Vaccines and rabies immunoglobulin for humans

■■ Chile (2003) ■■ Dominican Republic (2009) ■■ El Salvador (2009) ■■ Mexico (1995) ■■ Nicaragua (2005) ■■ Paraguay (2006)

6.2

WHO prequalification of human rabies vaccines

Vaccines supplied through United Nations agencies such as UNICEF should be prequalified by WHO. This is an established procedure, initiated voluntarily by vaccine manufacturers, for initial and continuous evaluation by WHO of nationally licensed vaccines. After initial prequalification, products are reassessed at regular intervals to ensure continuing quality. A revised procedure for WHO prequalification of vaccines was endorsed by the WHO Expert Committee on Biological Standardization in October 2010 and has been in effect since 1 February 2012 (4). National regulatory authorities can assume responsibility for regulatory control of a vaccine, and a vaccine must be licensed in the country of manufacture as a prerequisite to prequalification. WHO prequalification ensures the quality, safety and efficacy of vaccines and their suitability for use in national immunization programmes in low- and middle-income countries. The vaccine characteristics must be suitable for use in such programmes with regard to e.g. potency, thermostability, presentation, labelling and cold chain volume. The producer must then meet international standards of quality and comply with international standards of good manufacturing practice. Prequalification involves a review of the production process and quality control procedures, testing the consistency of lots, an audit of the manufacturing facilities by WHO with observers from the responsible national regulatory authority, assurance of continued acceptability and reassessments at regular intervals. Continued compliance is monitored. In 2012, only three rabies vaccines were prequalified for intramuscular use: purified Vero cell rabies vaccine, purified chick embryo cell vaccine and purified duck embryo vaccine. The list, which is updated when necessary, can be found at http://www.who.int/immunization_standards/vaccine_quality/ prequalification_vaccine_list_en/en/. The WHO Consultation encourages rabies vaccine manufacturers to enter the WHO prequalification process and Member States to purchase WHO prequalified vaccines.

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6.3

Requirements for human rabies vaccines

6.3.1 Potency requirements, tests and standards The minimal acceptable potency of CCEEVs is 2.5 international units (IU) per intramuscular dose, as determined in the mouse protection potency test (5,6). Alternative assays based on serum neutralization (7), ELISAs (8), fewer animals (9), peripheral challenge (10) and others (11) are being explored. The efficacy of these alternative tests should be established in multicentre studies carried by WHO collaborating centres, national regulatory authorities and control laboratories, in collaboration with manufacturers. The international standard for rabies vaccine is used in standardizing the mouse protection test and in vitro assays for glycoprotein content. In 2008, a candidate vaccine was calibrated against the fifth international standard in a collaborative study and became the sixth international standard for rabies vaccine. When used in mouse protection tests, this standard contains 8 IU per ampoule, i.e. 8 IU/ml, when reconstituted in 1 ml of distilled water. Other units are used in in vitro assays, such as enzyme immunoassays and single radial immunodiffusion tests, to determine the rabies virus glycoprotein antigen content (12).

6.3.2 Characterization and evaluation of rabies vaccines More than a dozen species or genotypes of Lyssavirus have been described as causative agents of rabies (see section 2). Lyssavirus genomes vary considerably, rabies virus being by far the commonest causative virus for human rabies and the only virus used to date in vaccines. Current vaccines may not protect against lyssaviruses other than those in phylogroup I (see section 2). The virus strains used for vaccines must be carefully selected, and the antigenic identity of the virus strains and the identity and purity of the cell lines used for production should be evaluated periodically. Comprehensive genetic characterization by full genome sequencing of vaccine virus strains is recommended. General principles for nonclinical and clinical evaluation of inactivated rabies vaccines have been published by WHO (4). Preclinical testing is a prerequisite for the initiation of clinical trials in humans and includes immunogenicity studies (proof of concept) and safety testing in animals. Clinical development of rabies vaccines should include evaluation of their use for pre- and post-exposure prophylaxis, with various vaccination schedules and routes of administration, the onset, extent and duration of protection, and the requirement for and timing of booster vaccination. Clinical trials should adhere to the principles described in the WHO Guidelines for good clinical practice (13) and to those for the design, conduct and analysis of vaccine clinical trials, described in the WHO Guidelines for clinical evaluation of vaccines (4). All clinical trials should be approved by the relevant national regulatory authority.

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6.4

Routes of vaccine administration

Current rabies vaccines are produced as individual doses for intramuscular injection. CCEEVs reconstituted with 0.5 or 1 ml of solvent in one intramuscular dose vial with a potency of ≥2.5 IU/dose can be used for both pre- and postexposure prophylaxis. The cost of cell culture-based vaccines for intramuscular administration limits, however, their widespread use in many areas where rabies is present. Intradermal administration of these vaccines is an equally safe and immunogenic alternative. Only one or two vials of vaccine are required to complete a full course of post-exposure prophylaxis by the intradermal route, thereby reducing the volume used and the direct cost of vaccine by 60–80% in comparison with standard intramuscular injection (14–18). There is no evidence that vaccines administered intradermally must be more potent than those recommended for intramuscular administration (3,19,20). Intradermal vaccination results in an equivalent immune response at a lower dose, thus sparing vaccine in pre- and post-exposure prophylaxis. Appropriate training should be given to ensure full intradermal instillation of the vaccine and to avoid accidental subcutaneous injection. An intradermal dose of 0.1 ml per site represents one fifth to one tenth of the intramuscular dose, depending on its volume after reconstitution. Although antibody titres are higher and more sustained after intramuscular injection, both routes induce rapid recall responses upon booster immunization. Intradermal vaccination is not recommended in immunocompromised individuals (21,22), as the underlying disease appears to impair transport of antigen-presenting dendritic cells to draining lymph nodes and thereby the magnitude of the antibody response. Once opened, vials should be stored for no longer than 6 h, resulting in some wastage, particularly in centres where the number of patients injected daily is small. Nevertheless, intradermal administration remains cost-effective for both pre- and post-exposure prophylaxis (23). Only two of the three WHO prequalified vaccines—purified Vero cell rabies vaccine and purified chick embryo cell vaccine—have been shown to be safe and effective when administered intradermally at a dose of 0.1 ml in a WHOrecommended pre- or post-exposure prophylaxis regimen. Vaccine manufacturers should provide clinical evidence that new products are immunogenic, effective and safe when given intradermally. Administration should adhere to WHO guidance for that route and prior approval by the national health authorities. In particular, the vaccine should have been compared with a vaccine of known immunogenicity, efficacy and safety and should have undergone serological testing with the rapid fluorescent focus inhibition test, and the results should have been published in an international, peer-reviewed journal. In countries where intradermal administration is an approved route for pre- or post-exposure prophylaxis, manufacturers of vaccines proven to be

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safe and effective when given by this route should register their product for intradermal use and state in the product insert that their vaccine can be used intradermally.

6.5

Adverse events after active immunization

In general, CCEEVs are safe and well tolerated. Adverse events may occur, however, depending in part on the purity of the inactivated rabies virus, which may vary among lots (24). In 35–45% of vaccinated people, minor, transient erythema, pain or swelling occurs at the site of injection, particularly after intradermal administration of a booster. Mild systemic adverse events, such as transient fever, headache, dizziness and gastrointestinal symptoms, have been observed in 5–15% of vaccinated people. Serious adverse events are rare and include Guille-Barre syndrome and allergic reactions (25).

6.6

Duration of immunity

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CCEEVs establish immunological memory that presumably persists for the life of the individual even after titres of neutralizing antibodies decline. Clinical data confirm that vaccinated people respond to booster immunization (26–28), even if the initial course of pre- or post-exposure prophylaxis was administered years previously and regardless of the route of priming or booster immunization (intramuscular or intradermal) and the presence or absence of detectable titres of rabies virus-specific antibodies at the time of the booster. In addition, published data indicate that periodic booster doses of vaccine are not required after primary rabies vaccination (29,30), except as an additional precaution for people whose occupation puts them at continual or frequent risk of exposure (see section 8.4). Nevertheless, all vaccinated individuals subsequently exposed to rabies, according to the WHO definition of exposure, should receive an abbreviated course of postexposure prophylaxis, as specified in section 8.

6.7

Rabies vaccine and full post-exposure prophylaxis failures

Post-exposure prophylaxis failures, when a patient dies despite having received the correct protocol in a timely manner, are very rare among the estimated 20 million people who receive post-exposure prophylaxis each year. Although such cases are certainly underreported, only a few have been notified, all in developing countries and most involving deviations from the WHO-recommended prophylaxis protocol (31,32). Most deviations from the recommended protocol leading to death are: delay in seeking rabies prophylaxis; lack of or improper administration of rabies immunoglobulin (e.g. failure to inject all bite sites); lack of or improper primary wound care, and/or poor-quality rabies vaccine (33).

Vaccines and rabies immunoglobulin for humans

6.8

Rabies immunoglobulins

In order to protect people from developing rabies, those who were previously unvaccinated or incompletely vaccinated, in category III of exposure or severely immunocompromised (e.g. AIDS patients or transplant recipients) people with category II exposure should receive both an effective rabies vaccine and rabies immunoglobulin (34). Rabies immunoglobulins should preferably be administered into and around the wound site to neutralize the rabies virus still present (see section 8). Three classes of biological product are available for passive immunization: human rabies immunoglobulin, equine rabies immunoglobulin and highly purified F(ab´)2 fragments produced from equine immunoglobulin (35). In this latter preparation the deletion of the Fc fragment might reduce the immunological functions of the antibody preparation, including its immunogenicity and thus the reactogenicity of the product. The second international standard preparation of human immunoglobulin is held and distributed on request by the WHO International Laboratory for Biological Standards at the National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, United Kingdom (12). The current WHO reference serum for standardization contains 30 IU per ampoule. Rabies immunoglobulin should be given with the first dose of vaccine into and around the wound site. Human immunoglobulin should be given at 20 IU/kg of body weight, while equine immunoglobulin has a shorter half-life in humans, and 40 IU/kg of body weight are required. Equine immunoglobulin is considerably less expensive than the human product, and most of the new equine preparations are potent, highly purified and safe, with few adverse events. Serum sickness can occur 1 week after administration of highly purified equine rabies immunoglobulin in <1–3% of recipients. The risk for anaphylactic reaction is low (1/150 000), and the reaction is generally treatable. Skin tests are not recommended before administration of equine rabies immunoglobulin, as such tests poorly predict severe adverse events and should not be the basis for not giving equine immunoglobulin if it is needed. Equine immunoglobulin should be administered under conditions that would allow management of an anaphylactic reaction. Rabies immunoglobulins are in short supply throughout the world. New technology may lead to use of monoclonal antibodies in post-exposure prophylaxis. WHO has recommended the use of monoclonal antibody ‘cocktails’ containing at least two antibodies against rabies virus, as alternatives for rabies immunoglobulins in post-exposure prophylaxis (36). A fully human monoclonal antibody cocktail is being evaluated for clinical safety and efficacy (37), and WHO is designing a humanized mouse monoclonal antibody cocktail for use in post-exposure prophylaxis for developing countries (38). WHO monoclonal 43

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antibodies have been licensed to a number of development partners for commercialization, and one initiated a phase-I clinical evaluation of a cocktail in 2012. These products are therefore expected to become available in the near future.

6.9 References 1. WHO position paper on rabies vaccines. Weekly Epidemiological Record, 2010, 85:309–320. 2. WHO Expert Committee on Biological Standardization. Fifty-sixth report, Annex 2. Geneva, World Health Organization, 2007 (WHO Technical Report Series, No. 941).

3. Human and dog rabies prevention and control: report of the WHO/Bill & Melinda Gates Foundation consultation, Annecy, France, 7–9 October 2009. Geneva, World Health Organization, 2010 (WHO/HTM/ NTD/NZD/2010.1) (http://whqlibdoc.who.int/hq/2010/WHO_HTM_ NTD_NZD_2010.1_eng.pdf). 4. WHO Expert Committee on Biological Standardization. Fifty-third report, Annex 1. Geneva, World Health Organization, 2004 (WHO Technical Report Series, No. 924). (http://www.who.int/immunization_standards/ vaccine_quality/pq_revision2010/en/index.html). 5. Seligmann EB Jr. Laboratory techniques in rabies: the NIH test for potency. Monograph Series. Geneva, World Health Organization, 1973, 23:279–286 WHO Technical Report Series No. 982, 2013

6. Wilber LA, Aubert MFA. The NIH test for potency. In: Meslin FX, Kaplan MM, Koprowski H, eds. Laboratory techniques in rabies, 4th ed. Geneva, World Health Organization, 1996:360–368. 7. Kamphuis E et al. Potency testing of inactivated rabies vaccines using a serological method. Developments in Biologics (Basel), 2012, 134:23–27.

8. Nimmagadda SV et al. Recombinant diabody-based immunocapture enzyme-linked immunosorbent assay for quantification of rabies virus glycoprotein. Clinical and Vaccine Immunology, 2010, 17:1261–1268. 9. de Moura WC et al. Potency evaluation of rabies vaccine for human use: the impact of the reduction in the number of animals per dilution. Journal of Virological Methods, 2009, 158:84–92.

Management of patients before and after death

10. Wunderli PS et al. The rabies peripheral challenge test: more accurate determination of vaccine potency. Vaccine, 2006, 24:7115–7123. 11. Stokes W et al. Report on the international workshop on alternative methods for human and veterinary rabies vaccine testing: state of the science and planning the way forward. Biologicals, 2012, 40:369–381.

12. National Institute for Biological Standards and Control. WHO international standard. Sixth international standard for rabies vaccine (NIBSC code: 07/162. Instructions for use, Version 1.0, dated 10/11/2008) (http://www.nibsc.ac.uk/products/biological_reference_materials/ product_catalogue/detail_page.aspx?catid=07/162). 13. WHO Expert Committee on the Use of Essential Drugs. Sixth report, Annex 3. Geneva, World Health Organization, 1995 (WHO Technical Report Series, No. 850). 14. Warrell MJ et al. Economical multiple-site intradermal immunisation with human diploid-cell-strain vaccine is effective for post-exposure rabies prophylaxis. Lancet, 1985, i:1059–1062. 15. WHO Expert Committee on Rabies. Eighth report. Geneva, World Health Organization, 1992 (WHO Technical Report Series, No. 824). 16. Briggs DJ et al. Antibody response of patients after postexposure rabies vaccination with small intradermal doses of purified chick embryo cell vaccine or purified Vero cell rabies vaccine. Bulletin of the World Health Organization, 2000, 78:693–698.

17. Quiambao BP et al. Reducing the cost of post-exposure rabies prophylaxis: efficacy of 0.1 ml PCEC rabies vaccine administered intradermally using the Thai Red Cross post-exposure regimen in patients severely exposed to laboratory-confirmed rabid animals. Vaccine, 2005, 23:1709– 1714. 18. Ambrozaitis A et al. Rabies post-exposure prophylaxis vaccination with purified chick embryo cell vaccine (PCECV) and purified Vero cell rabies vaccine (PVRV) in a four-site intradermal schedule (4-0-2-0-1-1): an immunogenic, cost-effective and practical regimen. Vaccine, 2006, 24:4116–4121. 19. Beran J et al. Potency requirements of vaccines administered intradermally using the Thai Red Cross regimen: investigation of the immunogenicity of serially diluted purified chick embryo cell rabies vaccine. Vaccine, 2005, 23:3902–3907. 45

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20. Sudarshan MK et al. Assessing the relationship between antigenicity and immunogenicity of human rabies vaccines. Results of a meta-analysis. Human Vaccines, 2005, 1:187–190.

21. Kopel E et al. Inadequate antibody response to rabies vaccine in immunocompromised patient. Emerging Infectious Diseases, 2012, 18:1493–1495. 22. Tantawichien T et al. Failure of multiple-site intradermal postexposure rabies vaccination in patients with human immunodeficiency virus with low CD4+ T lymphocyte counts. Clinical and Infectious Diseases, 2001, 33:E122–E124. 23. Hampson K, Cleaveland S, Briggs D. Evaluation of cost-effective strategies for rabies post-exposure vaccination in low-income countries. PLoS Neglected Tropical Diseases, 2011, 5:e982. 24. Finke S et al. Assessment of inactivated human rabies vaccines: biochemical characterization and genetic identification of virus strains. Vaccine, 2012, 30:3603–3609.

25. Grading of scientific evidence. Table III. Safety of cell-culture-based rabies vaccines. Geneva, World Health Organization, 2010 (http://www.who. int/entity/immunization/rabies_grad_safety.pdf). 26. Suwansrinon K et al. Survival of neutralizing antibody in previously rabies vaccinated subjects: a prospective study showing long lasting immunity. Vaccine, 2006, 24:3878–3880. 27. Brown D et al. Intradermal pre-exposure rabies vaccine elicits long lasting immunity. Vaccine, 2008, 26:3909–3912. 28. Naraporn N et al. Immune response to rabies booster vaccination in subjects who had postexposure treatment more than 5 years previously. Journal of Travel Medicine, 1999, 6:134–136. 29. Strady A et al. Antibody persistence following preexposure regimens of cell-culture rabies vaccines: 10-year follow-up and proposal for a new booster policy. Journal of Infectious Diseases, 1998, 177:1290–1295. 30. The immunological basis for immunization series, module 17: Rabies. Geneva, World Health Organization, 2011. 31. Wilde H et al. Failure of postexposure treatment of rabies in children. Clinical and Infectious Diseases, 1996, 22:228–232.

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32. Wilde, H. Failures of post-exposure rabies prophylaxis. Vaccine, 2007, 25:7605–7609. 33. Rupprecht CE et al. Evidence for a 4-dose vaccine schedule for human rabies post-exposure prophylaxis in previously non-vaccinated individuals. Vaccine, 2009, 27:7141–7148. 34. Guide for post-exposure prophylaxis. Geneva, World Health Organization, 2012 (http://www.who.int/rabies/human/postexp/en/). 35. Lang J et al. Evaluation of the safety, immunogenicity, and pharmacokinetic profile of a new, highly purified, heat-treated equine rabies immunoglobulin, administered either alone or in association with a purified, Vero-cell rabies vaccine. Acta Tropica, 1998, 70(3):317–333.

36. Consultation on a rabies monoclonal antibody cocktail for rabies post-exposure treatment. Geneva, 23–24 May 2002. Geneva, World Health Organization (available at www.who.int/rabies/vaccine/en/ mabs_final_report.pdf; accessed March 2013). 37. Bakker AB et al. First administration to humans of a monoclonal antibody cocktail against rabies virus: safety, tolerability, and neutralizing activity. Vaccine, 2008, 26(47):5922–5927.

38. Müller T et al. Development of a mouse monoclonal antibody cocktail for post-exposure rabies prophylaxis in humans. PLoS Neglected Tropical Diseases, 2009, 3(11):e542. Erratum in: PLoS Neglected Tropical Diseases, 2009,3(11):10.1371/annot at ion/df98339d-6b db-40e d-af83 cc38b249264a.

Vaccines for animals

Veterinary vaccines have been developed for use against rabies in domestic mammals and wildlife. These vaccines are either inactivated (killed), modifiedlive or biotechnology-derived products. Whatever the method for vaccine production, the quality of the source material and standards (e.g. virus master seed, specific pathogen-free eggs, cell seed) should be clearly documented, particularly with regard to sterility and safety. Rabies vaccines for animals should be approved by the competent state authorities and comply with national requirements for vaccines. When there are no adequate national regulations for veterinary biologicals (pre- and post-marketing requirements) with regard to potency, sterility, safety and efficacy, reference should be made to the relevant international standards (1–8). Vaccine strains should be genetically characterized, preferably by full genome sequencing. 47

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7.1 Vaccine types Vaccines should be administered by or under the supervision of a competent person, such as a veterinarian, according to the producer’s recommendations for e.g. minimum age, route of administration (oral, intramuscular or subcutaneous), duration of immunity and time between doses. Scientific evidence and the absence of contraindications may, however, allow adaptation of the vaccination schedules recommended by the producer, such as parenteral vaccination of animals younger than 3 months during mass programmes, in order to optimize herd immunity (see section 9).

7.1.1 Vaccines for domestic animals Injectable modified-live virus vaccines

Modified-live vaccines are produced from a modified egg-adapted strain of virus (e.g. Flury strain) serially passaged in embryonated chicken eggs. They can also be produced from strains adapted to cell culture (e.g. SAD/ERA). These vaccines are no longer considered safe because of their inherent ability to cause rabies, and their use in domestic animals should be discontinued. Injectable inactivated vaccines (monovalent or in combination)

WHO Technical Report Series No. 982, 2013 48

The vaccines most commonly used in domestic animal species are inactivated (killed) injectable vaccines, which are safe and inexpensive. The safety, potency and purity of such vaccines should be assessed by validated methods before use. Inactivated vaccines are produced in cell culture with either primary cells or continuous cell lines infected with an adapted strain of rabies virus. Various methods of inactivation are used, beta propiolactone or ultraviolet light being the most frequent. An adjuvant is recommended; one of the commonest is aluminium hydroxide. Inactivated rabies vaccines are available in either liquid or lyophilized form. Inactivated rabies vaccines can be used in combination with bacterins (e.g. Leptospira) and other viral antigens (polyvalent), such as canine distemper virus, canine adenovirus type 2 and canine parvovirus. Combined vaccines currently available for cats include various antigens, such as feline panleukopenia virus, feline calicivirus and feline herpesvirus. A combined rabies and footand-mouth disease vaccine is available for use in cattle, sheep and goats. An inactivated rabies vaccine combined with bacterin against Potomac fever (caused by Ehrlichia risticii) is available for horses. Injectable live recombinant vectored vaccine (monovalent or in combination)

A canarypox virus expressing the rabies virus glycoprotein has been licensed in the USA as a parenteral vaccine for cats. The commercially available vaccine

Vaccines for animals

combines the rabies–canarypox with feline panleukopenia, feline calicivirosis and feline herpesvirus components. Live replication-competent vaccines for oral use

The parenteral route is preferred for dog vaccination, although oral vaccination may be appropriate under specific conditions. The oral route should be used as a complement to parenteral mass vaccination campaigns, to improve vaccination coverage of the dog population by targeting individuals that are inaccessible for injectable vaccines (see section 9). The liquid vaccine usually contained in a sachet or blister pack should be incorporated in a bait, the taste, size, texture of which should be adapted to dogs. To maximize its use and to prevent the occurrence of vaccine-related untoward events in humans, WHO has established requirements for the safety and efficacy of candidate oral vaccines and for the design, testing and distribution of dog baits (9,10). Only vaccines with the lowest known residual pathogenicity should be used in dogs. To date, one attenuated vaccine (11) and one recombinant vaccine (12) have met the WHO minimum recommendations (10). Only one oral vaccine for dogs has been licensed so far. Use of oral vaccines in domestic species should be evaluated case by case, on the basis of preliminary knowledge of the structure (owned, ownerless) and accessibility of the dog population to interventions (13–15). As oral rabies vaccines are costly and safe distribution tends to be time-consuming, the cost–benefit ratio of administering these vaccines to dogs should be carefully assessed.

7.2

Potency requirements for animal rabies vaccines

7.2.1 Inactivated animal rabies vaccines The rabies vaccines used to immunize wild animals are usually live vaccines delivered by the oral route. The liquid vaccine usually contained in a sachet or blister pack should be incorporated in a bait preferably adapted to the target species with regard to taste, size and texture. Inactivated vaccines licensed for domestic animals can also be used for wildlife in trap–vaccinate–release programmes Modified-live virus vaccines

All attenuated vaccines currently used are derived from the original ERA/SAD (Street Alabama Dufferin) strain, with various levels of attenuation after passage in cell cultures. Several vaccines are attenuated by serial in-vitro selection on cloned baby hamster kidney cells or by passaging in mice in vivo. One vaccine was developed by using rabies virus glycoprotein monoclonal antibodies to select an attenuated virus carrying two mutations in position 333 (11,16). Use of rabies virus strains that can cause rabies in wildlife species is not recommended. 49

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Live recombinant vaccines

Several recombinant vaccines have been developed: a recombinant vaccinia virus and, more recently, a human adenovirus vector, both expressing the glycoprotein gene of rabies virus (17,18). Recombinant vaccines based on rabies virus with site-directed mutagenesis (reverse genetics) are also available. Some of these constructs have been approved by competent national authorities

7.2.2 Animal rabies vaccines for oral vaccination A batch-release titre is established before marketing release, which represents the lowest titre of vaccine that can protect 100% of the target experimental animals against a virulent rabies challenge. The batch-release titre in the vaccine bait should correspond to at least 10 times the minimum 100% protective dose found during the challenge test (3,21). National control laboratories or government institutions involved in licensing vaccines or evaluating oral rabies vaccination programmes may verify the viral titre of all batches of vaccine bait before and during a campaign (7,21,22). Such tests should be conducted in qualified laboratories with documented, validated methods and appropriate standards.

7.3 Safety of animal vaccines 7.3.1 Vaccines for parenteral use An effective pharmocovigilance system should be in place to detect vaccineassociated problems during post-marketing authorization (field use). Safety tests should be conducted by intracerebral inoculation of mice or, preferably, if validated, in cell culture (2,5,7,19).

7.3.2 Vaccines for oral use The safety of vaccines is assessed in target and non-target species, i.e. relevant wild rodents and other wild and domestic species that live in the area and may consume baits, as well as in non-human primates (10). The vaccine should not induce any adverse signs in either target or non-target species. Some modified-live rabies virus oral vaccines used in the field for wildlife may have residual pathogenicity, depending on the level of attenuation of the viral strain. Therefore, any rabies virus isolated from animals in the area of vaccination should be characterized with monoclonal antibodies or molecular techniques to ensure that no vaccine-induced rabies has occurred. In the event of accidental human exposure to attenuated rabies virus vaccines, medical attention should be sought and post-exposure prophylaxis considered. The potential risk to animals, humans and the environment of recombinant vaccines, such as those containing live pox or adenovirus vectors, WHO Technical Report Series No. 982, 2013 50

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should be assessed, and methods for mitigation or treatment, particularly in humans, should be identified early in research and development (10,23). When oral vaccines are used to vaccinate dogs, the risk for vaccine virus transmission among target and non-target species (including humans) should be assessed by testing for rabies virus in saliva and faecal samples from dogs up to 7 days after administration. No viable virus should be detectable after vaccination, as it would suggest replication and excretion. Any virus that is recovered should be characterized with molecular techniques or monoclonal antibodies.

7.4 Parenteral rabies vaccination Specific conditions may apply to the use of veterinary vaccines in mass vaccination programmes to control dog rabies. The implementation and monitoring of mass vaccination campaigns for dogs are described in section 9. To preserve the immunological properties of rabies vaccines, the manufacturers’ recommendations for storage should be respected. Particularly, prolonged breaks in the cold chain, exposure to sunlight and temperature fluctuations should be avoided. Opened vials should be used within 2–3 days (inactivated vaccines), provided sterile techniques are used to withdraw vaccine from multidose vials. If possible, dogs should also be vaccinated against other diseases, dewormed, spayed or neutered, to improve their health. Such a ‘visible’ effect might prompt people to bring their dogs for booster vaccinations in the future. A peak in rabies virus neutralizing antibodies is generally reached 4–6 weeks after initial antigenic stimulation. Thereafter, the levels of antibodies decrease rapidly and may be below the threshold of detection as soon as several weeks after vaccination. In dogs vaccinated several times, including those vaccinated twice 12 months apart, antibodies titres are generally higher, irrespective of the date of the serum test (24). Depending on the competent national authorities, the duration of efficacy of inactivated vaccines is considered to be 1–3 years.

7.5 References 1. WHO Expert Committee on Rabies. Eighth report. Geneva, World Health Organization, 1992 (WHO Technical Report Series, No. 824).

2. Meslin FX, Kaplan MM, Koprowski H, eds. Laboratory techniques in rabies, 4th ed. Geneva, World Health Organization, 1996. 3. WHO Expert Consultation on Rabies. First report. Geneva, World Health Organization, 2005 (WHO Technical Report Series, No. 931). 51

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4. Guidelines on nonclinical evaluation of vaccines, Annex 1. Geneva, World Health Organization, 2005 (WHO Technical Report Series, No. 927). 5. Rabies. In: Manual of diagnostic tests and vaccines for terrestrial animals (mammals, birds and bees), 7th ed. Paris, World Organisation for Animal Health, 2012:263–282. 6. Principles of veterinary vaccine production. In: Manual of diagnostic tests and vaccines for terrestrial animals (mammals, birds and bees), 7th ed. Paris, World Organisation for Animal Health, 2012:52–63. 7. Code of Federal Regulations, Title 9, Parts 1–199 (1-1-12 edition). Washington DC, Government Printing Office, United States Department of Agriculture–Animal and Plant Health Inspection Service, 2012.

8. Brown CM et al. Compendium of animal rabies prevention and control, 2011. Journal of the American Veterinary Medicine Association, 2011, 239(5):609–617. 9. Matter HC. Suggestion for the development of a research project for the field evaluation of several vaccine-bait delivery techniques to vaccinate dogs orally against rabies. Geneva, World Health Organization, 1993 (WHO/Rab.Res./93.40). 10. Guidance for research on oral rabies vaccines and field application of oral vaccination of dogs against rabies. Geneva, World Health Organization, 2007. WHO Technical Report Series No. 982, 2013

11. Cliquet F et al. The safety and efficacy of the oral rabies vaccine SAG2 in Indian stray dogs. Vaccine, 2007, 25:3409–3418. 12. Blancou J et al. Innocuité et efficacité d’un vaccine antirabique recombinant vaccine virus rabique administré par voie orale au renard, chien et chat [Safety and efficacy of an antirabies vaccine consisting of recombinant vaccinia-rabies virus administered orally to the fox, dog and cat.] Annales de Recherche Vétérinaire, 1989, 20(2):195–204. 13. Field application of oral rabies vaccines for dogs: report of a WHO consultation organized with the participation of the Office International des Epizooties, Geneva, Switzerland, 20–22 July 1998. Geneva, World Health Organization, 1998 (WHO/EMC/ZDI/98.15).

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14. Matter HC, Fico R. Accessibility of dogs to oral and parenteral vaccination against rabies in Tunisia and Turkey. Geneva, World Health Organization, 1992 (WHO/Rabies/93.206).

15. Matter H et al. Field evaluation of two bait delivery systems for the oral immunization of dogs against rabies in Tunisia. Vaccine, 1998, 16(7): 657–665. 16. Cliquet F et al. Eliminating rabies in Estonia. PLoS Neglected Tropical Diseases, 2012, 6(2):1–17. 17. Rosatte RC et al. Prevalence of tetracycline and rabies virus antibody in raccoons, skunks and red foxes following aerial distribution of V-RG baits to control raccoon rabies in Ontario Canada. Journal of Wildlife Diseases, 2008, 44:946–964. 18. Yarosh OK et al. Human adenovirus type 5 vectors expressing rabies glycoprotein. Vaccine, 1996, 14:1257–1264. 19. Rabies vaccines (inactivated) for veterinary use. In: European Pharmacopoeia. Strasbourg, Council of Europe, European Directorate for the Quality of Medicines and Health Care, 2010:734–736. 20. Servat A et al. In vivo potency tests of rabies vaccines for veterinary use. A 2-year retrospective analysis of data according to the criteria of the European Pharmacopoeia. Pharmeuropa, 2008, 20(4):655–664. 21. The oral vaccination of foxes against rabies. Report of the Scientific Committee on Animal Health and Animal Welfare. Luxembourg, European Commission, 2002. 22. Rabies vaccines (live, oral) for foxes. In: European Pharmacopoeia. Strasbourg, Council of Europe, European Directorate for the Quality of Medicines and Health Care, 2008:736–743.

23. Human vaccinia infection after contact with a raccoon rabies vaccine bait—Pennsylvania, 2009. Morbidity and Mortality Weekly Report, 2009, 58:1204. 24. Cliquet F et al. Neutralising antibody titration in 25,000 sera of dogs and cats vaccinated against rabies in France, in the framework of the new regulations that offer an alternative to quarantine. Revue Scientifique et Technique (International Office of Epizootics), 2003, 22(3):857–866.

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Prevention of human rabies

Rabies is almost always fatal. Thus, it is important to prevent it by immunization before and after suspect or proven exposure to the virus. The rabies vaccines and immunoglobulins used for prophylaxis should comply with WHO recommendations for production and control and for immunogenicity and safety for use by both the intramuscular and the intradermal route (see section 6).

8.1 General considerations After suspected or proven exposure to rabies virus, prompt use of CCEEVs with proper wound management and simultaneous administration of rabies immunoglobulin is almost invariably effective in preventing rabies, even after severe exposure. Assessment of potential exposure can be complex and confusing. When in doubt, post-exposure prophylaxis should be initiated, and the attending physician should consult an infectious disease specialist with expert knowledge of rabies. Pre-exposure prophylaxis for people who are at risk of exposure to lyssaviruses because of their job, residence or travel is strongly recommended. Vaccines can be administered intramuscularly or intradermally at certain sites. For intramuscular administration, the vaccine should be injected into the deltoid muscle for adults and children aged ≥2 years; for children aged <2 years, the anterolateral thigh is recommended. Rabies vaccine should not be administered in the gluteal area, as induction of an adequate immune response is less reliable. For intradermal administration, the recommended sites include the deltoids, lateral thighs or suprascapular areas (see Annex 4). The site is selected on the basis of the level of privacy that can be provided and sociocultural acceptance. Devices are available to facilitate intradermal injection. WHO Technical Report Series No. 982, 2013 54

8.2 Pre-exposure prophylaxis Pre-exposure prophylaxis is recommended for anyone who is at continual, frequent or increased risk for exposure to the rabies virus, as a result of their residence or occupation, such as laboratory workers dealing with rabies virus and other lyssaviruses, veterinarians and animal handlers. Travellers in high-risk areas should be vaccinated after a risk assessment. Children living in or visiting rabies-affected areas are at particular risk and should be given pre-exposure prophylaxis on an individual basis or in mass campaigns when there are no economic, programmatic or logistical obstacles (see also section 8.8).

Prevention of human rabies

As far as possible, the vaccination series listed below must be completed in the stipulated time; however, there is no need to restart the series if the doses are not given on the exact schedule (1). Intramuscular administration: One intramuscular dose is given on each of days 0, 7 and 21 or 28. Day 0 is the date of administration of the first dose of vaccine. Intradermal administration: One intradermal injection of 0.1 ml is given on each of days 0, 7 and 21 or 28. To maximize savings, sessions of intradermal preexposure prophylaxis should involve enough individuals to use all opened vials within 6 h.

8.3 Post-exposure prophylaxis Post-exposure prophylaxis consists of: ■■ local treatment of the wound as soon as possible after exposure, ■■ a course of potent, effective rabies vaccine that meets WHO recommendations and ■■ administration of rabies immunoglobulin, if indicated. Factors that should be taken into consideration in deciding to initiate postexposure prophylaxis include the epidemiological likelihood that the implicated animal was rabid, the severity of exposure (see section 8.3.2), the clinical features of the animal, its vaccination status (particularly for dogs and cats) and its availability for observation and laboratory testing. All exposures determined to represent a risk for rabies require post-exposure prophylaxis. Prophylaxis should be instituted immediately. If possible, the suspect animal should be identified, quarantined for observation (for healthy dogs and cats) or euthanized for laboratory examination. Prophylaxis should be continued while awaiting laboratory results or during the observation period. If the laboratory tests are positive, an immediate retrospective risk assessment should be conducted to identify all people who may have been exposed, and they should be given postexposure prophylaxis. Prophylaxis should be completed if the suspect animal is not available for testing or observation but may be discontinued if the animal is proved by appropriate laboratory examination to be free of rabies. When the domestic dog, cat or ferret at the origin of human exposure is healthy, properly vaccinated (at least two documented vaccinations with a potent vaccine) and easily accessible for observation for 10 days, proper wound management should be ensured and 55

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booster vaccination can be deferred, especially if the patient had received preexposure prophylaxis or previous post-exposure prophylaxis in the past 3 months (2). All bite victims and other people with suspect animal contacts presenting at a health care facility should immediately be reported to a veterinary expert to conduct an investigation of the animal and ensure laboratory examination if it is suspected of having rabies or to monitor the animal’s state of health if it is under observation. When animal bites, scratches and other contacts (excluding contacts with bats) occur in an area free of carnivore rabies and where there is adequate rabies surveillance, post-exposure prophylaxis may not be required. The decision should be based on a risk assessment conducted by a medical expert knowledgeable in the local epidemiology of rabies. In areas where canine and/or wildlife rabies is enzootic, prophylaxis should be instituted immediately after a suspected exposure, unless adequate laboratory surveillance is in place and data from laboratory and field sources indicate that the species involved is not a vector of rabies; for example, bites by rodents, rabbits and hares do not routinely require post-exposure prophylaxis. The recommendations given here are a general guide; they might be modified in certain situations, such as when a reliable exposure history cannot be obtained (e.g. from infants or mentally challenged people). This is particularly true in areas where rabies is enzootic and follow-up observation of the biting animal and/ or laboratory testing are not readily available. A careful risk assessment should ideally be conducted by a qualified medical professional on every patient exposed to an animal suspected of being rabid. WHO Technical Report Series No. 982, 2013 56

8.3.1 Local treatment of wounds Prompt local treatment of all bite wounds and scratches is an important step in post-exposure prophylaxis. The recommended first-aid procedures include immediate, thorough flushing and washing of the wound with soap and water, detergent, povidone iodine or other substances with virucidal activity. If soap or a virucidal agent is not available, the wound should be thoroughly and extensively washed with water. People who live in areas endemic for rabies should be taught simple local wound treatment and warned not to use procedures that may further contaminate or enlarge the wound. A bleeding wound at any site indicates potentially severe exposure and must be infiltrated with either human or equine rabies immunoglobulin. Most severe

Prevention of human rabies

bite wounds are best treated by daily dressing, followed by secondary suturing when necessary. If suturing after wound cleansing cannot be avoided, the wound should first be infiltrated with human or equine rabies immunoglobulin and suturing delayed for several hours to allow diffusion of the immunoglobulin through the tissues before minimal sutures are applied. Secondary sutures are less likely to become infected and present better cosmetic results if carried out under optimal conditions. An infected bite wound is no contraindication to injection of rabies immunoglobulin (3). Bites on the finger or toe tip, ear lobe or nasal area can be safely injected with rabies immunoglobulin, provided excessive pressure is not applied, as this can cause compression syndromes (4). Other treatments, such as administration of antibiotics and tetanus prophylaxis, should be applied as appropriate for potentially contaminated wounds.

8.3.2 Categories of exposure and post-exposure prophylaxis (Annex 5) In countries or areas enzootic for rabies, exposure to suspected or confirmed rabid (domestic or wild) animals is categorized as follows: ■■ category I: touching or feeding animals, licks on intact skin, contact of intact skin with secretions or excretions of a rabid animal or human. These are not regarded as exposures, and no post-exposure prophylaxis is required. ■■ category II: nibbling of uncovered skin, minor scratches or abrasions without bleeding. Vaccine should be injected as soon as possible. ■■ category III: single or multiple transdermal bites or scratches, licks on broken skin, contamination of mucous membrane with saliva from licks and exposure to bats. Vaccine and rabies immunoglobulin should be administered at distant sites as soon as possible. Immunoglobulin can be administered up to day 7 after injection of the first dose of vaccine. For categories II and III, thorough local wound treatment (see 8.3.1) is of paramount importance. Post-exposure prophylaxis, including rabies immunoglobulin, should always be administered when category III exposure is recognized, even months or years after contact. When it is not possible to complete post-exposure prophylaxis with the same cell culture-based or embryonated egg-based vaccine, a rabies cell culture-based vaccine that fulfils WHO requirements should be used. Thus should, however, be an exception.

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8.3.3 WHO-recommended post-exposure prophylaxis regimens Day 0 is the date of administration of the first dose of vaccine. It is important to complete the initial three doses within 1 week. Intramuscular administration The recommended regimen consists of either a five-dose (1-1-1-1-1) or a fourdose schedule (2-0-1-0-1 or 2-1-1): ■■ The five-dose ‘Essen’ regimen (1-1-1-1-1) consists of one dose administered on each of days 0, 3, 7, 14 and 28. A reduced, four-dose vaccine schedule (1-1-1-1-0) for healthy people is supported by the peer-reviewed literature, unpublished data, epidemiological reviews and expert opinion. This shortened Essen regimen, consisting of one dose on each of days 0, 3, 7 and 14, may be used as an alternative for healthy, fully immune competent, exposed people provided they receive wound care plus rabies immunoglobulin in category III as well as in category II exposures and a WHO-prequalified rabies vaccine (5). ■■ The four-dose ‘Zagreb’ regimen (2-0-1-0-1 or 2-1-1) consists of two doses of vaccine injected on day 0 (one into each of the two deltoid or thigh sites) followed by one dose on each of days 7 and 21. Intradermal administration The updated two-site Thai Red Cross regimen (2–2–2–0–2) consists of injections of 0.1 ml of vaccine at two different intradermal sites on each of days 0, 3, 7 and 28 (6). This regimen can be used for people with category II or III exposure in countries in which the intradermal route has been endorsed by the national health authorities. WHO Technical Report Series No. 982, 2013

8.3.4 Short post-exposure prophylaxis for previously vaccinated individuals Exposed or re-exposed patients who can document previous complete preexposure prophylaxis or complete post-exposure prophylaxis with rabies CCEEVs should receive: ■■ one dose of vaccine intramuscularly or intradermally at one site on both days 0 and 3. Rabies immunoglobulin is not indicated in such cases. This regimen can also be given to people vaccinated against rabies who have detectable rabies virus neutralizing antibody. ■■ As an alternative to this regimen, the patient may be offered a ‘one visit four-site’ intradermal regimen consisting of four injections of 0.1 ml equally distributed over the left and right deltoids, thigh or suprascapular areas at a single visit (7,8).

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For people who have received complete pre- or post-exposure prophylaxis within a maximum delay of 3 months before exposure or re-exposure to a bite or other contact, proper wound management should be ensured, and booster vaccination can be safely deferred, if the biting dog or cat is healthy, vaccinated and available for an observation period of 10 days (2). People with category III exposure who have received complete pre- or postexposure prophylaxis with a vaccine of unproven potency, including nerve tissue vaccines, or an incomplete course of pre- or post-exposure prophylaxis should receive a full post-exposure vaccination course, including rabies immunoglobulin.

8.4 Requirements for periodic booster injections Periodic booster doses of rabies vaccine are not necessary for people living in or travelling to high-risk areas who have received a complete primary series of pre- or post-exposure prophylaxis with rabies CCEEVs. Only people whose occupation puts them at continual or frequent risk of exposure should receive periodic booster injections as an extra precaution in the absence of recognized exposure. If available, monitoring of rabies virus neutralizing antibody in personnel at risk is preferred to routine boosters. For people potentially at high risk for laboratory exposure to high concentrations of live rabies virus, neutralizing antibody titration should be done every 6 months. If the titre falls below 0.5 IU/ml of serum, one booster dose of vaccine should be given intramuscularly or intradermally. Professionals who are not at continual risk of exposure, such as certain categories of veterinarians and animal health officers, should undergo serological monitoring every 2 years. As vaccine-induced immunological memory persists in most cases for years, a booster is recommended only if the rabies virus neutralizing antibody titre has dropped below 0.5 IU/ml.

8.5 Vaccination of immunocompromised individuals Several studies of patients with HIV/AIDS have shown that those with very low CD4 counts mount a significantly lower or no detectable neutralizing antibody response to rabies virus. In these patients and others in whom the presence of immunological memory is no longer assured, proper, thorough wound treatment and antisepsis accompanied by local infiltration of human or equine rabies immunoglobulin and a complete series of five intramuscular doses of rabies CCEEV is required for category II and III exposures. When feasible, the rabiesvirus neutralizing antibody response should be determined 2–4 weeks after vaccination to assess whether an additional dose of vaccine is required. When in doubt, consult an infectious disease specialist with expert knowledge of HIV/ AIDS and rabies prevention. 59

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8.6 Rabies immunoglobulin for passive immunization The role of rabies immunoglobulin in passive immunization is to provide neutralizing antibodies at the site of exposure before patients can begin producing their own antibodies physiologically after vaccination. Therefore, rabies immunoglobulin should be administered to all patients presenting with category III exposure. Rabies immunoglobulin is administered only once, preferably at or as soon as possible after initiation of post-exposure vaccination. It is not indicated beyond the seventh day after the first dose of rabies vaccine, regardless of whether the day 3 and day 7 doses were received, because an active antibody response to the CCEEV has already started, and there may be interference between active and passive immunization. The dose of human rabies immunoglobulin is 20 IU/kg of body weight, while that of equine immunoglobulin and F(ab’)2 products is 40 IU/kg of body weight. All the immunoglobulin, or as much as anatomically possible (but avoiding possible compartment syndrome), should be administered carefully into or around the wound site or sites. The remaining product, if any, should be injected intramuscularly at a site distant from the site of vaccine administration. Use of the same syringe or mixing rabies vaccine and rabies immunoglobulin must be avoided. For severe and multiple wounds, which require more immunoglobulin than the calculated dose, the product may be diluted with sterile normal saline to a volume sufficient for effective, safe infiltration of all wounds (8). Post-exposure prophylaxis, including rabies immunoglobulin for category III exposure, should be administered after exposure is recognized, even months or years later.

8.7 Contraindications and precautions WHO Technical Report Series No. 982, 2013 60

As rabies is fatal, there are no contraindications to post-exposure prophylaxis, and it should be given as indicated by the nature of the exposure in a setting in which staff are adequately trained in its administration and in the management of possible adverse reactions. There are no contraindications for post-exposure prophylaxis in infants, pregnant women or immunocompromised individuals, including children with HIV/AIDS. People taking chloroquine for malaria treatment or prophylaxis may have a reduced response to intradermal rabies vaccination and should receive the vaccine intramuscularly. As with all vaccinations, recipients should be kept under medical supervision for at least 15–20 min after vaccination. A previous severe reaction to any component of a vaccine (except rabies immunoglobulin) is a contraindication to use of the same vaccine for pre- or post-exposure prophylaxis.

Prevention of human rabies

8.8 Travellers to and residents of rabies-affected countries and areas, and indications for pre-exposure prophylaxis Travellers to and residents of rabies-affected countries and areas should avoid contact with free-roaming animals, especially dogs and cats, and with wild, freeranging or captive animals. For people who participate in spelunking, casual exposure to cave air is not a concern, but cavers should be warned not to handle bats. Contact with bats should be followed by post-exposure prophylaxis. The map in Figure 1 shows four categories of countries or areas, from those at no risk to those at low, moderate and high risk. The categorization is based on the major animal host/transmitter and lyssavirus species involved, and the availability of reliable, laboratory-based surveillance data on these reservoir species. Access to proper medical care and the availability of CCEEVs and other rabies biological products were also taken into consideration. ■■ category 1, no risk: lyssavirus risk-free countries or areas. ■■ category 2, low risk: countries or areas with either only rabies-related lyssaviruses circulating in bats or rabies virus circulating in bats (non haematophagous) and other wildlife. In both groups of countries or areas, proper medical care, CCEEVs and other rabies biological products are easily accessible, and reliable laboratorybased surveillance data are available. ■■ category 3, moderate risk: countries or areas where rabies virus circulates in bats (non haematophagous) and other wildlife. ■■ category 4, high risk: countries or areas with sustained dog-to-dog transmission of the rabies virus and/or where vampire bat rabies is reported (9). Advice to travellers and residents according to level of risk: No risk: No need for pre-exposure prophylaxis. Low risk and moderate risk: People involved in any activities that might bring them into direct contact with non haematophagous bats and other wild animals, especially carnivores (for example, wildlife professionals, researchers, veterinarians and adventure travellers visiting areas where bats and other wildlife are commonly found) should receive pre-exposure prophylaxis. High risk: People travelling to rural areas or involved in activities such as running, bicycling, camping or hiking should receive pre-exposure prophylaxis. Prophylaxis is also recommended for people with significant occupational risks, such as veterinarians, and residents of areas with a significant risk for exposure to domestic animals, particularly dogs and cats as well as wildlife including vampire 61

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Figure 1 Four categories of countries or areas, from those at no risk to those at low, moderate and high risk

bats. Children should be preventively immunized as they are at higher risk. When potentially exposed in a low, moderate or high risk country or area people who have received pre-exposure prophylaxis should receive booster vaccination (see section 8.3.4) and people who have not been previously vaccinated should consult a physician and if indicated receive post-exposure prophylaxis within the shortest possible delay (see section 8.3.3). Suggested certificates of pre- and post-exposure vaccination against rabies are shown in Annex 6. WHO Technical Report Series No. 982, 2013

8.9 References 1. Recommendations for routine immunization. Summary tables. Geneva, World Health Organization, 2012 (www.who.int/immunization/policy/ immunization_tables/en; accessed March 2013). 2. Sudarshan MK, Ravish HS, Ashwath Narayana DH. Time interval for booster vaccination following re-exposure to rabies in previously vaccinated persons. Asian Biomedicine, 2011, 5(5):589–593. 3. Wilde H et al. Is injection of contaminated animal bite wounds with rabies immune globulin a safe practice? Transactions of the Royal Society of Tropical Medicine and Hygiene, 1992, 86:86–88.

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4. Suwansrinon K et al. Is injecting a finger with rabies immunoglobulin dangerous? American Journal of Tropical Medicine and Hygiene, 2006, 75:363–364. 5. Rupprecht CE et al. Use of a reduced (4-dose) vaccine schedule for postexposure prophylaxis to prevent human rabies, recommendations of the Advisory Committee on Immunization Practices. Morbidity and Mortality Weekly Report, 2010, 59(RR02):1–9. 6. Madhusudana SN et al. Comparison of safety and immunogenicity of purified chick embryo cell rabies vaccine (PCECV) and purified Vero cell rabies vaccine (PVRV) using the Thai Red Cross intradermal regimen at a dose of 0.1 ml. Human Vaccines, 2006, 2(5):200–204. 7. Rabies vaccines: WHO position paper. Weekly Epidemiological Record, 2010, 85:309–320. 8. Human and dog rabies prevention and control, report of the WHO/Bill & Melinda Gates Foundation Consultation, 2009, Annecy, France. Geneva, World Health Organization, 2010 (WHO/HTM/NTD/NZD 2010.1).

9. Schneider MC. et al Rabies transmitted by Vampire bats to humans: an emerging zoonosis in Latin America? Pan American Journal of Public Health, 2009, 25(3):260–269.

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Canine rabies can be eliminated, as demonstrated in North America, western Europe, Japan and many areas of South America and parts of Asia. It is, however, still widespread, occurring in over 80 countries and territories, predominantly in the developing world. In more than 99% of all cases of human rabies, the virus is transmitted via dogs; half the global human population lives in canine rabiesendemic areas and is considered at risk for contracting rabies. Controlling and eventually eliminating the disease in dogs would have major benefits for human health by prevention at source of most human deaths from this cause. Animal vaccines that provide a considerable duration of immunity are commercially available, and mass parenteral vaccination programmes are the mainstay of canine rabies control. In recent years, rabies control and elimination programmes through mass vaccination of dogs have resulted in marked reductions or elimination of human rabies cases (1–4). In a few instances, rabies vaccination coupled with sterilization of dogs has resulted in local elimination of cases (5) or has been predicted to lead to elimination of human rabies (6). The contribution of sterilization, over and above vaccination alone, to the control of dog rabies has not been fully evaluated. 63

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Vaccination programmes should take into account the local ecology of the dog population, including the degree of ownership (owned and confined, owned and roaming, community-owned or ownerless). This knowledge is essential to ensure that the method of vaccination delivery maximizes access to dogs and in order to provide culturally appropriate education. The success of vaccination campaigns in Latin America was due to the central coordinating role of the public health sector and the involvement of communities in rabies control. In addition, as rabies control programmes must involve multiple agencies and sectors, including animal and public health, they require a ‘one health’ approach, with effective interministerial cooperation. While mass dog vaccination has repeatedly been shown to be effective for controlling canine rabies, there is no evidence that removal of dogs has a significant impact on the dog population density or the spread of rabies. Mass culling of dogs should not be an element of a rabies control strategy: it is ineffective and can be counterproductive to vaccination programmes. Euthanasia of a dog suspected of being rabid reduces human health risks and prevents further animal suffering. A list of the classical clinical signs of rabies in dogs is given in section 11. When the diagnosis is unclear, the dog can be quarantined and observed; however, if the signs progress, euthanasia should be performed (7).

9.1 Canine mass parenteral vaccination campaigns To achieve control and eventual elimination of rabies, programmes must ensure recurrent (usually annual) campaigns and achieve a vaccination coverage of at least 70% (8,9). This coverage should be sufficient to maintain the required level of herd immunity in the vaccinated population in spite of dog population turnover (births, deaths, emigration, immigration) in the period between campaigns (8,10). Latin America is an example of a region in which several countries have successfully controlled rabies. Since their formal pledge in 1983 to eliminate human deaths from rabies transmitted by dogs, the countries of the region have had a decrease of over 90% in rabies in dogs and hence a similar decrease in human deaths (9,12). This has been achieved predominately by mass vaccination of over 45 million dogs annually, with concurrent appropriate treatment of people potentially at risk for rabies (pre- and post-exposure prophylaxis) and epidemiological surveillance. Other recent examples are KwaZulu-Natal (South Africa), the Visayas (the Philippines) and Bali (Indonesia). The KwaZulu-Natal province of South Africa had been plagued by dog rabies for several decades. During 1983–2007, 79% of laboratory-confirmed human cases in South Africa occurred in this

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province, with a human population estimated at 10.6 million. The dog rabies elimination project illustrates the effectiveness of collaboration between the provincial government, donors (e.g. the Bill & Melinda Gates Foundation) and WHO. More than 1.5 million dogs have been vaccinated since the beginning of the project in 2009. In 2012, more than 630 000 dogs were vaccinated, the highest number immunized in a year by the provincial veterinary services. The occurrence of animal rabies has been halved in 3 years, with an initial decrease in human cases (11): for the first time in 20 years, KwaZulu-Natal reported in 2010– 2011 a continuous 12-month period without a single human case (12). Despite many challenges, the project is now being extended across southern Africa, with renewed support and momentum. The regional programme for rabies elimination in the Visayas is part of the national rabies programme jointly implemented by the departments of agriculture, health and education and chaired by the agriculture department’s Bureau of Animal Industry on the basis of the National Rabies Act 9482. The ‘rabies-free Visayas’ project is being carried out in collaboration with partners such as WHO, the Bill & Melinda Gates Foundation, the Global Alliance for Rabies Control (GARC) and the Optimus Foundation. The project involves vaccination of more than 3 million dogs over 5 years, and campaigns have been conducted in the Western Visayas, parts of Central Visayas including the island of Bohol (13) and the Eastern Visayas. Intensive information and education campaigns are conducted to strengthen community support and volunteer engagement in order to increase dog vaccination and responsible pet ownership and improve clinical management of human rabies and surveillance and diagnostic capability. The number of human deaths from rabies in the Visayas has decreased significantly, from 48 cases in 2008 to 13 in 2012, a 70% reduction (12). Rabies was introduced to Bali in 2008 and spread rapidly throughout the island, causing 141 human deaths by the end of 2012. Initial attempts to contain the spread of the disease involved indiscriminate mass culling of dogs. Since the introduction of mass canine vaccination as the main strategy from late 2010, the numbers of human and animal cases of rabies have dropped dramatically: the number of human cases decreased by 72% between 2010 and 2011 and by 90% between 2010 and 2012. Two mass vaccination campaigns have been completed, and a third campaign is nearing completion. The feasibility of an islandwide vaccination campaign was demonstrated by a local nongovernmental organization, the Balinese Animal Welfare Association, with funding from the World Society for the Protection of Animals. The Government of Indonesia with technical assistance from the FAO assumes responsibility for the second and subsequent campaigns. The reasons for the success of the programme have been: a clear operational goal of vaccinating 70% of dogs in each locality on the island during each campaign; daily reporting of vaccination and post-vaccination 65

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survey results, by SMS and on paper; daily, weekly and monthly Government coordination meetings during vaccination campaigns; and campaign-specific standard operating procedures, with in-service training of field staff (14,15).

9.2 Strategic planning and management of vaccination campaigns Vaccination campaigns must be strategically planned, well managed and adequately resourced and funded. The ‘rabies blueprint’ prepared by the Partners for Rabies Prevention provides guidance on planning and implementing dog vaccination campaigns (16).

9.2.1 Studies of dog ecology To plan a vaccination campaign, the dog population must be estimated and dog-keeping practices ascertained in order to calculate the resources required and the appropriate methods for accessing dogs for vaccination (17). The dog population can be estimated from the human:dog ratio, but these ratios vary widely by community. Low levels of reported dog ownership and variable ownership patterns in urban areas make it difficult to estimate urban dog populations accurately. Other methods for estimating dog populations include questionnaire surveys, which provide information on owned dogs only, and capture–mark–recapture approaches, which cover the free-roaming population (18). Details of these methods are available from the International Companion Animal Management Coalition (19) and the Partnership for Rabies Prevention (20). Such surveys are often usefully combined with post-vaccination surveys to evaluate vaccination coverage, and population estimates can be revised for future campaigns. Information from dog registries can be useful, but, as these do not include unregistered or ownerless dogs, use only of this source will lead to underestimates of the total dog population.

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9.2.2 Vaccination and immunization coverage Low or patchy vaccination coverage of the target population is directly correlated with the persistence of rabies and hence jeopardizes the prospects of elimination over an entire region, even is coverage elsewhere is high. Vaccination may be more effective if carried out comprehensively in a small contiguous area than in many separate areas. Models of rabies transmission are helpful in identifying the best strategy for such situations (21). Reactive vaccination is not recommended unless increased surveillance shows that the incidence has been reduced to low levels in a few remaining foci. Reactive strategies take longer to control rabies and are less likely to lead to successful control than systematic vaccination in an entire area.

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The required immunization coverage can be achieved by well-designed educational campaigns, intersectoral and interdisciplinary cooperation, community participation, local commitment to planning and execution, the availability of high-quality vaccine, media support, and effective general coordination and supervision of activities by the appropriate authorities.

9.3 Implementing and monitoring dog vaccination campaigns 9.3.1 Target animals and vaccination methods During mass campaigns, all dogs should be vaccinated, regardless of age, weight or state of health. Although the aim should be to vaccinate as many dogs as feasible, herd immunity is achieved by vaccinating at least 70% of the population. As cats are important vectors of rabies to humans, cats should also be vaccinated when presented at vaccination campaigns. A common reason for low coverage is the misperception that puppies should not be vaccinated (22–24). In many countries endemic for canine rabies, young dogs comprise a large proportion of the population, and owners and vaccination teams must be made aware that puppies, including newborns, should also be vaccinated to ensure adequate population coverage. Three basic approaches have been used, either alone or in combination, for accessing dogs for vaccination campaigns: house-to-house visits, fixed vaccination posts in well-recognized sites within a community, and temporary vaccination posts set up by mobile teams. Such posts are usually sufficiently attended only when they are at less than 500 m or about a 10-min walk (25). The choice of approach depends on the community and should be made at local level. A combination of approaches may be required.

9.3.2 Timing of campaigns Rabies vaccination campaigns are generally conducted annually, but more frequent campaigns may be conducted in areas where population turnover is high. Intensive vaccination campaigns lasting from 1 day to 1 month have been effective in rabies control, most notably in Latin America. Campaigns must, however, reach at least 70% of the dog population, and coverage should not be compromised in pursuit of speed. Campaigns might be organized on weekends or during school holidays to improve turnout, as children often bring their dogs for vaccination.

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9.3.3 Monitoring vaccination campaigns Registration and permanent identification of vaccinated dogs is recommended; however, effective methods of identification are not widely available, and further research is required. Lack of resources or capacity to permanently identify dogs should not obviate implementation of a vaccination campaign. The use of coloured tags or plastic collars as temporary marking has proven to be useful in identifying vaccinated dogs (25) and motivates owners to take their pets for vaccination. Identification of vaccinated dogs is necessary in order to evaluate the vaccination coverage rate and to differentiate unvaccinated dogs for follow-up vaccination. For example, in Bali, red collars or red spray paint (for puppies that were still growing) were used to mark every dog that was vaccinated. A survey was then conducted within 3 days of the campaign to assess the numbers of marked and unmarked dogs; where coverage was calculated to be less than 70%, a revaccination campaign was organized to access unvaccinated dogs. Routine serological monitoring in the context of mass dog vaccination campaigns is not recommended if: ■■ a reputable vaccine has been used (defined as a vaccine that has been demonstrated to confer protection for 2 years or more after a single injection against a virulent challenge, killing at least 80% of controls); ■■ vaccination teams have been trained and have used proper injection technique, dog handling and vaccine vial management; and ■■ the cold chain has been maintained throughout. If repeated annual vaccination campaigns that reach the targeted coverage are not resulting in a decrease in the number of animal rabies cases, one or more of the above elements may not have not been complied with. Well-designed serological and other studies (e.g. vaccine potency, cold chain monitoring) may then be warranted.

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9.3.4 Cost–effectiveness of dog vaccination Several theoretical studies have indicated that dog vaccination in combination with post-exposure prophylaxis is more cost–effective for preventing human deaths from rabies than post-exposure prophylaxis alone (26,27). This conclusion remains uncertain, however, as the costs of different campaigns vary widely, and the operational costs can be substantially higher than those used in modelling studies, such as US$ 1.73–5.50 in rural United Republic of Tanzania (20,24). Furthermore, the demand for post-exposure prophylaxis does not invariably decrease with a decrease in the incidence of dog rabies; this relation requires further investigation.

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Little research has been done on the effect of requiring owners to contribute to registration or campaign costs, especially for vaccine, or on the options for differential contributions based on owners’ capacity to pay, and further evaluation of this approach would be beneficial. When dog owners are unwilling or unable to pay and this jeopardizes a critical level of immunization coverage, the intervention (i.e. registration, marking, vaccination, certificate delivery) must be provided free of charge and the costs balanced against the public health benefits.

9.3.5 Vaccines to be used As vaccines are susceptible to extremes of temperature, including freezing, care should be taken to ensure that the cold chain is maintained within an acceptable temperature range. Long-acting vaccines with a minimum duration of immunity of 2 years should be used in annual campaigns to revaccinate all dogs. Revaccination has no adverse effects, and annual campaigns provide a simple, effective message. Turning people and their dogs away could confuse this message, while the direct costs of revaccination are marginal in comparison with campaign costs. All members of a vaccination team who handle dogs should receive preexposure prophylaxis before the campaign. Adequate post-exposure prophylaxis should be available for people exposed during the campaign.

9.4 Increasing access to dogs for vaccination When the usual approaches for accessing dogs for parenteral vaccination are deemed insufficient, other measures can be used. Increased community engagement and mobilization can improve the turn-out for vaccination campaigns, their cost–effectiveness and sustainability, and the surveillance and management of rabies cases. When a proportion of the dog population cannot be handled by their owners or when no single owner claims responsibility for vaccination, expert dog handlers can be used to catch and restrain dogs humanely for vaccination. Various methods are available for dog catching. Expert dog handlers require suitable training to ensure they can catch dogs efficiently, reliably and humanely; inexpert handling can injure both catcher and dog and may make future catching for vaccination more difficult. Oral vaccination of dogs may improve coverage in situations in which dogs cannot be restrained or caught. Further field studies are required to evaluate the cost–effectiveness of oral vaccination for achieving target coverage in different settings with different delivery strategies (28).

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9.5 Supplementary measure: humane dog population management Humane management of dog populations is achieved mainly by responsible dog ownership and provision of sterilization services and basic dog health care (29). The objective of dog population management in the context of canine rabies control is to improve and maintain vaccination coverage and reduce risky dog behaviour. As there is no evidence that rabies transmission depends on the density of dog populations, reducing the population size through humane means may not be the most important factor, although it may have other benefits (e.g. with regard to dog welfare or nuisance behaviour). Dog population management may therefore be beneficial in canine rabies control. Work on the impact of humane dog population management programmes on rabies (and other associated benefits) has been relatively limited (5,6,29) and further evaluation of this approach would be beneficial. Humane dog population management is an effective strategy for reducing dog population turnover and creating a healthy, sustainable population. As the status and composition of dog populations varies from country to country, no one intervention will work in all situations. Authorities should work with people who know the local dog population in order to understand ownership, demographics and the attitude of the local community towards dogs. This information can form the basis for a tailored package of humane dog population management tools for long-term, sustainable management (16,19,29). India has an unusually high proportion of ownerless dogs. Dog population management has been used for canine rabies control in animal birth control programmes, in which free-roaming dogs are caught, sterilized and vaccinated before being released. Several locations have reported reductions in the number of human deaths from rabies during such programmes (5,30,31). The contribution of sterilization, over and above vaccination alone, to the control of dog rabies has, however, not been fully evaluated.

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9.6 Main components of a dog rabies control programme The Consultation recommended that the following components be included in a dog rabies control programme: ■■ Establish national focal points and national rabies elimination committees to prepare, implement and monitor long-term plans for management of people at risk with targeted pre- and post-exposure prophylaxis regimens, mass vaccination of dogs and humane management of dog populations.

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■■ Strengthen surveillance and diagnostic facilities to include rapid diagnostic measures. ■■ Ensure sustainable community, district, national and regional rabies control programmes. ■■ Develop effective cross-border collaboration for rabies control and elimination. ■■ Promote through campaigns and child education programmes increased awareness in the general public of the benefits of responsible dog ownership, basic care of suspected rabid bites and avoiding animal exposure ■■ Foster cooperation among all relevant sectors, including veterinary services, public health, wildlife management and ecologists, to develop evidence-based approaches to human and animal rabies elimination. ■■ Support integration of rabies control activities at all levels of the health services, aligning them with other public health programmes, such as those for bacterial (e.g. tuberculosis), parasitic (e.g. neurocysticercosis, cystic echinococcosis) and vector-borne diseases (e.g. human African trypanosomiasis, leishmaniasis). Synergies among programmes improve the logistics of use of human, material and financial resources. ■■ Seek funding from bilateral and multilateral agencies and other donors in the framework of technical cooperation or humanitarian aid. ■■ Strengthen coordination and collaboration among international organizations, such as WHO, the Food and Agriculture Organization of the United Nations (FAO), OIE with their specialized networks of collaborating centres and reference laboratories and nongovernmental global and regional organizations (such as GARC, the World Veterinary Association, the Commonwealth Veterinary Association, the World Society for the Protection of Animals, and other international animal welfare organizations and coalitions). ■■ Stimulate cooperation with the pharmaceutical industry and institutions for the provision of vaccines, both human and veterinary, and technical cooperation to ensure proper vaccine storage, delivery and administration. 71

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9.7

Operational research for dog rabies control

Operational research on dog rabies control is conducted during interventions (e.g. vaccination, population control), taking advantage of the fact that animals are handled and can be inspected and marked. In operational research, efforts should be made to adhere to proper protocols, and ensure rigorous statistical standards and unbiased sampling. When possible, controls should be included. The main areas in which further operational research is needed are as follows. ■■ Questionnaire surveys on owned dogs and to elicit opinions about ownerless dogs should be conducted to determine dog population size (per person, per household, per surface area), demography, dynamics, and distribution before and after interventions (32,33). Vaccination and other veterinary interventions can provide the opportunity to apply a visual mark temporarily or permanently, such as a collar, ear notch, ear tag or tattoo for recapture studies. Better methods are needed for marking dogs rapidly and cost–effectively and for subsequent mark–recapture analyses, which include, for example, information on short-term movements of dogs (home range for 1 day to 1 week). Methods are needed to integrate questionnaire surveys and wildlife census methods to better determine the numbers of truly ownerless dogs that may not be readily accessible for vaccination. ■■ Questionnaire surveys can also be used to collect information on awareness about rabies, social attitudes to dogs and methods of dog population control as part of an education programme. WHO Technical Report Series No. 982, 2013

■■ Direct observation and questionnaire surveys should be used to collect data on the extent of supervision, which must be clearly defined. This information can be used to estimate the accessibility of dogs for veterinary interventions, which depend on the extent of supervision, culture, habitat and ecology (climate, meteorology). ■■ Methods for marking dogs that are sterilized or vaccinated temporarily (e.g. collars, stains, microchips) or permanently (e.g. tattoos, ear tags) should be explored. ■■ Better methods are needed for recording the absolute numbers and proportions of the dog population in different classes (e.g. age, sex,

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treatment) with hand-held devices, positioning devices or computer software and for managing the results for rapid use to assess the incidence of disease in relation to vaccination coverage. ■■ Means could be explored for classifying and recording dog condition, diseases and parasites (e.g. during veterinary interventions, field surveys, household visits) to determine the health of the dog population. This would allow assessment of the effects of diseases on dog population dynamics and management of dog health. ■■ Applied research is required on the economics of dog vaccination, the sustainability of programmes, willingness to pay, cost–effectiveness and cost–benefit analyses in different cultural, ecological and economic contexts (13,26,27,34,35), including large-scale and regional models. The research should include socioeconomic barriers to programme implementation, translation of research into policy and practice and potential integration of dog rabies control into programmes for other dog-borne zoonoses, such as echinococcosis and leishmaniasis. ■■ The relevant information and methods of disseminating it should be assessed. The impact of education campaigns can be judged by analysing questionnaire surveys conducted before and after a campaign. Other means that can be used to evaluate education include changes in the numbers of dog bites, hospital visits and free-roaming dogs. ■■ As dog population management moves towards surgical or nonsurgical sterilization or contraception, questions remain on the impact of fertility control on dog population size and rabies control, including effects on population dynamics, social behaviour and disease transmission (5,36). Research should conducted to assess whether fertility control reduces the contact rate, home range and aggressiveness (particularly of males) and the disease transmission rate. ■■ Recently developed nonsurgical sterilants and contraceptives, such as immunocontraceptives and intratesticular sterilants, should be tested when dogs can be closely monitored to determine their humaneness, the longevity of the effect at population level and the feasibility of using and delivering these drugs (37). ■■ The cost, feasibility and sustainability of combining surgical or nonsurgical sterilization with rabies vaccination should be assessed. In parallel, cost–benefit analyses should be carried out to compare dif73

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ferent dog population management options and to determine whether and how fertility control could be used as an adjunct to optimize rabies elimination programmes in some contexts.

9.8 References 1. Lembo T et al. Renewed global partnerships and redesigned roadmaps for rabies control. Veterinary Medicine International, 2011 (doi:10.4061/2011/923149). 2. Lembo T et al. Zoonoses prevention, control, and elimination in dogs. In: Macpherson CNL, Meslin F-X, Wandeler AI, eds. Dogs, zoonoses and public health, 2nd ed. Wallingford, Oxon., CAB International, 2013:205– 258. 3. Nel L, Le Roux K, Atlas R. Meeting the rabies control challenge in South Africa. Microbe, 2009, 4(2):61–65. 4. Wandeler AI et al. Dogs and rabies. In: Macpherson CNL, Meslin F-X, Wandeler AI, eds. Dogs, zoonoses and public health, 2nd ed. Wallingford, Oxon., CAB International, 2013:43–66.

5. Reece JF, Chawla SK. Control of rabies in Jaipur, India, by the sterilisation and vaccination of neighbourhood dogs. Veterinary Record, 2006, 159:379–383. 6. Totton SC et al. Stray dog population demographics in Jodhpur, India following a population control/rabies vaccination program. Preventive Veterinary Medicine, 2010, 97:51–77.

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7. Terrestrial animal health code. Chapter 7.7. Stray dog control. Paris, World Organisation for Animal Health, 2011 (http://www.oie.int/index. php?id=169&L=0&htmfile=chapitre_1.7.7.htm; accessed 29 November 2012). 8. Coleman PG, Dye C. Immunization coverage required to prevent outbreaks of dog rabies. Vaccine, 1996, 14:185–186. 9. Cleaveland S et al. Dog rabies vaccination campaign in rural Africa: impact on the incidence of dog rabies and human dog-bite injuries. Vaccine, 2003, 21:1965–1973.

10. Tamayo H et al. Case report (4) Americas. Elimination of human rabies transmitted by dogs in Latin America and the Caribbean: achievements. In: OIE Global Conference on Rabies Control, Republic of Korea, 7–9 September 2011 (http://www.oie.int/eng/A_RABIES/presentations.htm; accessed 29 November 2012).

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11. Report of the 4th meeting of the international coordination group of the Gates Foundation/WHO project for human and dog rabies elimination in low-income countries, 2–4 October 2012, Cebu, Philippines. Geneva, World Health Organization, 2013 (http://www.who.int/rabies/bmgf_who_ project/en). 12. Report of the 3rd meeting of the international coordination group of the Gates Foundation/WHO project for human and dog rabies elimination in low-income countries, 19–21 October 2011, PieterMaritzburg, KwaZulu Natal, South Africa. Geneva, World Health Organization, 2011 (http:// www.who.int/rabies/bmgf_who_project/en/). 13. Lapiz SMD et al. Implementation of an intersectoral programme to eliminate human and canine rabies. The Bohol Rabies Prevention and Elimination Project. PLoS Neglected Tropical Diseases, 2012, 6(12):e1891. 14. Suseno PS et al. Dog vaccination and campaign management for effective rabies control: the Bali experience. In: International Conference on Emerging Infectious Diseases, 11–14 March 2012, Atlanta, Georgia. Atlanta, Georgia, United States Centers for Disease Control and Prevention, 2012. 15. Suseno PP et al. Integrated bite case management for rabies in Bali: putting one health into action. In: International Conference on Emerging Infectious Diseases, 11–14 March 2012, Atlanta, Georgia. Atlanta, Georgia, Centers for Disease Control and Prevention, 2012.

16. Lembo T et al. The blueprint for rabies prevention and control: a novel operational toolkit for rabies elimination. PLoS Neglected Tropical Diseases, 2012, 6(2):e1388. 17. Report of a WHO consultation on dog ecology studies related to rabies control. Geneva, World Health Organization, 1988 (WHO/Rab. Res./88.25). 18. Hiby LR et al. A mark–resight survey method to estimate the roaming dog population in three cities in Rajasthan, India. BMC Veterinary Research, 2011, 7:46. 19. Humane dog population management guidance. International Companion Animal Management Coalition, 2008 (http://www.wsava.org/PDF/2008/ Misc/AWC_ICAM_Coalition.pdf). 20. Blueprint for rabies prevention and control [canine rabies blueprint]. Partners for Rabies Prevention (www.rabiesblueprint.com; accessed March 2013). 75

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21. Townsend SE et al. Surveillance guidelines for disease elimination: a case study of canine rabies. Comparative Immunology, Microbiology and Infectious Diseases, 2012 (http://dx.doi.org/10.1016/j.cimid.2012.10.008).

22. Suzuki K et al. Rabies vaccination coverage and profiles of the owned dog population in Santa Cruz de la Sierra: Bolivia. Zoonoses and Public Health, 2008, 55(4):177–183. 23. Flores-Ibarra M, Estrella-Valenzuela G. Canine ecology and socioeconomic factors associated with dogs unvaccinated against rabies in a Mexican city across the US–Mexico border. Preventive Veterinary Medicine, 2004, 62(2):79–87. 24. Kaare M et al. Rabies control in rural Africa: evaluating strategies for effective domestic dog vaccination. Vaccine, 2009, 27:152–160.

25. Kappeler A, Wandeler A. Dog population studies related to a vaccination campaign against rabies in Lalitpur City, Nepal. Report to WHO. Geneva, 1989 (whqlibdoc.who.int/Kappeler_Wandeler_Nepal_Report_1989_ eng; accessed 18 February 2012). 26. Boegel K, Meslin FX. Economics of human and canine rabies elimination: guidelines for programme orientation. Bulletin of the World Health Organization, 1990, 68:281–291. 27. Zinsstag J et al. Transmission dynamics and economics of rabies control in dogs and humans in an African city. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106:14996– 15001. 28. Oral vaccination of dogs against rabies: guidance for research on oral rabies vaccines and field application of oral vaccination of dogs against rabies. Geneva, World Health Organization, 2007 (http://www.who.int/ rabies/vaccines/veterinary_ vaccines/en/index.html; accessed May 2012). 29. Hiby E. Dog population management. In: Macpherson CNL, Meslin F-X, Wandeler AI, eds. Dogs, zoonoses and public health, 2nd ed. Wallingford, Oxon., CAB International, 2013:177–204. 30. Chinny Krishna S. Control of rabies—Has the ABC programme been a success in India? Indian Journal of Environmental Education, 2003, 2:5–8. 31. Tenzin, Ward MP. Review of rabies epidemiology and control in South, South East and East Asia: past, present and prospects for elimination. Zoonoses and Public Health, 2012 (doi: 10.1111/j.1863-2378.2012.01489.x).

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32. Jackman J, Rowan AN. Free-roaming dogs in developing countries: the benefits of capture, neuter, and return programs. In: Salem DJ, Rowan AN, eds. The state of the animals IV. Washington DC, Humane Society Press, 2007:55–64.

33. Lembo T et al. The feasibility of canine rabies elimination in Africa: dispelling doubts with data. PLoS Neglected Tropical Diseases, 2010, 4:e626 34. Knobel DL et al. Re-evaluating the burden of rabies in Africa and Asia. Bulletin of the World Health Organization, 2005, 83:360–368. 35. Kayali U et al. Cost-description of a pilot parenteral vaccination campaign against rabies in dogs in N’Djaména, Chad. Tropical Medicine and International Health, 2006, 11:1058–1065. 36. Carroll MJ et al. The use of immunocontraception to improve rabies eradication in urban dog populations. Wildlife Research, 2010, 37:1–12. 37. Massei G. Fertility control in dogs. In: Macpherson CNL, Meslin F-X, Wandeler AI, eds. Dogs, zoonoses and public health, 2nd ed. Wallingford, Oxon., CAB International, 2013:205–258.

10. Prevention and control of rabies in wild animals In the past, rabies was seen predominantly in domestic dogs, although there were occasional reports indicating the involvement of wildlife. Strict implementation of dog mass vaccination and other measures resulted in the virtual disappearance of dog-mediated rabies in Europe and North America during the 1940s, but the disease unexpectedly re-emerged in wildlife. With progress in molecular approaches to the identification and phylogeny of virus variants, understanding of lyssavirus epidemiology has improved significantly. Rabies is a viral zoonosis associated with many species of Carnivora and Chiroptera, which are the primary hosts of the rabies virus; only Chiroptera species are the primary hosts of almost all other lyssaviruses (see section 2).

10.1 Epidemiology and ecology of rabies in carnivore species 10.1.1 Africa The cosmopolitan lineage of canine rabies virus is believed to have spread across the African continent during the European colonization. Domestic dogs remain the major primary hosts of rabies virus in Africa (1). Although sporadic cases of rabies in wildlife have been documented across the African continent, convincing 77

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evidence for the circulation of rabies in populations of wild carnivores has been found only in southern Africa, where wild canids, such as jackals (Canis adustus and C. mesomelas) and bat-eared foxes (Otocyon megalotis) are assumed to be primary hosts of rabies virus (2,3). Additionally, members of the Herpestidae family (e.g. mongooses) appear to be responsible for transmission of a distinct variant of rabies virus in southern Africa (4). Infection with a canid rabies virus has been shown to be the cause of significant mortality among kudus (Tragelaphus strepsiceros) in Namibia, and direct oral transmission of infective saliva from kudu to kudu is suspected (5,6). Spillover rabies virus from dogs is threatening endangered wild African canids such as the Ethiopian wolf (C. simensis) and the African wild dog (Lycaon pictus) (7–10).

10.1.2 Middle East and Asia While dog rabies predominates in central and tropical Asia, rabies is maintained by wild canids in the forest–steppe and steppe zones of continental Asia, primarily by the red fox (Vulpes vulpes) and in the Russian far east by the raccoon dog (Nyctereutes procyonoides) (11,12). In southern China, the ferret badger (Melogale moschata) has been associated with human rabies for several years and is considered to be a primary host in this region (13). Although occasional cases of rabies have been reported in wild carnivores in a number of countries in the Middle East and central, South and SouthEast Asia, it is unclear whether wildlife rabies is independent of the dog rabies transmission cycle in these regions. Fox rabies is present in Israel, the West Bank and Gaza Strip and has emerged in Turkey, where most cases of cattle rabies result from contacts with rabid foxes (14). Furthermore, certain countries in the Middle East region are reporting increasing numbers of cases of wildlife rabies, including the Islamic Republic of Iran, Oman, Saudi Arabia and Yemen. Red foxes and golden jackals (C. aureus) are usually implicated in those regions (15–17).

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10.1.3 Europe Wildlife rabies emerged in Europe after dog rabies was eliminated, the new primary host being the red fox (V. vulpes). Coming from the east, fox rabies spread inexorably across the continent within a few decades. By the mid-1980s, large parts of central and western Europe were affected. The westward expansion came to a halt in areas such as France and northern Italy, where foxes were treated with oral rabies vaccine (17). Infected foxes are responsible for maintaining rabies virus within the fox population and also for transmission to other wildlife species and domestic animals. In affected areas, rabies is detected in a wide variety of species at different

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frequencies. The animals most likely to come into contact with rabid foxes, such as roe deer, cattle and other domestic ruminants, represent most of the victims. There are indications that the raccoon dog (Nyctereutes procyonoides) may act as another primary wildlife host, as it is the second most frequently reported infected species in central and Baltic Europe (18). Presently, fox-mediated rabies is still prevalent in eastern and southeastern Europe, while large parts of western and central Europe have been freed from fox rabies by implementation of national and regional oral rabies vaccination programmes (19). Some southern and insular Mediterranean countries were never affected by the fox rabies epizootic, although a case was reported in northern Greece in October 2012 (20). Other countries have never had fox rabies, e.g. Sweden and the United Kingdom (17).

10.1.4 North America With successful elimination of canine rabies in Canada and the USA in the middle of the 20th century and substantial progress in prevention and control of canine rabies in Mexico, wildlife rabies began to emerge in North America, as in Europe. In contrast to other parts of the world, wildlife rabies in temperate North America involves many primary host cycles, often with overlapping geographical ranges, making animal rabies control a major challenge. The commonest primary hosts are red foxes (V. vulpes) in parts of Alaska and Canada and raccoons (Procyon lotor) in the east. While the North American fox rabies epizootic extended its range in Canada, a different rabies virus variant emerged in raccoons in Florida (USA) and spread to neighbouring states. The spread was accelerated by translocation of rabid raccoons into the mid-Atlantic area in the 1970s, and the outbreak extended south and north as far as Quebec. Whereas the epizootic of fox rabies in southeastern Canada was eventually eliminated towards the end of the twentieth century, largely as a result of oral rabies vaccination, raccoon rabies still poses a serious problem in the region (21–23). The Arctic fox (Alopex lagopus) is a primary host in the polar regions of the continent, and the striped skunk (Mephitis mephitis) is a major host throughout the central plains and in California (22,23). In addition, grey foxes (Urocyon cinereoargenteus) are involved, particularly in southwest USA, and several species of skunk (Spilogale spp.) are recognized as primary hosts in Mexico. Each wild species maintains one predominant host-adapted rabies virus variant but can also harbour distinct spillover variants of rabies virus from other primary host species. Spillover to other wild and domestic animals is frequent in all areas. To date, oral rabies vaccine has played a major role in the prevention and control of rabies in red foxes and raccoons and in the elimination of rabies in coyotes and grey foxes in Texas. 79

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10.1.5 South America Rabies has been documented in wild carnivores in several areas, and phylogenetic studies of the genomes of rabies virus isolates from a variety of species indicate the presence of several distinct wildlife primary hosts, including the marmoset (Callithrix spp.) and the crab-eating fox (Cerdocyon spp.). Surveillance of wildlife for rabies is, however, generally inadequate to allow major epidemiological inferences. Information on the presence of rabies can be obtained from the Pan American Health Organization (http://new.paho.org/rabies).

10.1.6 Caribbean islands The small Indian mongoose (Herpestes auropunctatus), which was introduced from South Asia to many Caribbean islands in the second half of the 19th century for rodent control, is a primary rabies host in parts of the Caribbean. For example, mongoose rabies is currently reported in Cuba, the Dominican Republic, Grenada and Puerto Rico. Other Caribbean islands are considered free of rabies among domestic and wild carnivores.

10.1.7 Eurasian and American arctic and subarctic regions Arctic foxes (Alopex lagopus), domestic dogs and red foxes participate in the propagation of arctic rabies or ‘polar madness’, although the epidemiology is not well understood in these thinly populated areas with incomplete surveillance. Interestingly, arctic-like rabies virus lineages are also found in central and SouthEast Asia.

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Lyssaviruses have been detected in bats throughout the world, although different species are present in different regions (24; see also Table 1 in section 2). Bats have been identified as vectors for all Lyssavirus species except Mokola virus and Ikoma lyssavirus (see section 2), for which the true primary host is yet to be found. This observation strongly suggests that bats are true primary hosts for lyssaviruses. Bats have several traits (e.g. small size, long life, low intrinsic population growth rates and a variety of well-defined ecological niches) that are different from those of carnivore rabies hosts. Consequently, the properties of the lyssaviruses adapted to bats must be different from those that cause rabies in carnivores. The factors involved in maintenance of lyssaviruses in bats are insufficiently explored.

10.2.1 Lyssaviruses in Africa, Australia and Eurasia At least four lyssavirus species are known to circulate in populations of insectivorous and frugivorous African bats (see Table 2, section 2). Lagos bat

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virus, a lyssavirus predominantly associated with various large African fruit bat species (Megachiroptera) was originally isolated from Eidolon helvum in Nigeria in 1956 and later from other bat species in the Central African Republic, Senegal and South Africa. An epizootic that resulted in significant mortality among Epomophorus bats was observed in Natal, South Africa, where the virus is still occasionally isolated. Lagos bat virus has also occasionally been isolated from the insectivorous Gambian slit-faced bat (Nycteris gambiensis). No human cases have been confirmed to date, perhaps due to insufficient surveillance and virus characterization. Spillover of Lagos bat virus to other mammals has been reported infrequently (1,25). Duvenhage virus was first isolated in 1970 from a person in Transvaal, South Africa, who died of rabies encephalitis after being bitten by an insectivorous bat reported to be associated with Miniopterus spp. Two further cases of rabies due to Duvenhage virus in humans have been reported, one in South Africa and the other in the Netherlands, the latter infection having been contracted in Kenya (1). In 2009, a bat-associated lyssavirus called Shimoni bat virus was isolated from the insectivorous Commerson leaf-nosed bat (Hipposideros commersoni) in Kenya. With Mokola virus and Lagos bat virus, it belongs to phylogroup II (26) (see section 2.3). In 1996, Australian bat lyssavirus was isolated from fruit-eating bats (flying foxes, Pteropus alecto) on the eastern coast of Australia, a country considered to be ‘rabies-free’ since 1867. Two human deaths due to rabies caused by Australian bat lyssavirus were confirmed in 1996 and 1998. Australian bat lyssavirus has been isolated from all four species of frugivorous megabat (genus Pteropus, family Pteropodidae) in Australia and from an insectivorous bat species, the yellow-bellied sheath-tailed bat (Saccolaimus flaviventris) (27,28). In Europe, sporadic cases of rabies have been diagnosed in bats in the past 60 years. Most cases are in serotine bats (Eptesicus serotinus), the viruses being identified as European bat lyssavirus type 1, while those from Myotis bats (M. dasycneme and M. daubentonii) are characterized as European bat lyssavirus type 2 (29,30). Cases of bat rabies appear to be less frequent in Europe than in the New World; however, the level of surveillance in Europe is still very heterogeneous, despite international recommendations. In total, four autochthonous human rabies cases transmitted by bats have been confirmed in Europe: two in the Russian Federation (1977 and 1985), one in Finland (1985) and one in Scotland (2002) (24). In 2002, a common bent-wing bat (Miniopterus schreibersii) was captured in the Russian Federation near the Georgian border and subsequently tested positive for lyssavirus infection. The virus, named West Caucasian bat virus, was a genetically divergent bat-derived member of the Lyssavirus genus, representing a member of phylogroup III, with no serological cross-reactivity to other lyssaviruses (31). 81

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Bokeloh bat lyssavirus, isolated from a Natterer bat (Myotis nattereri) in Germany in 2010 and France in 2012, has been shown to differ from all previously known lyssaviruses occurring in Europe but to be antigenically and genetically close to European bat lyssavirus type 2 and Khujand virus (32,33). In 2012, an Ikoma lyssavirus-like virus was detected in Miniopterus schreibersi on the Iberian Peninsula (34). In central Asia, three bat-associated lyssaviruses have been isolated. In 1991, an apparently healthy lesser mouse-eared bat (Myotis blythi) captured in the Aravan district, Kyrgyzstan, tested positive for rabies by the mouse inoculation test. Ten years later, near the town of Khujand, Tajikistan, a whiskered bat (Myotis mystacinus) also tested positive. Subsequent characterization of the isolated viruses revealed two new lyssavirus species: Aravan virus and Khujand virus (35). In 2002, a virus from Murina spp., commonly known as tube-nosed bats, was classified as a lyssavirus and named Irkut virus after a village in Irkutsk Province. One human case of rabies reported in Far East Russia in 2007 was due to infection with a virus similar to the original Irkut virus (31). Little is known about the epidemiology of bat lyssaviruses that have been isolated only once.

10.2.2 Rabies in insectivorous bats in the Americas To date, all bat lyssaviruses in the Americas have been categorized as rabies virus. Many genetically and antigenically distinct variants circulate in bat species, several occurring within a single species, and the geographical distribution of variants overlaps. There is, however, an inverse correlation between cross-species transmission and phylogenetic distance among insectivorous bat species (36,37). Spillover to other animals is observed frequently. Although the incidence of human rabies is low in temperate North America, nearly 50% of cases are caused by bat-associated rabies virus (38). The silver-haired bat (Lasionycteris noctivagans) and the eastern tri-coloured bat (Parasrellus subflavus) play key roles in transmitting bat rabies to humans.

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10.2.3 Vampire bat rabies

Vampire bat rabies is a major public health problem in the subtropical and tropical areas of the Americas, from Mexico to Argentina. A rabies virus variant related to the other American bat viruses is maintained in haematophagous bats, mainly by Desmodus rotundus (37) and is transmitted frequently to domestic animals and humans. Vampire bat-transmitted bovine paralytic rabies has a significant economic effect on the livestock industry. Currently, most cases of human rabies in Amazonia are caused by haematophagous bats (39).

10.3 Rabies in rodents Testing of tens of thousands of wild and synanthropic rodents in areas endemic for rabies across the world has revealed only exceptional instances of dead-

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end spillover of rabies virus infection, indicating that these animals are neither primary hosts nor play a role in the epidemiology and transmission of the disease.

10.4 Wildlife species of special concern Rabies has emerged as a threat to conservation after outbreaks in highly endangered populations of Ethiopian wolves (C. simensis) in the Bale Mountains National Park, in African wild dogs (Lycaon pictus) in eastern and southern Africa and in the Blanford fox (V. cana) in Israel. Ethiopian wolves and African wild dogs are among the world’s most highly endangered carnivore species, and transmission of rabies virus from more abundant primary hosts (such as domestic dogs) is considered a threat for extinction of several populations. Rabies has been recorded in wolves (C. lupus) everywhere in the northern hemisphere where rabies occurs in wildlife, and they are therefore often believed to play a major role in transmission. Although wolves are susceptible and readily succumb to the disease, they cannot sustain circulation of rabies virus independently of other wildlife, as wolf population densities and dynamics do not support epizootics, and the highly territorial nature of wolves prevents ready spread of the disease from one pack to another. Once a pack member is infected, the disease can decimate the pack because of its highly social nature, with regular contact among the animals. The genetic make-up of rabies virus isolated from wolves is identical to those found in more abundant carnivore primary hosts in their vicinity (either domestic dog or wild species). Although wolves are more a victim of the disease rather than a true primary host, they can transmit rabies virus to other naive, susceptible hosts. Rabies in wolves is often experienced as a dramatic event, particularly if people are involved. Because they migrate over long distances, wolves that are incubating rabies virus are believed to be able to reintroduce wildlife rabies into freed areas.

10.5 Elimination of rabies in wild carnivores 10.5.1 Reduction of animal populations Rabies virus transmission within wild carnivore populations that are capable of sustaining an infection cycle is considered to be density-dependent. Conventional methods of rabies control with drastic decimation of wild carnivore populations have failed to eliminate rabies (17,40). The resilience of Carnivora to elimination, their high reproductive potential and the capacity of the environment to provide food, water and shelter often make population control efforts futile. Consideration of humane, economical and ecological aspects will prevent inefficient large-scale culling campaigns. 83

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10.5.2 Immunization Mass vaccination of the principal wildlife hosts is a more effective control method than culling. This method emerged independently in Europe and North America (22,40). Since the late 1970s, the oral rabies vaccination strategy originally developed for foxes has been used to eliminate fox rabies in large parts of western and central Europe, Canada and the USA. Its success was due to research on and development of tools including effective, safe vaccines, machine-made baits that are attractive to a variety of species, automated, computer-supported aerial bait distribution, adequate vaccination strategies and strong political commitment (22,41). An oral rabies vaccination strategy that works for one carnivore primary host species will not necessarily work for others. Adapted oral vaccine strategies have been used quite successfully not only for red foxes but also for other primary wildlife hosts, including coyotes, grey foxes and raccoon dogs, although they require optimization for raccoons (23). Different strategies are needed for other primary wildlife hosts. As oral rabies vaccination programmes are designed to eliminate rabies from a defined area or to prevent spread of the disease by creating an immunological barrier (containment, cordon sanitaire), they should result in sufficient herd immunity to reduce transmission (i.e. the effective reproductive rate of the disease falls below 1) in the target primary wild host. The level of herd immunity required varies with the transmission dynamics of the disease in particular target species and populations and with local conditions. Vaccines used in the field must fulfil the requirements of national or international regulatory authorities for biological products, i.e. efficacy, safety and stability, and be licensed or registered (see section 7). Baits must be designed for each target wild animal to ensure that the vaccine is released onto a susceptible target tissue (oropharyngeal mucosa or tonsils) to elicit an immune response. The bait casing must fulfil three functions: to carry the attractant for the target species, to contain a biomarker (usually tetracycline) of bait uptake by the target population, and to protect the vaccine blister, capsule or sachet from ultraviolet light to ensure the stability of the virus titre. The requirements for bait casings are laid down in relevant standards (42–46). The bait must be thermostable to guarantee its palatability, and it should be tested before marketing authorization at different temperatures (44). As the majority of rabies vaccine baits are consumed within 7 days of distribution in the field, the bait casing should protect the vaccine sachet or blister for this time under local weather conditions. Warnings should be printed on the blister or bait matrix. Bait uptake and herd immunity in the target population depend on, e.g., vaccine efficacy and stability, the bait casing and attractiveness, the baiting method, the spatial distribution of baits, timing of oral rabies vaccination

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campaigns and the abundance of bait competitors. The mode of bait distribution should guarantee that most of the target species has access. Oral rabies vaccine campaigns are usually conducted twice a year, in spring and in autumn in Europe and once a year in North America, with bait delivered mainly from fixed-wing aircrafts or helicopters (23,44). Manual distribution should complement aerial distribution or may be the only way to distribute bait in densely populated areas.

10.5.3 Planning, implementing and evaluating oral rabies vaccination programmes Oral rabies vaccine has become the essential tool for preventing geographical spread, control and elimination of rabies when the primary host is wildlife. As oral rabies virus vaccines and baits developed for one primary host species may not work for others, vaccine efficacy, bait design and attractiveness should be evaluated for each new target species. Evaluation of oral vaccination programmes should include a cost–benefit analysis for public health. The basic requirements for planning, implementing and evaluating large-scale vaccination campaigns or field trials have been published (43,44) and were revised recently (45). Epidemiological data based on reliable surveillance and laboratory studies of rabies cases in target and non-target species (wild and domestic) must be available before an oral rabies vaccination programme or field trial is initiated. Planning Strong political commitment is a prerequisite for an oral rabies vaccination programme, as the legal framework, planning, organization and evaluation are vital to its success. A national rabies committee should be constituted that includes all stakeholders. An effective programme is based on a comprehensive plan, outlining the justification (benefits), the objectives, roles (which agencies should be involved), responsibilities (who is responsible for what) and chains of command as well as infrastructure (laboratory requirements and equipment, cold chain), estimated costs (budgetary requirements) and funding. The plan must also include information on the areas to be covered in consecutive years, taking into account the patterns of movement of wildlife populations, the geographical characteristics of the area, the rabies situation in neighbouring countries, details of the vaccination strategy (timing, mode of bait distribution, bait density, flight line distance), safety considerations, surveillance and monitoring of campaigns. The size of the target population should be estimated, with baseline levels of the biomarker (if applicable) in the target species before vaccination. As a general rule, an oral rabies vaccination programme should consist of two phases: an attack phase (elimination) and a maintenance phase. A long-term, large-scale approach is the most effective, and there must be a guarantee that the programme can be sustained in the long term. The plan should be distributed to 85

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competent authorities well in advance for consideration and evaluation. Upon request, WHO can provide the necessary expertise. Implementation Delivery of oral rabies vaccine requires infrastructure and logistics that ensure the integrity of the bait and the vaccine (maintenance of cold chain) and that allow distribution of adequate numbers of baits (airports, aircrafts, other personnel) to cover large areas evenly. Initial meetings should be organized by the national rabies committee for all stakeholders, including hunters, trappers, wildlife service staff, forest officers, physicians, veterinarians and local authorities, to discuss the programme in detail and to agree on the responsibilities of each stakeholder. Responsible authorities and personnel should be trained in rabies surveillance, database management, data analysis and interpretation to monitor the progress of the intervention; reporting and dissemination of information to the competent authorities; the vaccine bait, the target species and the human component, and sampling of specimens under appropriate conditions. Trained personnel and laboratory facilities should be available to carry out the recommended standard tests for routine diagnosis of rabies (see section 4) and for monitoring (biomarker detection, serology, virus titration, characterization of rabies virus isolates) the campaign in a quality assurance system. The awareness of hunters, trappers, the general public and medical and veterinary practitioners about the campaign should be raised, so that they can take appropriate measures in case of accidental exposure to the vaccine. A medical or veterinary advisory group should be established. Assignment of specialists is strongly encouraged to investigate the prevailing and changing epidemiological situation in both humans and animals and to evaluate the campaign and report regularly to the responsible authorities. National meetings should be held regularly with all stakeholders to discuss the progress of the campaign and any adaptations required for future campaigns. Evaluation Surveillance and monitoring of oral rabies vaccination campaigns are essential for evaluating their success. They require a sustained, constant, intensive approach. Adequate surveillance is important, as the incidence of rabies is the index of the impact of a programme. A risk-based sampling scheme should be used, focused on so-called ‘indicator animals’ that are ill, suspected of being rabid, show abnormal behaviour, found dead or involved in human exposure. Although the exact number of animals required cannot be predetermined, it should be sufficient to demonstrate an acceptable statistical degree of certainty (18). Surveillance

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should generally be conducted before, during and after administration of vaccine, not only in the vaccination areas but also in neighbouring areas, particularly those free of rabies, to detect spread of the epizootic or re-infection as early as possible to allow a swift response and countermeasures (45). Rabies viruses isolated from animals in the vaccination areas should be characterized. The Consultation stressed the importance of reinforced surveillance in vaccination areas and beyond and requested governments to consider and adopt the above guidelines. Monitoring the efficacy of an oral rabies vaccination programme (bait uptake, seroconversion) requires adequate sampling of hunted or trapped animals of the target species. The suggested sampling size is four target animals per 100 km2 and year (18); however, experience has shown that this sample size can be difficult to achieve, depending on the topographical features of the vaccination area, infrastructure and logistics. If this number of samples cannot be taken, a reference area in which the sample size could be reached may be selected. Basic or denominator data, e.g. species, date of finding and submission, location (Gauss–Krueger coordinates or lowest national unit of territories), age, sex, results of laboratory investigations (fluorescent antibody or tissue culture infection test, virus characterization, biomarker detection, serology) should be collected for all animals found for stratification and proper epidemiological (temporal and spatial) analysis. To eliminate rabies in wildlife, ‘progressive control pathways’ and procedures for international certification of rabies-free status should be established. International cooperation International cooperation and coordination in planning, implementing and evaluating oral rabies vaccination programmes at all levels is necessary for success and cost–effectiveness. Preliminary contact should be made with neighbouring countries when the policy is decided, and these contacts should be maintained until elimination of the disease. Regular multilateral meetings with representatives of the public health and veterinary authorities of neighbouring regions and countries ensure coordination of activities along common borders and transparency. The involvement of WHO collaborating centres and of other international organizations is recommended. The results of vaccination programmes should be presented at international conferences, as a presence on the international stage can help pressure national governments to remain heavily committed to rabies elimination. Other options Besides oral vaccination, strategic trapping of wild carnivores and releasing them after parenteral vaccination (trap–vaccinate–release) has been used with 87

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apparent success in some areas of North America, primarily for skunks and raccoons (46,47).

10.6 Bat rabies control The goal of eliminating the disease in bats is challenged by the plethora of lyssavirus species and the substantial role of Chiroptera in global ecology, such as in seed dispersal, pollination and arthropod predation. Elimination of bat rabies is therefore not possible at the present time. The public health risk associated with bat rabies (except that transmitted by vampire bats) is lower than those associated with carnivores rabies, although the consequences of infection are also severe. Therefore, any method that indiscriminately destroys bats should be avoided, especially as bats are protected in most countries. Education of the public is the key to preventing bat-transmitted human rabies. It should include basic information on avoiding potentially infectious contact with bats, seeking proper medical attention after exposure and preventing bats from establishing colonies in sensitive buildings (e.g. hospitals and schools). Vampire bat-transmitted paralytic rabies of cattle can be controlled by vaccinating cattle. The approaches to controlling vampire bat-transmitted rabies by culling the primary host species with an anticoagulant, by direct application on the backs of captured bats or by intramuscular injection of cattle, is questionable and obsolete. Strict application of post-exposure prophylaxis is recommended in cases of human exposure to vampire bats. Preventive immunization of populations living in highly enzootic areas with limited access to anti-rabies biologicals should be considered.

10.7 Other public health measures WHO Technical Report Series No. 982, 2013

The general public should be better informed about avoiding direct contact with wildlife in general and with abnormally behaving and sick animals in particular. Any person bitten by a wild or domestic animal, particularly in areas where wildlife rabies is endemic, should seek medical attention (see section 7). Translocation of wildlife for any purpose except conservation should be banned or strongly discouraged.

10.8 References 1. Weyer J et al. Epidemiology of human rabies in South Africa, 1983–2007. Virus Research, 2011, 155(1):283–290.

2. Sabeta CT et al. Molecular epidemiology of rabies in bat-eared foxes (Otocyon megalotis) in South Africa. Virus Research, 2007, 129(1):1–10.

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3. Zulu GC et al. Molecular epidemiology of rabies: focus on domestic dogs (Canis familiaris) and black-backed jackals (Canis mesomelas) from northern South Africa. Virus Research, 2009, 140(1–2):71–78. 4. Van Zyl N et al. Evolutionary history of African mongoose rabies. Virus Research, 2010, 150(1–2):93–102. 5. Scott T et al. Rabies in kudu (Tragelaphus strepsiceros). Berliner und Munchener tierarztliche Wochenschrift, 2012, 125(5–6):236–241. 6. Mansfield K et al. A molecular epidemiological study of rabies epizootics in kudu (Tragelaphus strepsiceros) in Namibia. BMC Veterinary Research, 2006, 2:2. 7. Haydon DT et al. Low-coverage vaccination strategies for the conservation of endangered species. Nature, 2006, 443:692–695.

8. Johnson N et al. A new outbreak of rabies in rare Ethiopian wolves (Canis simensis). Archives of Virology, 2010, 155(7):1175–1177. 9. Hofmeyr M et al. Rabies in African wild dogs (Lycaon pictus) in the Madikwe Game Reserve, South Africa. Veterinary Record, 2000, 146(2):50–52. 10. Woodroffe R et al. Contact with domestic dogs increases pathogen exposure in endangered African wild dogs (Lycaon pictus). PLoS One, 2012, 7(1):e30099.

11. Gruzdev KN. The rabies situation in Central Asia. Developments in Biologics (Basel), 2008, 131:37–42. 12. Shao XQ et al. Genetic evidence for domestic raccoon dog rabies caused by Arctic-like rabies virus in Inner Mongolia, China. Epidemiology and Infection, 2011, 139(4):629–635. 13. Liu Y et al. Ferret badger rabies origin and its revisited importance as potential source of rabies transmission in Southeast China. BMC Infectious Diseases, 2010, 10:234.

14. Vos A et al. Rabies in foxes, Aegean region, Turkey. Emerging Infectious Diseases, 2009, 15(10):1620–1622. 15. Seimenis A. The rabies situation in the Middle East. Developments in Biologics (Basel), 2008, 131:43–53. 16. World Health Organization Mediterranean Zoonoses Control Programme and World Organisation for Animal Health. Inter-country expert workshop on protecting humans from domestic and wildlife rabies 89

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in the Middle East, 23–25 June 2008, Amman, Jordan. Paris, 2008 (www. oie.int/doc/ged/D6490.pdf; accessed 3 December 2012).

17. King AA et al., eds. Historical perspectives of rabies in Europe and the Mediterranean Basin. Paris, World Organisation for Animal Health, 2004. 18. Cliquet F et al. Development of harmonised schemes for monitoring and reporting of rabies in animals in the European Union. Brussels, European Food Safety Agency, 2010 (http://www.efsa.europa.eu/en/scdocs/ scdoc/67e.htm). 19. Friedrich-Loeffler-Institut, Bundesforschungsinstitut für Tiergesundheit. WHO rabies bulletin for Europe. Greifswald-Insel Riems (www.who rabies-bulletin.org). 20. World Organisation for Animal Health. Rabies, Greece. Paris, 2012. (http://www.oie.int/wahis_2/public/wahid.php/Reviewreport/; accessed 23 October 2012).

21. MacInnes CD et al. Elimination of rabies from red foxes in eastern Ontario. Journal of Wildlife Diseases, 2001, 37(1):119–132. 22. Rupprecht CE et al. (2008) Can rabies be eradicated? Developments in Biologics (Basel), 2008, 131:95–121. 23. Slate D et al. Oral rabies vaccination in north America: opportunities, complexities, and challenges. PLoS Neglected Tropical Diseases, 2009, 3(12):e549. WHO Technical Report Series No. 982, 2013

24. Banyard AC et al. Bats and lyssaviruses. Advances in Virus Research, 2011, 79:239–289.

25. Markotter W et al. Epidemiology and pathogenicity of African bat lyssaviruses. Developments in Biologics (Basel), 2008, 131:317–325. 26. Kuzmin IV et al. Shimoni bat virus, a new representative of the Lyssavirus genus. Virus Research, 2010, 149:197–210. 27. Gould AR. et al. Characterisation of a novel lyssavirus isolated from Pteropid bats in Australia. Virus Research, 1998, 54:165–187. 28. Gould AR et al. Characterisation of an Australian bat lyssavirus variant isolated from an insectivorous bat. Virus Research, 2002, 89:1–28. 29. Schatz J et al. Current state of bat rabies surveillance in Europe. Zoonoses and Public Health, 2012 (doi: 10.1111/zph.12002).

30. McElhinney LM et al. Molecular epidemiology of bat lyssaviruses in Europe. Zoonoses and Public Health, 2012 (doi: 10.1111/zph.12003).

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31. Kuzmin IV et al. Phylogenetic relationships of Irkut and West Caucasian bat viruses within the Lyssavirus genus and suggested quantitative criteria based on the N gene sequence for lyssavirus genotype definition. Virus Research, 2005, 111:28–43. 32. Freuling CM et al. Novel lyssavirus in Natterer’s bat, Germany. Emerging Infectious Diseases, 201, 17(8):1519–1522. 33. Picard-Meyer E et al. Short item: Isolation of the novel BBLV Lyssavirus in Natterer’s bat in France. Bulletin Epidémiologique—Santé animale, alimentation, 2012. (http://www.anses.fr/bulletin-epidemiologique/). 34. Aréchiga N et al. Novel lyssavirus from a Miniopterus schreibersii bat in Spain. In: Twenty-third Rabies in the Americas Conference, São Paulo, Brazil, 14–18 October 2012 (abstract CO.04 at http://acontecimento.com. br/rita2012/rita_2012_abstract.pdf; accessed March 2013). 35. Kuzmin IV et al. Bat lyssaviruses (Aravan and Khujand) from Central Asia: phylogenetic relationships according to N, P and G gene sequences. Virus Research, 2003, 97:65–79. 36. Streicker DG et al. Host phylogeny constrains cross-species emergence and establishment of rabies virus in bats. Science, 2010, 329:676–679.

37. Streicker DG et al. Ecological and anthropogenic drivers of rabies exposure in vampire bats: implications for transmission and control. Proceedings of the Royal Society B. Biological Sciences, 2012, 279:3384– 3392. 38. De Serres G et al. Bat rabies in the United States and Canada from 1950 through 2007: human cases with and without bat contact. Clinical Infectious Diseases, 2008, 46(9):1329–1337. 39. Schneider MC et al. Rabies transmitted by vampire bats to humans: an emerging zoonotic disease in Latin America? Revista Panamericana de Salud Pública, 2009, 25(3):260–269. 40. WHO Expert Consultation on Rabies. First report. Geneva, World Health Organization, 2005 (WHO Technical Report Series, No. 931). 41. Müller T et al. Rabies elimination in Europe—a success story. In: Compendium of the OIE Global Conference on Rabies Control, Seoul, Korea, 7–9 September 2012.

42. Manual of diagnostic tests and vaccines for terrestrial animals. Chapter 2.1.13. Rabies. Paris, World Organisation for Animal Health, 2012 (http://www.oie.int/international-standard-setting/terrestrial-manual/ access-online/; accessed 4 December 2012). 91

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43. Report of a WHO seminar on wildlife rabies control, Geneva, Switzerland, 2–5 July 1990. Geneva, World Health Organization, 1990 (WHO/CDS/ VPH/90.93). 44. Report of the WHO/APHIS consultation on baits and baiting delivery systems for oral immunization of wildlife against rabies. Geneva, World Health Organization, 1990 (WHO/Rab. Res./90.36). 45. Blueprint for rabies prevention and control [fox rabies blueprint]. Partners for Rabies Prevention (www.rabiesblueprint.com; accessed March 2013). 46. Rosatte RC et al. Trap–vaccinate–release and oral vaccination for rabies control in urban skunks, raccoons and foxes. Journal of Wildlife Diseases, 1992, 28(4):562–571.

47. Slavinski S et al. Trap–vaccinate–release program to control raccoon rabies, New York, USA. Emerging Infectious Diseases, 2012, 18(7):1170– 1172.

11. Rabies surveillance Surveillance is the systematic, continuous collection, analysis and interpretation of data and their dissemination to appropriate people in order that action be taken (1). Its aim is to demonstrate the absence of disease or to identify its presence or distribution in order to allow timely dissemination of information for integrated action among different sectors (2). Surveillance is distinct from monitoring, which is defined by the OIE as intermittent performance and analysis of routine measurements and observations to detect changes in the environment or health status of a population. Monitoring in rabies control may include assessment of vaccination coverage through household surveys, observation of marks applied to dogs during mass parenteral vaccination (see section 7) and bait uptake in oral vaccination campaigns for wildlife. Further details on the surveillance of rabies in wild animals and monitoring of oral vaccination programmes are given in section 8. For rabies, surveillance therefore involves measuring the incidence of the disease in both humans and animals. Measures of incidence are essential in rabies control and prevention to ensure appropriate management of cases and outbreaks, to monitor trends in order to evaluate the effectiveness of interventions and to estimate the burden of disease. Rabies surveillance also includes sharing data through appropriate channels, such as the World Animal Health Information System and Database (WAHIS and WAHID), the Global Early Warning System (GLEWS), the Empress embedded data system, the Rabies Bulletin Europe and official regional databases. Rabies should be a notifiable disease in national health and veterinary services. Timely responses to surveillance activities and results

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will motivate field and hospital staff to continue reporting cases (see standard case definitions in 4.1 and clinical diagnosis in humans in 4.2). The response should include, at a minimum, prompt acknowledgement of reports, feedback on the results of diagnostic tests and advice on management of cases and outbreaks. Communication with medical and veterinary staff in the field ensures appropriate management and follow-up of cases and improves case detection rates. To be effective, rabies surveillance must be based on diagnostic confirmation of human and animal suspected and probable cases. It is recommended that countries that lack or have inadequate diagnostic facilities improve their capacity through OIE laboratory twinning projects and links with WHO collaborating centres. The involvement of private and public veterinarians, animal health workers, game wardens and other such professionals is essential, as they are the most likely professionals to see a clinically rabid dog. They should be aware of the clinical signs in a suspected case, the method of sample collection and the process for reporting. Lack of infrastructure and resources for collecting and submitting samples is often a greater impediment to rabies surveillance than lack of diagnostic facilities (3). Animal rabies surveillance should be based on risk and therefore focused on investigation and diagnosis of suspected cases. Rabies may be suspected when animals show clinical signs of rabies, unprovoked bites have been reported and animals are morbid or found dead. The clinical signs of rabies in animals vary widely. The classical signs include abnormal behaviour, altered vocalization, pica, hypersexuality, drooling saliva, aimless wandering, ‘fly-snapping’, ‘bone-inthe-throat’ syndrome, aggression, incoordination, paralysis and convulsions. In rabies-endemic areas, loss of inhibition and abnormal behaviour in wild animals (such as activity of nocturnal animals during the day) should raise suspicion of rabies. In a dead animal, soiling of the mouth can indicate abnormal biting behaviour. Hyperaesthesia is not a feature of rabies in animals. Surveillance should be maintained even in countries that have successfully eliminated canine rabies. The recent emergence of canine rabies in several rabiesfree islands in Indonesia (4,5) and costly outbreaks in Europe of rabies transmitted by illegally imported pets and companion animals from rabies-endemic areas (6) show the importance of such surveillance. Routine characterization of virus isolates from cases and outbreaks is encouraged in order to identify animal host origins, sources of infection and geographical origin (7,8), particularly in view of increased international travel and animal movement. Measurement of rabies-specific antibodies is not recommended for routine rabies surveillance. In addition to laboratory-confirmed cases, the numbers of suspected and probable animal cases, animal bites and people seeking and receiving post-exposure prophylaxis should be recorded and reported. This 93

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information should be shared with the medical and veterinary sectors to facilitate management of animal bites, outbreak investigation and implementation of control measures.

References 1. Making surveillance work [modules 1–4]. Geneva, World Health Organization Department of Vaccines and Biologicals, 2001 (V&B/00.08 to 00.11).

2. Terrestrial animal health code [Chapter 1. Animal disease diagnosis, surveillance and notification, section 1.4. Surveillance]. Paris, World Organisation for Animal Health, 2012 http://www.oie.int/international standard-setting/terrestrial-code/access-online/; accessed 26 November 2012). 3. Halliday J et al. Bringing together emerging and endemic zoonoses surveillance: shared challenges and a common solution. Philosophical Transactions of the Royal Society of London B, 2012, 367:2872–2880.

4. Windiyaningsih C et al. The rabies epidemic on Flores Island, Indonesia (1998–2003). Journal of the Medical Association of Thailand, 2004, 87(11):1389–1393. 5. Susilawathi NM et al. Epidemiological and clinical features of human rabies cases in Bali 2008–2010. BMC Infectious Diseases, 2012, 12:81.

6. Lardon Z et al. Imported episodic rabies increases patient demand for and physician delivery of antirabies prophylaxis. PLoS Neglected Tropical Diseases, 2010, 4(6):e723. WHO Technical Report Series No. 982, 2013

7. Bourhy H et al. The origin and phylogeography of dog rabies virus. Journal of General Virology, 2008, 89:2673–2681.

8. Talbi C et al. Phylodynamics and human-mediated dispersal of a zoonotic virus. PLoS Pathogens, 2010, 6(10):e1001166.

12. Rabies-free countries or areas To assist public health authorities in assessing the risk for contracting rabies after contact with animals, this Consultation defined three types of risk-free countries or areas: dog-rabies free, wildlife (excluding bats) rabies-free and Lyssavirus-free. These definitions differ from the current OIE definition of rabies-free countries for the purpose of animal movement (1).

Rabies-free countries or areas

The following requirements apply to all three definitions: ■■ Rabies in all animal species and humans is notifiable, and a continuous, effective surveillance system is in operation. ■■ The system has or has ready access to one rabies laboratory in which WHO (2) or OIE-recommended techniques (3) for rabies diagnosis are used. ■■ An adequate number of samples from suspected cases in the main susceptible domestic and wild animal species in the country are tested. The level of statistical significance used to define sample size should be set by the suitable national authority. ■■ National authorities should ensure that samples are collected throughout the country. ■■ An effective import policy, i.e. measures to prevent the importation of rabies, especially those in section 13, is in place. A country or area that is free of risk for dog rabies is defined as one in which: ■■ No case of indigenously acquired infection due to a dog rabies virus has been confirmed in humans, dogs or cats or any other animal species at any time during the previous 2 years. ■■ Any autochthonous positive case must be shown by molecular characterization to be a spillover from wildlife. If an imported case in carnivores is confirmed, the status of the country or area shall not be affected if molecular characterization confirms the nonindigenous source of the virus and epidemiological tracing backwards and forwards reveals no evidence of secondary dog infections. A country or area that is free of risk for wild carnivore rabies is defined as one in which: ■■ No case of indigenously acquired infection due to a wild carnivore virus has been confirmed in humans or any domestic or wild species, at any time during the previous 2 years. ■■ Any autochthonous positive case must be shown to be a spillover from bats or dogs.

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■■ If an imported case is confirmed, the status of the country or area shall not be affected if molecular characterization confirms the nonindigenous source of the virus and epidemiological tracing backwards and forwards reveals no evidence of secondary infections in any wild or domestic carnivores. ■■ Serological evidence of infection in some wild animals (e.g. mongoose) should be considered an indicator of the presence of rabies. A country or area that is free of risk for Lyssavirus rabies is defined as one in which: ■■ No case of indigenously acquired infection with any Lyssavirus has been confirmed in humans or any domestic or wild species, including bats, at any time during the previous 2 years. ■■ If an imported case is confirmed, the status of the country or area shall not be affected if molecular characterization confirms the nonindigenous source of the virus and epidemiological tracing backwards and forwards reveals no evidence of secondary infections in any species. ■■ Serological evidence of infection in bats should be considered an indicator of the presence of rabies. In countries or areas in any of the above categories, additional measures may be in place, such as vaccination of dogs and other pets. Reporting of several cases over time at the borders of a previously defined Lyssavirus risk-free country or area should be sufficient for the national authorities to suspect that rabies is likely to have been acquired indigenously rather than to have been imported. In deciding whether to use human pre- or post-exposure prophylaxis, reference should be made to section 8. WHO Technical Report Series No. 982, 2013

References 1. Terrestrial animal health code [vol. 2, chapter 8.10: Rabies]. Paris, World Organisation for Animal Health, 2011 (http://www.oie.int/index.php?id =169&L=0&htmfile=chapitre_1.8.10.htm; accessed 21 September 2012). 2. Laboratory techniques in rabies, 4th ed. Geneva, World Health Organization, 1996.

3. Manual of diagnostic tests and vaccines for terrestrial animals [vol. 1, chapter 2.1.12]. Paris, World Organisation for Animal Health, 2011 (http://www.oie.int/fileadmin/Home/eng/Health_standards/ tahm/2.01.13_RABIES.pdf; accessed 21 September 2012).

International movement of animals

13. International movement of animals Regulations for importing domestic, captive wild and wild mammals from rabiesfree countries or from countries considered infected with rabies should comply with OIE standards (1), including presentation of a valid international veterinary certificate (2). International standards depend on the rabies status of the country of origin and the animal species involved.

13.1 International transport of dogs, cats and ferrets from rabiesinfected countries or areas National importing authorities should require an international veterinary certificate attesting that the animal was not showing signs of rabies at time of shipment, was permanently identified, vaccinated or revaccinated and subjected to a positive serological test prior to shipment. OIE international standards should be followed. A model of international rabies vaccination certificate is set out as Annex 7.

13.2 International transport of livestock and animals for zoos, research, shows and other activities from rabies-infected countries or areas These animals should comply with OIE standards (3), which include a veterinary certificate for domestic animals, laboratory rodents/lagomorphs and wildlife, permanent identification and optional vaccination for domestic ruminants, equids, camelids and suids and a statement that the animals showed no sign of rabies on the day of shipment and particularly for laboratory and wild animals that they were kept in quarantine, or other relevant isolation, for 6 months before shipment with no rabies case detected in the isolation establishment for at least 12 months prior to shipment. Countries that are free from rabies may either prohibit the importation of certain species of mammals, in particular Carnivora and Chiroptera, or permit their entry only under license, subject to quarantine in premises and under conditions approved by the government veterinary service. Entry may be permitted for limited periods or for life. In view of the increase in the number of reported rabies cases in wild animals acquired as pets, national authorities should control the trade in such animals. Keeping such animals as pets should be discouraged.

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13.3 Special exemption of guide dogs for people with disabilities and of other service dogs Certified guide dogs for people with disabilities and other service dogs (e.g. military and search dogs) in rabies-free countries should be permitted to accompany their owners into rabies-infected countries if the dogs are vaccinated with a cell-culture vaccine that fulfils WHO and OIE standards and are shown to have an adequate virus-neutralizing antibody titre by one of the methods recommended by the OIE (3) and WHO (4). These dogs must be identifiable by means of a microchip. Provided that the owners confirm that they were kept confined, on a leash or under permanent visual supervision while abroad in a rabies-infected country, the dogs should be allowed to remain outside the country for a maximum of 6 months with no requirement for re-entry other than reconfirmation of the antibody titre.

13.4 References 1. Terrestrial animal health code [vol. 2, chapter 8.10: Rabies]. Paris, World Organisation for Animal Health, 2011.

2. Terrestrial animal health code [vol. 1, chapter 5.11: Model international veterinary certificate for dogs and cats originating from rabies infected countries]. Paris, World Organisation for Animal Health, 2012 (http:// www.oie.int/index.php?id=169&L=0&htmfile=chapitre_1.5.11.htm; accessed 21 September 2012). 3. Manual of diagnostic tests and vaccines for terrestrial animals [vol. 1, chapter 2.1.12]. Paris, World Organisation for Animal Health, 2011 (http://www.oie.int/fileadmin/Home/eng/Health_standards/ tahm/2.01.13_RABIES.pdf; accessed 21 September 2012). 4. WHO Expert Consultation on Rabies. First report. Geneva, World Health Organization, 2005 (WHO Technical Report Series, No. 931).

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14. Global and regional activities on rabies Many activities on rabies have taken place at international, regional and national levels since publication of the first report of the WHO Expert Consultation on Rabies (1). A growing number of partners (intergovernmental and nongovernmental organizations, public and private institutions and foundations) are contributing to the prevention, control and elimination of human and animal rabies at global, regional and national levels, such as FAO, OIE, the Association

Global and regional activities on rabies

of South-East Asian Nations (ASEAN), the South Asian Association for Regional Cooperation, GARC, the Commonwealth Veterinary Association, Humane Society International, the Rabies in Asia Foundation, Vets Beyond Borders, the World Society for the Protection of Animals, and the Bill & Melinda Gates Foundation. These partners have prepared global standards and policies, helped in resources mobilization, provided regional coordination or directly supported national programmes. The list does not pretend to be exhaustive.

14.1 WHO global and regional activities 14.1.1 WHO headquarters The WHO World Survey of Rabies, created in 1990, was subsequently enhanced by a computerized data management system to process data collected online at country level, known as ‘Rabnet’. The system was improved from 2000 onwards with the addition of new features, such as the production of interactive maps at global and country levels and customized charts, graphs and maps. The database was designed to analyse global trends in the disease as well as regional and national changes. The system was, however, closed down in 2010, as too few individual reports were entered annually into the system by designated national rabies focal points to make analysis of the data meaningful. WHO, the WHO regional offices and collaborating centres on rabies and GARC are studying alternative ways of collecting data and producing annual reports on human and animal rabies. Information on rabies in humans, domestic animals and wildlife should be shared across sectors. The concept of ‘neglected zoonotic diseases’ emerged at a meeting held at WHO headquarters in September 2005 (2) and was reinforced at international conferences held in 2007 (3) and 2010 (4). The term ‘neglected’ for this group of diseases indicates that they are insufficiently addressed by governments and the international community, and that they are best defined by the people and communities they affect most: poor people living in remote rural areas or urban slums of the developing world. The term is now well accepted internationally. Rabies has unfortunately all the features of a neglected zoonotic disease. It is, however, the disease most amenable to control, as the tools are available. It is the first zoonosis on the list of neglected diseases targeted for regional and eventually global elimination. An interagency meeting proposed investment in a ‘priority neglected zoonotic diseases portfolio’, comprising regional elimination of human–dog transmitted rabies in Latin America and Asia (5). A first costing indicated that about US$ 10 million per annum in external funding for the next 5 years will be required to achieve the expected outcomes by 2016. Rabies is discussed at length in the first and second WHO reports on neglected tropical diseases (6,7) and is included in the shorter list of targeted 99

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diseases for regional elimination in the executive summary of the ‘roadmap for implementation’ published in 2012 (8). Rabies is also one of the main viral zoonoses in the technical report of the Disease Reference Group on Zoonoses of the Special Programme for Research and Training in Tropical Diseases (TDR) published by WHO in 2012 (9,10). Recommendations and a map are available and updated regularly on the risk for contracting rabies (see section 6.8) in the WHO publication International travel and health, to inform international travellers about the necessity for preexposure prophylaxis, depending on their destination (11). Since the last Expert Consultation in 2004 and in accordance with its mandate to provide guidance to Member States on rabies prophylaxis, WHO has issued a position paper on rabies vaccines in a series of regularly updated position papers on vaccines and vaccine combinations against diseases of international public health importance (12). This position paper, issued in 2010, was based on the outcome of a WHO consultation on rabies prevention and control in humans and animals held in Annecy, France, in 2009 (13). The position paper, which replaced one issued in 2002, was reviewed and endorsed by WHO’s Strategic Advisory Group of Experts on vaccines and immunization (12). Position papers are designed for use mainly by national public health officials and immunization programme managers and are of interest to international funding agencies, the vaccine manufacturing industry, the medical community, the scientific media and the public. Since 2002, WHO has maintained a website that provides information on rabies in humans and animals, human and animal vaccines and pre- and postexposure prophylaxis. It also contains selected WHO reports and peer-reviewed articles. Since 2009, the site has provided information on progress made in implementation of the 5-year (2009–2013) pilot project for human and dog rabies elimination in selected developing countries (KwaZulu-Natal in South Africa, southwestern United Republic of Tanzania and the Visayas in the Philippines) funded by the Bill & Melinda Gates Foundation and managed by WHO (14,15).

14.1.2 WHO regional offices Asia The WHO Regional Office for South-East Asia has been proactive in preparing standards and guidelines, issuing recommendations and providing technical support to Member States for the prevention and control of human and animal rabies in the region. It advocates use of cost–effective intradermal vaccination to improve the availability and affordability of modern rabies vaccines, and phasing out of the production and use of nerve tissue vaccine. The production and use of this vaccine has been abandoned since 2005 in Bangladesh, Cambodia, India, the Lao People’s Democratic Republic, Nepal and Viet Nam. The WHO Collaborating

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Centre for Rabies Diagnostics in Bangalore, India, has introduced direct rapid immunohistochemistry tests into the region in collaboration with the WHO Collaborating Centre for Reference and Research on Rabies at the Centers for Disease Control and Prevention in Atlanta, Georgia, USA, and regional hands-on training in rabies diagnosis was organized in Bangalore in 2010 to train laboratory professionals in the use of these tests. To consolidate achievements in the control of zoonoses, particularly rabies, in Member States, the regional office has organized meetings (16,17) and prepared a regional strategy for elimination of human rabies transmitted by dogs. The aim is to eliminate human rabies by progressive control of dog rabies and human prophylaxis in rabies-endemic countries and to maintain the status of rabies-free areas in the region by 2020 (17). Latin America The programme for elimination of humans rabies transmitted by dogs is led by the Veterinary Public Health unit of the Pan American Health Organization/WHO Regional Office for the Americas in Rio de Janeiro, Brazil. The objectives of the plan for eliminating rabies from the principal cities of Latin America, initiated in 1983, were extended in 1992 to elimination of dog-transmitted rabies in small conglomerates and rural areas. Since 1983, the occurrence of dog-transmitted rabies has diminished steadily, with a reduction of approximately 90% in human and canine cases. A series of inter-American meetings on health and agriculture at ministerial level is organized by the Veterinary Public Health unit to discuss intersectoral policies and include the regional rabies elimination programme. Every 2 years, the unit also convenes a meeting of the directors of national rabies programmes, at which the epidemiological situation and strategies for prevention of rabies are discussed and updated. The conclusions and recommendations are submitted to ministers of health and agriculture during the interministerial meetings for their consideration and endorsement. The eleventh meeting of the directors of national programmes, held in Brasilia in 2006, recommended elimination of human rabies transmitted by dogs from the hemisphere by 2012, and the fifteenth interministerial meeting, held in Rio de Janeiro in 2008, committed the ministers of health and of agriculture to this goal (18). In 2009, the forty-ninth Directing Council of the Pan American Health Organization in Resolution CD49.R9 proposed 2015 as the target date for regional elimination of all neglected diseases and other poverty-related infections, including rabies (19). The Regional Information System for Epidemiologic Surveillance of Rabies in the Americas (http://siepi.panaftosa.org.br/) produces reports on human and animal rabies based on official data entered into the system by health and agriculture ministries in Member States. Data from 1970 onwards are available for on-line consultation. 101

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14.1.3 WHO network of collaborating centres on rabies A network of collaborating centres on rabies was established almost at the inception of WHO to support WHO activities at country, intercountry, regional, interregional and global levels. The collaborating centres also participate in strengthening the institutional capacity of Member States in terms of information, services, research and training for rabies-related activities such as diagnosis, surveillance, research and monitoring and evaluation of projects and programme for elimination of rabies in humans and animals. The centres are officially designated by WHO on the basis of a jointly agreed plan of work, usually for 4 years, renewable after annual evaluation of their performance by WHO. The plan of work depends on the expertise or specificity of the centre but usually covers: ■■ collection, collation and dissemination of information on rabies; ■■ standardization of rabies diagnostic reagents, prophylactic and therapeutic substances, as well as methods and procedures for their application; ■■ design and application of appropriate techniques; ■■ provision of reference substances and other services; ■■ participation in collaborative research under the Organization’s leadership; ■■ training, including research training; and ■■ coordination of activities carried out by several institutions. There are 12 designated WHO collaborating centres, most for reference and research on rabies. Five are in Asia, four in Europe and three in the USA (see Annex 8). The WHO Collaborating Centre for Rabies Surveillance and Research hosted by the Friedrich-Loeffler-Institute in Germany produces the WHO Rabies Bulletin Europe (see section 14.2.2).

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14.2 Examples of activities by partners Several global and regional initiatives for rabies control and eventual elimination began rapidly and continued to flourish in the past decade. Examples are described below.

14.2.1 Global activities Food and Agriculture Organization of the United Nations (FAO) FAO contributes to rabies control by raising awareness and providing policy advice and technical support for animal rabies control in a number of African and

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Asian countries. It supports animal health clubs in schools in Sierra Leone, and contributes to partnerships and alliances for preventing and controlling rabies, such as the Partnership for Rabies Prevention and GARC. FAO has organized global stakeholder consultations on dog population management for rabies control with the World Society for the Protection of Animals and, with OIE and WHO, is exploring a ‘progressive control pathway’ to rabies elimination focused on elimination of dog-transmitted human rabies (20). World Organisation for Animal Health (OIE) OIE is an intergovernmental organization that issues science-based standards, guidelines and recommendations for the control of infectious diseases in animals, including those that are transmissible to humans, such as rabies. Internationally agreed diagnostic laboratory methods and requirements for the production and control of animal rabies vaccines and other biological products are published in the OIE Manual of diagnostic tests and vaccines for terrestrial animals (21). The OIE Terrestrial animal health code (22) lists measures adopted internationally for the control of rabies. Through its network of reference laboratories (http://www. oie.int/en/our-scientific-expertise/reference-laboratories/list-of-laboratories) and collaborating centres (http://www.oie.int/en/our-scientific-expertise/ collaborating-centres/list-of-centres), the OIE provides policy advice, strategy design and technical assistance for the diagnosis, control and elimination of rabies in animals. In 2011, the OIE organized, with WHO and FAO, a global conference on rabies entitled ‘Towards sustainable prevention at the source’ in the Republic of Korea, which raised the awareness of responsible parties and decision-makers on the importance of tackling rabies at its animal source and re-emphasized the role of national veterinary services in preventing and controlling the disease (23). The Global Alliance for Rabies Control (GARC), Partners for Rabies Prevention and World Rabies Day GARC is the only registered charity working specifically on reducing the global burden of rabies. It has two branches: the Global Alliance for Rabies Control in the USA and the Alliance for Rabies Control, established in Scotland. GARC’s mission is to eliminate human deaths from rabies and to relieve the burden of rabies in animals, especially dogs (http://www.rabiescontrol.net/). GARC works with governments and communities in Africa and Asia to plan and conduct intersectoral (or ‘one health’), sustainable rabies control programmes, funded through partnerships between governments, international foundations, private donations and animal welfare organizations. GARC has established a repository for educational material that is available on the World Rabies Day website for individuals and organizations that require accurate material to improve awareness in their regions. 103

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GARC was instrumental in establishing and is a member of the Partners for Rabies Prevention (24). This informal group comprises the main international agencies involved in rabies: WHO, FAO, OIE, WHO rabies collaborating centres, research scientists, representatives of the Bill & Melinda Gates Foundation, the UBS Optimus Foundation and representatives of industry. The Partners for Rabies Prevention have published a blueprint for rabies prevention and control (25). World Rabies Day was initiated by GARC in 2006. It now involves all partners in human and animal health at international, national, state and local levels, veterinary, medical and other professional and student organizations, and corporate and non-profit partners. Its goal is to raise awareness and mobilize resources for human rabies prevention and animal rabies control. The inaugural campaign in September 2007 was attended by nearly 400 000 people in 74 countries. This response was an important step for rabies prevention and control and shows that the need for action to control this easily preventable disease is widely recognized. World Rabies Day events have been held in 150 countries, with education for 182 million people and vaccination of 7.7 million dogs.

14.2.2 Regional activities Africa The Southern and Eastern African Rabies Group was founded in 1992 for the control of dog rabies. Official meetings are held about every 2 years, for presentation of data on rabies in standardized country reports, which are subsequently published on an open access website (http://www.searg.info). The country reports describe rabies in humans, domestic animals and wildlife and outline requirements for vaccine purchase or production and vaccination strategies. These meetings help to improve diagnosis, surveillance and awareness and highlight the lack of knowledge of the true burden of rabies in Africa. The tenth meeting was held in Maputo, Mozambique, in 2011, and the next will be held in Dar es Salaam, United Republic of Tanzania, in 2013. The African Rabies Expert Bureau (http://www.afroreb.info/) is an informal network of rabies experts in French-speaking countries of Africa. It was established in 2008. Members meet regularly to review the rabies situation in their countries, share experience and discuss any problems encountered and potential solutions. Reports of their meetings are published in international journals. The Bureau provides a platform for French-speaking rabies experts to exchange information and to link with other networks of rabies experts.

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Asia FAO has provided technical support for animal rabies control in a number of Asian countries, particularly Indonesia. OIE advises national veterinary services in the Asia Pacific region on dog rabies control and dog population management and provides dog rabies vaccine to certain counties within a project supported by the European Union. The aim of this 4-year project (2009–2013), conducted by FAO, OIE and WHO, is to strengthen the capacity of countries and of the two main regional organizations, ASEAN and the South Asian Association for Regional Cooperation, in order to enhance regional cooperation on diseases of animal origin, including rabies. The Global Framework for the Progressive Control of Transboundary Animal Diseases in Asia and the Pacific has identified rabies as a priority at the human–animal interface and called for increased political commitment at national and regional levels. Member States of ASEAN and the South Asian Association for Regional Cooperation have also identified rabies as a priority public health problem, and governments have expressed concern and commitment for the elimination of human rabies. The ASEAN countries adopted a call for action to prevent and control rabies, with the goal of elimination by 2020 (26). The Rabies in Asia Foundation, at a conference in 2009, resolved to take seven steps to achieve human and dog rabies elimination by 2020 and requested the WHO regional committees of the South-East Asia and Western Pacific regions to meet the demands of Member States for technical assistance and technology transfer and to launch regional initiatives for dog rabies control and elimination in Asia in collaboration with regional organizations. ASEAN, FAO, OIE and WHO organized a rabies workshop in Chiang Mai, Thailand, in January 2012, which was attended by officials responsible for animal and human health from 12 Asian countries. Country progress was described, and the group decided to make a unified effort to eliminate rabies in the region, with a plan for control and eradication (27). The Asian Rabies Expert Bureau is an informal network of rabies experts established in 2004. Its members meet regularly to review the situation in their countries, share experience and discuss any problems encountered and their solutions. Meeting reports are available on their site (http://www.areb.info). Latin America An international conference on rabies in the Americas (http://www. rabiesintheamericas.org/) is organized annually to review and discuss rabies research and control in the region. The meeting has an international committee consisting of representatives of Brazil, Canada, Mexico and the USA. The twentythird meeting was held in Brazil in October 2012. 105

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Middle and Near East The Middle East and Eastern Europe Rabies Expert Bureau (http://www.meereb. info) is an informal network of rabies experts established in 2010. Members meet regularly to review the situation in their countries, share experience and discuss any problems and their potential solutions. Reports of their meetings are published in international journals (28). Europe A rabies reporting system, the WHO Rabies Bulletin Europe, was created in 1977 by WHO and the Friedrich-Loeffler Institute in Germany; it is hosted by the WHO Collaborating Centre for Rabies Surveillance and Research at the Institute. The system is continuously updated, and all data reported are automatically transferred to a database, summarized by administrative unit and aggregated per country. More than 40 European countries report officially confirmed rabies cases in both wild and domestic animal species and in humans on a quarterly basis. The Rabies Bulletin Europe is printed quarterly, and a free version is available electronically from www.who-rabies-bulletin.org/. The website also allows dynamic database queries. Since 1990, maps of rabies cases have been displayed online, and since 2009 surveillance data are also mapped. The Bulletin provides valuable information for both the general public and the scientific community. European Union member states exchange information on rabies regularly at meetings of the Standing Committee of Food Chain and Animal Health. In 2003, the European Union established a subgroup on rabies within a task force for monitoring animal disease eradication to assess co-financed oral rabies vaccination campaigns in member states and neighbouring non-member countries. The subgroup comprises private and governmental rabies experts, who visit member states at the request of the European Commission. Its conclusions and recommendations to improve oral rabies vaccination programmes are submitted to the European Commission and the respective member state for consideration. Its reports are publicly available (http://ec.europa.eu/food/animal/ diseases/eradication/taskforce_en.htm). The Food and Veterinary Office of the European Commission conducts on-the-spot inspections of the execution of co-financed rabies elimination programmes at all levels in member states. Its reports can be obtained from the website (http://ec.europa.eu/food/fvo/inspectprog/policy_papers/index_ en.htm). Since 2008, the European Union Reference Laboratory for Rabies in Nancy, France, has organized annual meetings of European national rabies laboratories in order to harmonize and standardize diagnostic techniques. The European Union also supports partner countries through Technical Assistance and Information Exchange, an instrument managed by the Directorate-General

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for Enlargement of the European Commission. Information on recent missions and workshops on rabies is available online (http://ec.europa.eu/enlargement/ taiex/dyn/taiex-events/index_en.jsp). Independent multilateral meetings are organized with representatives of public health and veterinary authorities of neighbouring countries that have oral rabies vaccination programmes. The European Centre for Disease Prevention and Control, which was established to strengthen the capacity of the European Union to prevent and control infectious diseases, organized a consultation in January 2009 with WHO participation to review the epidemiological situation of rabies in Europe, to identify approaches to administering post-exposure prophylaxis and to find solutions to the shortage of rabies biologicals, including the possibility of establishing a virtual stockpile (29). Human rabies cases and the epidemiology of rabies are presented and discussed in Eurosurveillance, a peer-reviewed scientific journal for articles on the epidemiology, surveillance, prevention and control of communicable diseases that are relevant to Europe. It is published by the European Centre for Disease Prevention and Control (http://www.eurosurveillance.org/).

14.3 References 1. WHO Expert Consultation on Rabies. First report. Geneva, World Health Organization, 2005 (WHO Technical Report Series, No. 931).

2. The control of neglected zoonotic diseases: a route to poverty alleviation. Report of a joint WHO/DFID-APHP meeting with the participation of FAO & OIE. Geneva, World Health Organization, 2006 (WHO/SDE/ FOS/2006.1). 3. Integrated control of neglected zoonotic disease in Africa: applying the ‘one health’ concept. Report of a joint WHO/EU/ILRI/DBL/FAO/OIE/ AU meeting. Geneva, World Health Organization, 2008 (WHO/HTM/ NTD/NZD/2008.1). 4. The control of neglected zoonotic diseases (NZDs): community-based interventions for prevention and control. Report of the third conference organized by WHO/ICONZ/DFID-RIU/SoS/EU/TDR/FAO with the participation of ILRI and OIE. Geneva, World Health Organization, 2011 (WHO/HTM/NTD/NZD/2011.1). 5. Interagency (FAO,OIE,WHO) meeting on planning NZDs prevention and control. Geneva, World Health Organization, 2011 (WHO/HTM/ NTD/NZD/2011.3). 107

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6. Working to overcome the global impact of neglected tropical diseases: first WHO report on neglected tropical diseases. Geneva, World Health Organization, 2010 (WHO/HTM/NTD/2010.1). 7. Sustaining the drive to overcome the global impact of neglected tropical diseases: second report on neglected tropical diseases. Geneva, World Health Organization, 2013 (WHO/HTM/NTD/2013.1). 8. Accelerating work to overcome the global impact of neglected tropical diseases: a roadmap for implementation. Geneva, World Health Organization, 2012 (WHO/HTM/NTD/2012.1). 9. Molyneux D et al. Zoonoses and marginalised infectious diseases of poverty: Where do we stand? Parasites and Vectors, 2011, 4(106):1–19.

13. Human and dog rabies prevention and control: report of the WHO/Bill & Melinda Gates Foundation consultation, Annecy, France, 7–9 October 2009. Geneva, World Health Organization, 2010 (WHO/HTM/ NTD/NZD/2010.1) (http://whqlibdoc.who.int/hq/2010/WHO_HTM_ NTD_NZD_2010.1_eng.pdf). WHO Technical Report Series No. 982, 2013

14. Report of the 4th meeting of the international coordination group of the Bill & Melinda Gates Foundation/WHO project for human and dog rabies elimination in low-income countries, 2–4 October 2012, Cebu, Philippines. Geneva, World Health Organization, 2013 (http://www.who.int/rabies/ bmgf_who_project/en). 15. Report of the 3rd meeting of the international coordination group of the Bill & Melinda Gates Foundation/WHO project for human and dog rabies elimination in low-income countries, 19–21 October 2011, PieterMaritzburg, KwaZulu-Natal, South Africa. Geneva, World Health Organization, 2011 (http://www.who.int/rabies/bmgf_who_project/en/). 16. Regional meeting on zoonotic diseases. Report of the meeting, Jakarta, Indonesia, 6–8 November 2007. New Delhi, WHO Regional Office for South-East Asia, 2008 (SEA-CD-174).

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17. Report of the informal consultation to finalize a regional strategy framework for the elimination of human rabies transmitted by dogs in the South-East Asia Region, June 2011, Bangkok, Thailand. New Delhi, WHO Regional Office for South-East Asia, 2012. 18. 15th inter-American meeting at ministerial level, on health and agriculture, Rio de Janeiro, Brazil, 11–12 June 2008. Washington DC, WHO Regional Office for the Americas/Pan American Health Organization, 2008. 19. Elimination of neglected diseases and other poverty-related infections. Pan American Health Organization and World Health Organization. 49th Directing Council. 61st session of the Regional Committee. Washington DC, 2009 [resolution CD49.R19]. (http://new.paho.org/hq/ dmdocuments/2009/CD49.R19%20(Eng.).pdf; accessed March 2013).

20. Rabies: a looming threat. Rome, Food and Agriculture Organization of the United Nations Animal Production and Health Division, 2010 (http://www.fao.org/ag/againfo/home/en/news_archive/AGA_in_ action/2010_rabies.html). 21. Manual of diagnostic tests and vaccines for terrestrial animals, 6th ed. Paris, World Organisation for Animal Health, 2011. 22. Terrestrial animal health code. Paris, World Organisation for Animal Health, 2011 (http://www.oie.int/index.php?id=169&L=0&.htm; accessed 29 November 2012).

23. Global conference on rabies control. Towards sustainable prevention at the source, Incheon-Seoul, Republic of Korea, 7–9 September 2011 [recommendations]. Paris, World Organisation for Animal Health, 2011 (http://www.oie.int/fileadmin/Home/eng/Conferences_Events/ docs/pdf/recommendations/A_Recommendation_Global%20 Rabies%20Conference%20Seoul_final.pdf). 24. Lembo T et al. Renewed global partnerships and redesigned roadmaps for rabies prevention and control. Veterinary Medicine International, 2011 (ID 923149, doi:10.4061/2011/923149).

25. Lembo T et al. The blueprint for rabies prevention and control: a novel operational toolkit for rabies elimination. PLoS Neglected Tropical Diseases, 2012, 6(2):e1388. 26. Call for action: towards the elimination of rabies in the ASEAN Member States and the Plus Three Countries. Jakarta, Association of Southeast Asian Nations, 2010 (http://www.aseanplus3-eid.info/ Rabies_Call_for_ Action; accessed 10 December 2012).

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27. Report of the ASEAN/FAO/OIE/WHO rabies workshop, January 2012, Chiang Mai, Thailand. Jakarta, Association of Southeast Asian Nations, 2012. 28. Report of the second meeting of the Middle East and Eastern Europe Rabies Expert Bureau (MEEREB), Paris, France, June 5–8, 2012. Lyon, 2012 (http://www.meereb.info/meetings-concrete-actions; accessed March 2013). 29. Meeting report: expert consultation on rabies post-exposure prophylaxis, Stockholm, 15 January 2009. Stockholm, European Centre for Disease Prevention and Control, 2009 (http://www.ecdc.europa.eu/en/ publications/Publications/0906_MER_Expert_Consultation_on_ Rabies_Post-exposure_Prophylaxis.pdf; accessed March 2013).

15. Research 15.1 Diagnostics Although new techniques and protocols have been proposed for the diagnosis of rabies, especially in humans, over the past 10 years, the number of laboratoryconfirmed human rabies cases reported is limited and represents an underestimate of the real impact of this neglected zoonotic disease, particularly in Africa and Asia. Better tests for rapid, economical diagnosis, with no loss of sensitivity or specificity, would therefore be welcome (1–3). For molecular methods, more universal primers, real-time RT-PCR and nested PCR assays, focus on viral genes other than N and G and improved sequencing protocols are needed, especially for developing countries where the diversity of lyssaviruses is poorly recognized. International standards are lacking for determining the sensitivity of these techniques, making comparisons difficult. Such standards should be prepared for use in local laboratories in rabies-endemic countries in order to ensure proper evaluation of the new molecular techniques (3). Proficiency testing should be organized at international level to ensure reliable data on diagnoses and the incidence of rabies. Lateral flow and other tests should be devised for rapid detection of rabies viral antigens in the field, with adequate validation according to international standards.

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15.2 Epidemiology The lack of accurate data on disease burden, which are required for setting regional and national priorities for research and control, results in a vicious circle of indifference and neglect (4). Better decentralized surveillance methods and more sensitive and specific laboratory techniques are therefore needed, including:

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■■ diagnostic approaches based on validated protocols and specimens and evaluated under field conditions; and ■■ epidemiological models to better estimate the incidence of rabies. Recent research showed that the incidence of rabies in some countries was as much as 15 times higher than that in official reports (5,6). Further work on the design and local implementation of these models is encouraged as well as more accurate field data for incorporation into the models (7). These methods and techniques should be used to generate: ■■ data on disease incidence, a critical input to epidemiological models of rabies and currently the main limitation to accurate estimates of disease burden; ■■ information on the epidemiology and population dynamics of rabies in natural mammalian host populations (8,9,10); ■■ information on the ecological patterns, frequency and extent of movement of infected animals in order to predict the spread of rabies; and ■■ extensive genomic and evolutionary analyses to establish the diversity of lyssavirus species and variants in order to identify the determinants of rabies spread. Integration of phylogeographical data with data on viral genetics is a powerful means for characterizing, predicting and ultimately preventing and controlling the spatial spread of rabies. Recent observations suggest that bats are important lyssavirus reservoirs, and the virus variants associated with Chiroptera may occasionally spill over to other mammals, with potential adaptation and establishment (11). Evidence of direct exposure to bats is sometimes lacking in human rabies infections, and research is required on the epidemiology of bat lyssaviruses (11) and potential pathogenic mechanisms in such spillover infections. There have been no recent comprehensive studies of relevant hosts and viruses or alternative routes and unusual settings.

15.3 Molecular, genetic and epidemiological characterization of new viral isolates Isolation of new viruses is being reported more and more frequently throughout the world (see section 2). Scientists who identify new lyssaviruses are encouraged to characterize the isolates promptly and to compare them with previously described species. It is particularly important to determine the epidemiology of new species (range of hosts, geographical distribution and significance 111

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for domestic animals and humans) and to verify whether commercial rabies biologicals, such as vaccines and antibodies, protect against them.

15.4 Biological medical products Currently, the recommended prophylaxis for people severely exposed to lyssaviruses is combined administration of rabies vaccine and immunoglobulins. Both products remain expensive for a significant portion of the target human population. Therefore, ways and means should be sought to decrease their cost and to find new therapeutic approaches. Moreover, although nearly all veterinary products are for pre-exposure use, there may be circumstances in which they would be useful for post-exposure prophylaxis of a naive animal. Validated protocols by product and species should be drawn up. Several new approaches have been proposed. Reverse genetics involves use of negative-stranded RNA viruses as cloning and expression vectors, and newer, safer, more effective recombinant viruses, based for example on adenoviruses, and DNA and plant-based vaccines continue to receive attention (12,13). All genetically engineered rabies vaccines must comply with national and international biosafety guidelines. If new lyssaviruses continue to be identified, especially in bats, vaccines with a broader protection spectrum will be needed. Production of multivalent vaccines by classical methods in cell culture or by molecular techniques (recombinant virus expressing chimeric G protein, insertion of various epitopes into the lyssavirus G protein) should be investigated. Activation of innate immune responses by novel vaccine carriers and adjuvants and their protection when used for post-exposure prophylaxis should be studied further. Rabies immunoglobulins are a critical element of human rabies postexposure prophylaxis, particularly after severe or multiple bites on the face by rabid carnivores. More research, development and assessment are needed of suitable immunoglobulins or alternatives, such as human monoclonal antibodies, in rabies prophylaxis (14) (see also section 6.8) to ensure wider access to passive immunization at a reduced cost. In addition to standard laboratory potency tests for rabies immunoglobulin and other products to determine the concentration of virus neutralizing antibodies per unit volume, some measure of expected efficacy is desirable. Reproducible animal models should be found for assessing the effectiveness of various immunoglobulins and other products (monoclonal antibody cocktail) for in situ virus neutralization after infection. The in vivo half-lives of antibody preparations in relevant target tissues should be established for new preparations. The levels of antibody required for passive immunization and their duration should be determined, particularly for those based on human monoclonal antibodies.

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The current mouse protection test for vaccine potency is fraught with difficulties, and more appropriate methods are needed to assess the antigenic content and its correlation with protection (see sections 6.3.1 and 7.2). Appropriate animal models should be found for studying the pathogenesis and intensive care of human rabies patients (see section 5). No commercial antiviral therapy is available. Investigation of therapeutic approaches based on blocking interactions among viral proteins, targeting the viral replication complex, may lead to the development of new small molecules. Current research on short interfering RNA (15) should be extended. A holistic approach should entail rapid intra-vitam diagnostics, intensive patient care, vaccination, administration of immunoglobulins, cytokines and antiviral therapy, as appropriate, and should be based on realistic animal models and inferences from successfully treated human cases. Current translational and operational research on inactivated vaccines for veterinary use and new modes of delivery should continue to provide better, easier means for controlling rabies in the animal reservoir in tropical and resource-poor areas. Focused research and development is required to produce live replication-competent vaccines for oral and other routes, which are more effective in wildlife primary hosts such as raccoons, mongooses and skunks and safe for non-target species, including humans.

15.5 Human rabies prophylaxis Shorter post-exposure prophylaxis regimens are being evaluated, such as a shortened Essen intramuscular regimen for immunocompromised patients and four-site intradermal regimens with 0.1 ml per site in association with rabies immunoglobulin (16–18). If they are found to be suitable, they will reduce the expense of travelling to clinics to receive multiple doses of rabies vaccine over extended periods and will probably improve compliance (19). Industry support of such regimens would be welcomed. WHO encourages the incorporation of rabies vaccination into infant and child immunization programmes in areas where canine rabies is a major public health problem and there are no economic, logistical or programmatic obstacles. Further studies are needed to find alternative routes of vaccine delivery, appropriate inexpensive devices and prefilled syringes to facilitate pre- and postexposure prophylaxis for rabies (20,21).

15.6 Pathobiology Lyssaviruses naturally infect neurons, resulting in dysfunction and death (22,23). Further studies are required to elucidate the molecular basis of the pathobiology of rabies virus in neurons and other tissues. Insights from pathobiological studies can be used in designing additional approaches for the therapy of rabies (24,25).

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Comprehensive understanding of the pathobiology of lyssaviruses is lacking. Many studies have addressed the nature of the relations between lyssaviruses and their hosts, but the roles of different viral proteins and how they affect the host cellular machinery remain largely a mystery (26,27). Retrospective and prospective comparisons of the pathogenic processes in lyssaviruses found in nature to which humans are differentially susceptible could help to resolve the mystery. Further research is needed on the factors that determine the ability of lyssaviruses to cross species barriers, from wild animal reservoirs to domestic animals and to humans, and their ability to spread in new animal host species. The role of innate immune responses in controlling host switching should also be investigated. Relevant cell lines are needed to better appreciate the immunobiology of the Chiroptera and their pathogens.

15.7 Host ecology Different species of mammals harbour different virus variants, and host identification is often difficult (e.g. among bat species). Means are needed for identifying different host species. The priorities for research include host identification, distribution and behaviour (e.g. in relation to disease transmission) and population dynamics in relation to disease persistence. Research should be conducted on innovative attractive baits and on improving vaccine bait delivery to species such as mongooses, skunks, raccoons and dogs. Development of cost–effective, large-scale oral rabies vaccination strategies in vast areas where rabies is endemic should be continued, taking into account the ecology of the primary host, bait density and the timing and frequency of campaigns. Novel immunocontraceptive products could improve the management of animal populations in mass vaccination strategies (28).

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15.8 References 1. Durr S et al. Rabies diagnosis for developing countries. PLoS Neglected Tropical Diseases, 2008, 2:e206. 2. Lembo T et al. Evaluation of a direct, rapid immunohistochemical test for rabies diagnosis. Emerging Infectious Diseases, 2006, 12:310–313. 3. Dacheux L et al. More accurate insight into the incidence of human rabies in developing countries through validated laboratory techniques. PLoS Neglected Tropical Diseases, 2010, 4(11):e765.

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4. Knobel DL et al. Re-evaluating the burden of rabies in Africa and Asia. Bulletin of the World Health Organization, 2005, 83:360–368.

5. Cleaveland S et al. Estimating human rabies mortality in the United Republic of Tanzania from dog bite injuries. Bulletin of the World Health Organization, 2002, 80:304–310. 6. Ly S et al. Rabies situation in Cambodia. PLoS Neglected Tropical Diseases, 2009, 3:e511. 7. Hampson K et al. Transmission dynamics and prospects for the elimination of canine rabies. PLoS Biology, 2009, 7:e53. 8. Zinsstag J et al. Transmission dynamics and economics of rabies control in dogs and humans in an African city. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106:14996. 9. Bourhy H et al. The origin and phylogeography of dog rabies virus. Journal of General Virology, 2008, 89:2673–2681. 10. Kuzmin IV et al. Molecular inferences suggest multiple host shifts of rabies from bats to mesocarnivores in Arizona during 2001–2009. PLoS Pathogens, 2012, 8(6):e1002786. 11. Streicker DG et al. Rates of viral evolution are linked to host geography in bat rabies. PLoS Pathogens, 2012, 8(5):e1002720. 12. Bahloul C et al. Field trials of a very potent rabies DNA vaccine which induced long lasting virus neutralizing antibodies and protection in dogs in experimental conditions. Vaccine, 2006, 24:1063–1072.

13. Wu X et al. Development of combined vaccines for rabies and immunocontraception. Vaccine, 2009, 27:7202–7209. 14. Bakker AB et al. First administration to humans of a monoclonal antibody cocktail against rabies virus: safety, tolerability, and neutralizing activity. Vaccine, 2008, 26:5922–5927. 15. Israsena N, Mahavihakanont A, Hemachudha T. Rabies virus infection and microRNAs. Advances in Virus Research, 2011, 79:329–344. 16. Sudarshan MK et al. Evaluation of a one week intradermal regimen for rabies post-exposure prophylaxis: results of a randomized open label, active controlled trial in healthy adult volunteers in India. Human Vaccines and Immunotherapeutics, 2012, 8(8):1–5.

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17. Prapimporn S et al. Postexposure rabies prophylaxis completed in 1 week: preliminary study. Clinical Infectious Diseases, 2010, 50(1):56–60. 18. Warrell M et al. A simplified 4-site economical intradermal postexposure rabies vaccine regimen: a randomised controlled comparison with standard methods. PLoS Neglected Tropical Diseases, 2008, 2:e224. 19. Hampson K, Cleaveland S, Briggs D. Evaluation of cost–effective strategies for rabies post-exposure vaccination in low-income countries. PLoS Neglected Tropical Diseases, 2011, 5:e982. 20. Laurent PE et al. Safety and efficacy of novel dermal and epidermal microneedle delivery systems for rabies vaccination in healthy adults. Vaccine, 2010, 28(36):5850–5856.

21. Program for Appropriate Technology in Health (PATH). Intradermal delivery of vaccines. Seattle, Washington, 2010–2013 (http://sites.path. org/deliverytech/id/). 22. Schnell MJ et al. The cell biology of rabies virus: using stealth to reach the brain. Nature Reviews Microbiology, 2010, 8:51–61. 23. Rieder M, Conzelmann KK. Interferon in rabies virus infection. Advances in Virus Research, 2011, 79:91–114. 24. Jackson AC. Update on rabies diagnosis and treatment. Current Infectious Disease Reports, 2009, 11:296–301. 25. Jackson AC. Therapy of rabies encephalitis. Biomedica, 2009, 29:169–176. 26. Thanomsridetchai N et al. Comprehensive proteome analysis of hippocampus, brainstem, and spinal cord from paralytic and furious dogs naturally infected with rabies. Journal of Proteome Research, 2011, 10(11):4911–4924. 27. Reinke SN et al. Metagenomic and metabolomic characterization of rabies encephalitis: new insights into the treatment of an ancient disease. Journal of Infectious Diseases, 2012 (doi: 10.1093/infdis/jis479). 28. Carroll MJ et al. The use of immunocontraception to improve rabies eradication in urban dog populations. Wildlife Research, 2010, 37:1–12.

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Concluding remarks

Concluding remarks The meeting was closed by Dr Hiroki Nakatani, Assistant Director-General of the WHO cluster for HIV/AIDS, Tuberculosis, Malaria and Neglected Tropical Diseases, and Dr F.X. Meslin. Dr Nakatani thanked the participants, observers and representatives of other international governmental and nongovernmental organizations on behalf of the Director-General of WHO, Dr Margaret Chan, for their work and support for a neglected zoonosis with a substantial public health and economic impact. In particular, he thanked the heads of the WHO collaborating centres and members of the WHO Expert Advisory Panel on Rabies. Dr Nakatani stressed the need for strong, effective, cross-sectoral collaboration on the human–animal interface for rabies control, and welcomed the participation of FAO and OIE in the Consultation. He noted with appreciation the interest of private manufacturers of human and animal rabies vaccines. Dr Nakatani described the significant health and economic burden represented by rabies, incurring use of 70 million doses of human rabies vaccines in an estimated 20 million people, mostly in developing countries; the societal cost of rabies worldwide is estimated to be in excess of US$ 6 billion including US$ 1.6 billion spent on post-exposure prophylaxis. Those figures will continue to escalate as the demand for safe cell-culture human vaccines increases. Dr Nakatani confirmed the conclusion of the Consultation that human dog-transmitted rabies is readily amenable to control, regional elimination in the medium term and even global elimination in the long term. He closed the Consultation, commenting that a resolution on major neglected tropical diseases, including rabies, was being prepared for submission to the World Health Assembly in May 2013 in the expectation of securing Member States’ commitment to the control, elimination or eradication of these diseases. Endorsement of the resolution would open the door for exciting advances in rabies prevention and control.

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Acknowledgements The Expert Consultation and the WHO Secretariat acknowledge the special contributions to drafting the background documents made by Dr K. Hampson, Dr I. Kuzmin, Dr T. Hemachuda, Dr C. Rupprecht, Professor S. Madhusudana, Dr D. Briggs, Dr H. Ertl, Dr H. Wilde, Dr F. Cliquet, Dr M.K. Sudarshan, Dr B. Quiambao, Dr A. Rahman, Dr E. Russell, Dr G. Massei, Dr A. Wandeler, Dr T. Müller, Professor S. Cleaveland, Dr M. Vigilato and Dr H. Bourhy. The financial contribution of the Bill & Melinda Gates Foundation is gratefully acknowledged.

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Annexes

Annexes Annex 1. List of participants Heads of WHO collaborating centres Dr Hervé Bourhy, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, Paris, France Dr Florence Cliquet, WHO Collaborating Centre on Research and Management on Zoonoses Control, AFSSA-LERPAS, Laboratoire d’études sur la rage et la pathologie, des animaux sauvages, Malzéville, France Dr Bernhard Dietzschold, WHO Collaborating Centre for Neurovirology, Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, USA Dr Hildegund Ertl, WHO Collaborating Centre for Reference and Research on Rabies, Immunology Program Leader, The Wistar Institute, Philadelphia,USA Dr Anthony Fooks, WHO Collaborating Centre for the Characterization of Rabies and Rabies-related Viruses, Animal Health and Veterinary Laboratries Agency, Weybridge, England Dr Thiravat Hemachudha, WHO Collaborating Centre for Research and Training on Viral Zoonoses, Member of the WHO Expert Advisory Panel on Rabies, Professor of Neurology, Neurology Division, Department of Medicine, Chulalongkorn University Hospital, Bangkok, Thailand Dr Rattan Lal Ichhpujani, WHO Collaborating Centre for Rabies Epidemiology, Member of the WHO Expert Advisory Panel on Rabies, Additional Director, Microbiology Department, Centre for AIDS and Related Diseases, National Centre for Disease Control, Delhi, India Professor S.N. Madhusudana, WHO Collaborating Centre for Reference and Research in Rabies, Member of the WHO Expert Advisory Panel on Rabies, Department of Neurovirology, National Institute of Mental Health and Neurosciences, Bangalore, India Dr Thomas Müller, Head, WHO Collaborating Centre for Rabies Surveillance and Research, Friedrich-Loeffler Institut, Federal Research Institute for Animal Health, GreifswaldInsel Reims, Germany

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Members of the WHO Expert Advisory Panel on Rabies Dr Ahmad Fayaz, Former Head, Rabies Laboratory, Pasteur Institute, Tehran, Islamic Republic of Iran Professor Louis Hendrik Nel, Professor of Virology, Department of Microbiology and Plant Pathology, Faculty of Natural and Agricultural Sciences, University of Pretoria, Hillcrest, South Africa, President of the South Eastern Africa Rabies Group (SEARG) (Chair) Dr Beatriz P. Quiambao, Chief, Clinical Research Division, Research Institute for Tropical Medicine, Philippines. President of the Rabies Asia Foundation (RIA) Dr Charles E. Rupprecht, Former Chief, Rabies Section, Centers for Disease Control and Prevention, Atlanta, USA Dr Naseem Salahuddin, Indus Hospital, Korangi, Karachi, Pakistan (Rapporteur) Professor Dr Mysore K. Sudarshan, Dean, Principal and Professor of Community Medicine, Kempegowda Institute of Medical Sciences, Bangalore, India. President of the Rabies Asia Foundation (RIA) Dr Alexander Wandeler, Former Head, rabies laboratory, Canadian Food Inspection Service, Scientist Emeritus, Carp, Canada

Mr Kevin Le Roux, Rabies Project Management, Veterinary Services, Pietermaritzburg, KwaZulu-Natal, South Africa Dr Giovanna Massei, Ecologist, Food and Environment Research Agency, York, England Dr Mathew Maziku, National Project Coordinator, Rabies, WHO Country Office, Dar es Salaam, United Republic of Tanzania Dr Maria P. Rebollo, Scientific Manager, Monitoring and Evaluation, Liverpool School of Tropical Medicine, Centre for Neglected Tropical Diseases, Liverpool, England Dr Graham Smith, Senior Researcher, Food and Environment Research Agency, York, England Dr Mathurin Cyrille Tejiokem, Epidemiologist, Epidemiology and Public Health Laboratory, Centre Pasteur du Cameroun, Yaoundé, Cameroon

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Dr Henry Wilde, Professor of Medicine, Senior Consultant, WHO Collaborating Centre for Zoonoses and Rabies, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

Representatives of other organizations Intergovernmental organizations World Organisation for Animal Health (OIE) Dr Marta Martinez, Veterinary Epidemiologist Dr Dietrich Rassow, Chargé de mission, WHO Nongovernmental organizations Association for the Prevention and Control of Rabies in India Dr A. Rahman, President, Commonwealth Veterinary Association for Prevention and Control of Rabies in India, Department of Community Medicine, Bangalore, India Dodet Science Dr Betty Dodet, Caluire et Cuire, France Global Alliance for Rabies Control Dr Kim Doyle, Trustee of the Alliance for Rabies Control, Global Alliance for Rabies Control c/o Balfour and Manson, Edinburgh, Scotland Ms Maylin Meincke, Development Studies, University of Helsinki, Helsinki, Finland Dr Elizabeth Miranda, Asian Coordinator, Global Alliance for Rabies Control, Laguna, Philippines Marwar Trust Mr Federico Spinola, Founder, Partnership for Animals, Geneva, Switzerland PATH Dr Darin Zehrung, Technical Officer, Portfolio Leader Vaccine Delivery Technologies, Seattle, USA World Society for the Protection of Animals Dr Elly Hiby, Scientific Advisor, World Society for the Protection of Animals, Cambridge, England Dr Esmée Russell, Campaign Manager, World Society for the Protection of Animals, London, England

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Observers Dr Michaël Attlan, Director, Traveler Endemic and Emerging Vaccines Franchise, SanofiPasteur, Lyon, France Dr Jac Bergman, Global Marketing Director, Small Animal Vaccines, Global Companion Animal Business Unit, Boxmeer, the Netherlands Dr Rachel Chikwamba, Technical Lead, Rabies Initiatives, Pretoria 0001, South Africa Dr Pradip Desai, Director, Span Diagnostics Ltd, Surat, India Dr Alexandra Giesen, Novartis Vaccines and Diagnostics, Global Medical Affairs, Munich, Germany Dr Reinhard Glueck, CSO, Zydus Cadila Healthcare, Zydus Research Centre, Gujarat, India Dr Françoise Guinet-Morlot, Project Director New Vaccines, Sanofi Pasteur, Marcy l’Etoile, France Dr Gaurav Gupta, Head, Viral Vaccines, Zydus Cadila, Zydus Research Centre, Gujarat, India Dr K. Jager, Intervet, Boxmeer, the Netherlands Dr Philipe Mahl, Rabies Programme Manager, Virbac, Carros, France Dr Joanne Maki, Veterinary Public Health, Global Public Health Director, Athens, Georgia, USA Dr Claudius Malerczyk, Head, Medical Affairs, Middle East and Africa, Novartis Vaccines and Diagnostics, Marburg, Germany Dr Stephanus Francois Marais, Commercialisation Manager, CSIR Biosciences, Pretoria, South Africa Dr Wilfred Marissen, Programme Director, Crucell Holland B.V., Leiden, the Netherlands Dr Anvar Rasuli, Medical Product Leader, Global Medical Affairs, Sanofi Pasteur, 2 Avenue Pont Pasteur, Lyon, France Dr Micha Roumiantzeff, Fondation M. Merieux, 1 rue Dangon, 69004 Lyon, France Dr Carolin Schumacher, Director, Corporate Public Affairs, Merial, Lyon, France Dr Daniela Todorova-Balvay, R&D Manager, Span Diagnostics SARL, Compiegne, France Dr Adriaan Vos, Head, Vaccine Development Technologies, IDT Biologika GmbH, DessauRosslau, Germany

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WHO Regional Office for Africa Dr Landry Bidé, Medical Officer, Neglected Tropical Diseases, Brazzaville, Congo

WHO Regional Office for the Americas/Pan American Health Organization Dr Alfonso Clavijo, Veterinary Public Health, Panamerican Centre for Foot-and-Mouth Disease, São Bento, Duque de Caxias, Rio de Janeiro, Brazil Dr Marco Vigilato, Veterinary Public Health, Panamerican Centre for Foot-and-Mouth Disease, São Bento, 25045-002 Duque de Caxias, Rio de Janeiro, Brazil 123

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WHO Regional Office for South-East Asia Dr Gyanendra Gongal, Scientist, Veterinary Public Health, Disease Surveillance and Epidemiology, New Delhi, India

Invited but unable to attend Dr Katinka de Balogh, Senior Officer, Veterinary Public Health, Food and Agriculture Organization of the United Nations, Rome, Italy Dr Philip Binu, Business Development, Zydus Research Centre, Gujarat, India Dr Deborah Briggs, Executive Director, Global Alliance for Rabies Control, c/o Balfour and Manson, Edinburgh, Scotland Dr Yu Hongjie, Director, Division of Infectious Disease Control, Centre for Disease Control, Beijing, China Dr Ivan V. Kuzmin, Rabies Program, Centers for Disease Control and Prevention, Atlanta, Georgia, USA Dr Tiziana Lembo, Institute of Comparative Epidemiology, Faculty of Veterinary Medicine, University of Glasgow, Glasgow, Scotland Dr Anastasia Pantelias, Bill & Melinda Gates Foundation, Seattle, Washingthon, USA Dr A. Rowan, Humane Society International, Washington, District of Columbia, USA WHO Technical Report Series No. 982, 2013 124

Dr Louis Taylor, PRP Coordinator, Global Alliance for Rabies Control, Manhattan, Kansas, USA

Annexes

Annex 2. Record form for cases of possible exposure to rabies Case no.: Date: Time: Patient Name: Age: Sex: Address: Telephone (home and mobile): Telephone (work): GP and Tel: Details of exposure Country and town: Date of exposure: Date of travel: Nature of exposure: bite/lick/saliva/scratch/other (to specify) Site of exposure: Was the skin broken? Yes/No Did the wound/s bleed? Yes/No Number of wounds: Depth of bite/s: superficial/deep Category of exposure: Details of animal Type of animal/species: Wild/domestic: Provoked/unprovoked (give details): Is the animal’s owner/home known? Yes/No: Were efforts made to trace the animal? Yes/No: When was the animal last seen alive? Animal’s vaccination status, if known: Previous rabies vaccination history of the patient Did s/he have a 3-dose intramuscular/intradermal pre-exposure rabies vaccination? Yes/No 125

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Details: Was anti-rabies post-exposure prophylaxis given previously? Yes/No Which rabies vaccine was given? Details (day/date etc.): Was rabies immunoglobulin given? Yes/No Locally/systemically: Other information Contact on-call virologist/physician for advice with above information If unavailable, contact: Recommended treatment ■■ 1. Wound washing using water/ soap/ antiviral agent. ■■ 2. Rabies vaccination: ■■ Modified course of treatment for those with previous pre-exposure prophylaxis: days 0 and 3 ■■ –– –– –– Standard course for unvaccinated: Intramuscular – days 0, 3, 7, 14, 28 or 0, 3, 7, 14 Intramuscular – days 0 (2 doses), 7, 21 Intradermal – days 0 (2 sites), 3 (2 sites), 7 (2 sites), 28 (2 sites)

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■■ 3. Rabies immunoglobulin: human rabies immunoglobulin, 20 IU/kg body weight; equine rabies immunoglobulin, 40 IU/kg body weight Injection site: Patient weight (kg): Volume recommended (IU and ml): Post-exposure course arranged? Yes/No General practitioner informed via letter/e-mail/phone/SMS text? Yes/No Name, telephone and signature of completing physician

Annexes

Annex 3. Four steps for replacing nervous tissue vaccine by modern rabies vaccines produced on cell culture or embryonated eggs Countries that are still producing or using neural tissue-based vaccines should follow this proposed four-step strategy to replace nerve tissue vaccines by modern vaccines. Step 1: Relevant national authorities, usually under the leadership of national health authorities, must make the final decision to change from nerve tissue vaccines to modern vaccines. After reviewing the safety, immunogenicity and efficacy of modern vaccines, the authorities should evaluate the local conditions, and assess the feasibility and cost of replacing nerve tissue vaccine. Consideration should be given to the use of the costsaving intradermal regimens for rabies pre- and post-exposure prophylaxis. Step 2: National guidelines should be formulated that give clear instructions on modern vaccines for pre- and post-exposure prophylaxis, including indications for their use and routes of administration; similarly, guidance should be given for rabies immunoglobulin and other products. The guidelines should be drawn up by technically competent experts on the basis of the recommendations in reports of the WHO Expert Advisory Group on Rabies, other WHO advisory groups, up-to-date scientific literature, the experience of international and national experts and observations. They should be disseminated to all centres that provide pre- and post-exposure prophylaxis. The guidelines must be based on clear policies concerning, e.g. vaccine subsidy (if any) and handling leftover vaccine, and should be regularly updated. Step 3: Rabies centres should receive a constant supply of safe, effective, WHOrecommended rabies vaccines and immunoglobulin from a central office. Once the decision is made to stop nerve tissue vaccine production and use, the procurement of modern vaccines should start, to avoid any gap in provision of treatment once the nerve tissue vaccine supplies run out. Coordination with regulatory bodies for registration of new rabies biologicals and for post-marketing surveillance of new rabies vaccines and rabies immunoglobulin is also important. Step 4: A network of specialized bite centres should be set up, in which the staff are trained in giving pre- and post-exposure prophylaxis and managing adverse reactions; adequate quantities of rabies biologicals at these centres must be ensured. A referral system should be established to maximize the benefit of the intradermal regimen and to reduce the amount of leftover vaccine. A quality assurance system should be instituted, with standards that are followed by all centres. Provincial and municipal governments should be involved in establishing new centres, ensuring a sustainable supply of rabies vaccines, immunoglobulin and other supplies and guaranteeing reporting, investigation of human rabies cases and monitoring of the rabies programme.

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Annex 4. Technique for intradermal administration of rabies vaccine and precautions to be taken Intradermal administration can be used in all countries in which the intradermal route has regulatory approval for pre- or post-exposure prophylaxis for rabies. The vaccines administered must be licensed for administration by this route and recommended by WHO (see section 5.1). Intradermal administration should not be used for immunocompromised individuals or individuals receiving chloroquine-based antimalarial treatment or long-term corticosteroid or other immunosuppressive therapy. As the volume of an intradermal vaccine dose is smaller than an intramuscular dose, the intradermal route is especially suitable for treating many patients at the same centre within a short time, i.e. within the recommended period of 6–8 h after reconstitution of the vaccine. As the currently available vaccines do not contain preservatives, they must be refrigerated after reconstitution and must be discarded after 6–8 h. The intradermal route is more cost–effective than the standard intramuscular route and is therefore appropriate when vaccine and money are in short supply and in centres where exposed patients are treated. Preliminary steps Before administering rabies vaccine intradermally: ■■ All staff must be adequately trained in the intradermal injection technique. ■■ If the vaccine is given as part of post-exposure prophylaxis, the additional steps should be followed; i.e. the wound must be washed and, if applicable, the appropriate dose of rabies immunoglobulin administered. WHO Technical Report Series No. 982, 2013

■■ An appropriate 1.0-ml syringe (insulin or tuberculin syringe) and a short, fine hypodermic needle should be used. More costs are saved if a fixed-needle syringe is used, as the void volume is reduced. ■■ The intradermal schedule should be selected. WHO recommends the 2-2-20-2 updated Thai Red Cross schedule (see section 8.3.3).

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Step 1 Aseptically reconstitute the vaccine immediately before administration with the appropriate volume of diluent provided by the manufacturer. Do not use a different diluent. Do not use a different amount of diluent. Draw enough vaccine into the syringe to inject a single patient, using appropriate sterile precautions. Carefully remove any air bubbles. Disinfect the injection site with antiseptic, then stretch the surface of the skin and insert the tip of the needle (bevelled edge facing upwards) into the upper layer of the skin (dermis), ensuring that the needle and syringe are almost parallel to the skin surface. Step 2 Begin injecting the vaccine. If the needle is in the correct position, there is considerable resistance. A raised papule, which looks like orange peel, will appear immediately, measuring 6–8 mm in diameter. If the vaccine is injected easily, or if the papule does not appear, it has been given subcutaneously, i.e. too deeply. In such cases, the correct injection should be repeated. Step 3 Once all doses of 0.1 ml of vaccine have been injected into the same patient, discard the needle and the syringe. Reconstituted vaccine can be used for more than one patient; however, a sterile syringe and needle must be used to draw up vaccine for each patient. The reconstituted vaccine must be stored in a refrigerator at 2–8 °C and used within 6–8 h.

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Annex 5. Recommended post-exposure prophylaxis according to type of exposure Category of exposure Type of exposure to a domestic or wilda animal suspected or confirmed to be rabid, or animal unavailable for testing Recommended post-exposure prophylaxis

Touching or feeding animals None, if reliable case history is available Licks on intact skin Contact of intact skin with secretions or excretions of a rabid animal or human case Nibbling of uncovered skin Administer vaccine immediatelyb Minor scratches or abrasions without Stop treatment if animal remains healthy throughout an observation period of 10 bleeding daysc or is proven to be negative for rabies by a reliable laboratory using appropriate diagnostic techniques. Single or multiple transdermal bitesd or scratches, licks on broken skin Contamination of mucous membrane with saliva (i.e. licks) Exposure to batse Administer rabies vaccine immediately, and rabies immunoglobulin, preferably as soon as possible after initiation of post-exposure prophylaxis. Rabies immunoglobulin can be injected up to 7 days after first vaccine dose administration. Stop treatment if animal remains healthy throughout an observation period of 10 days or is proven to be negative for rabies by a reliable laboratory using appropriate diagnostic techniques.

III

Exposure to rodents, rabbits or hares does not routinely require rabies post-exposure prophylaxis. If an apparently healthy dog or cat in or from a low-risk area is placed under observation, treatment may be delayed. This observation period applies only to dogs and cats. Except for threatened or endangered species, other domestic and wild animals suspected of being rabid should be euthanized and their tissues examined for the presence of rabies antigen by appropriate laboratory techniques. Bites especially on the head, neck, face, hands and genitals are category III exposures because of the rich innervation of these areas. Post-exposure prophylaxis should be considered when contact between a human and a bat has occurred, unless the exposed person can rule out a bite or scratch or exposure of a mucous membrane.

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Annex 6. Suggested rabies vaccination certificates for humans The vaccination certificates below are provided as models. The certificates should be kept carefully by the vaccinated person with his or her personal health documents. Blank certificates should be supplied by the manufacturer of the vaccines. Certificate of pre-exposure vaccination against rabies Name___________________________________________________________ Date of birth/Age (years)__________ Sex______Occupation__________________ Address__________________________________________________________ _______________________________________________________________ Tel. no. __________________ Signature ________________________________________________________ Primary vaccination Date of vaccination Vaccination centre/Place Type/Name of vaccine Manufacturer (batch no.)/Expiry date Dose (ml) Route of administration (intramuscular or intradermal) Site of vaccination Adverse event, if any Rabies virus neutralizing antibody titre, if done/Method Signature of physician Day 0 Day 7 Day 21 or 28

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Booster doses for people at high risk of exposure Date of booster vaccination Vaccination centre/Place Type/Name of vaccine Manufacturer (batch no.)/Expiry date Dose (ml) Route of administration (intramuscular or intradermal) Site of vaccination Adverse event, if any Rabies virus neutralizing antibody titre, if done/Method Signature of physician

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Certificate of post-exposure vaccination against rabies Name___________________________________________________________ Date of birth/Age (years)_____________Sex________ Occupation _____________ Address _________________________________________________________ _________________________________________________ Tel. no. ___________________________ Date of exposure_______WHO category of exposure _______Biting animal__________ Healthy/Sick _________Animal vaccination status________Rabies virus neutralizing antibody titre/Method_________ Observations after 10 days (when relevant) _________________________________ _______________________________________________________________ 1. Wound washed with water/soap/antiviral agent________________ 2. Rabies immunoglobulin: Date of treatment________________ Clinic/hospital name _____________ Place_________________ Name/Type of rabies immunoglobulin (human/equine) __________________ __________________________________________________________

Annexes

Manufacturer (batch no./Expiry date)_______________________________ Weight of patient____kg. Dose (IU)______Total volume (ml)_____________ Rabies immunoglobulin infiltrated into and around wound / intramuscular (ml) __________________________________________________________ Remaining immunoglobulin injected at site away from site of vaccine injection intramuscularly (ml)___________ 3. Rabies vaccine: Vaccine regimen: Five-dose Essen (1-1-1-1-1) or four-dose Essen (1-1-1-1-0) Zagreb (2-1-1) Updated Thai Red Cross two-site intradermal (2-2-2-0-2) Other Date of vaccination Vaccination centre/ Place Type/Name of vaccine Manufacturer (batch no.)/Expiry date Dose (ml) Route of administration (intramuscular or intradermal) Site of vaccination Adverse event, if any Rabies virus neutralizing antibody titre, if done/Method Signature of physician Day 0 Day 3 Day 7 Day 14 Day 21 Day 28

General remarks (if any)

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Annex 7. International rabies vaccination certificate for dogs, cats and ferrets The vaccination certificate below is provided as a model. It is based on the OIE certificate.1 Some countries may require additional information. Certificat international de vaccination antirabique pour chiens, chats et furets/ International rabies vaccination certificate for dogs, cats and ferrets I. Propriétaire/Owner Nom et adresse/Name and address ___________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ II. Signalement/Description Espèce/Species _________________________________________________ Age ou date de naissance (si possible)/Age or date of birth (when known) _____________________________________________________________ Sexe/Sex ______________________________________________________ Race/Breed ____________________________________________________ Robe/Coat colour ________________________________________________ Type de pelage et marques/signes particuliers/Coat type and marking/distinguishing marks ________________________________________________________ Numéro de micro chip/Microchip no.__________________________________ Type de lecteur du micro chip/Microchip scanner type _____________________ Emplacement du micro chip/Location of microchip________________________ Numéro et emplacement du tatouage (si présent)/Location and tattoo number (if applicable) ___________________________________________________ III. Vaccinations antirabiques/Rabies vaccinations Le soussigné certifie avoir vacciné contre la rage l’animal décrit à la page 1, comme il est indiqué ci-après. Au moment de la vaccination, l’animal a été reconnu en bonne santé. The undersigned declares herewith that she or he has vaccinated the animal described on page 1 against rabies, as shown below. The animal was found to be healthy on the day of vaccination. 1

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Terrestrial animal health code [vol. 2, chapter 8.10: Rabies]. Paris, World Organisation for Animal Health, 2011 (http://www.oie.int/index.php?id=169&L=0&htmfile=chapitre_1.8.10.htm; accessed 21 September 2012).

Annexes

(1) Date de vaccination/Vaccination date

(2) Nom du vaccin/ Name of vaccine

(3) Nom du fabricant/ Name of manufacturer

(4) Numéro de lot/ Batch no.

(5) Date d’expiration/ Expiry date

(6) signature et cachet du vétérinaire officiel/ Signature and stamp of official veterinary surgeon

(7) Valable jusqu’au/ Valid until

IV. Tests sérologiques antirabiques/Rabies serological tests Déclaration du vétérinaire/Veterinary declaration Je soussigné(e) certifie avoir pris connaissance des résultats officiels du test sérologique pratiqué sur l’animal décrit ci-dessus à la date du (jj/mm/aa)_________________, conduit par un laboratoire agréé confirmant que le titre d’anticorps neutralisants anti-rage était supérieur ou égal à 0.5 UI/ml. Période de validité: Date

Nom, date, et cachet du vétérinaire officiel I have seen an official record of the result of a serological test for the animal, carried out on a sample taken on (dd/mm/yy)___________________ and tested in an approved laboratory, which states that the rabies-neutralizing antibody titre was equal to or greater than 0.5 IU/ml. Period of validity: Name, date and signature of the authorized veterinarian: Tests supplémentaires/Further tests: Résultat/ Result Laboratoire agréé/ Approved laboratory Signature et cachet du vétérinaire/ Signature and stamp of veterinary surgeon

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V. Autres vaccinations/Other vaccinations Date Vaccin utilisé/ Type of vaccine Numéro de lot/ Signature et cachet du vétérinaire/ Batch no. Signature and stamp of veterinary surgeon

VI. Informations complémentaires/Additional information Pays d’origine/Country of origin_____________________________________ ____________________________________________________________ Pays dans lesquels l’animal a séjourné, selon les déclarations du propriétaire (indiquer les dates)/Countries visited by the animal as declared by the owner (give dates)____________________________________________________ ____________________________________________________________ ____________________________________________________________ Notes: Le présent certificat ne dispense pas de l’application des autres dispositions en vigueur pour l’entrée dans chaque pays. Prière de lire la section VII. This certificate may not be sufficient to meet all the requirements of the countries of destination. Please read Section VII. WHO Technical Report Series No. 982, 2013 136

Autorisation d’imprimer délivrée par (indiquer l’autorité nationale compétente): Printing authorized by (indicate the national responsible authority): Pour être valable, le présent certificat doit porter un numéro perforé à chaque page. To be valid, this certificate must bear a number perforated on each page. VII. Passage de frontière/Frontier crossing Le propriétaire de l’animal doit, avant de se rendre à l’étranger avec celui-ci, s’assurer des conditions sanitaires imposées par les autorités du pays de destination, le présent certificat ne dispensant pas de l’application des autres dispositions en vigueur dans certains pays.

Annexes

The owner of the animal must, before going abroad with it, make sure of the veterinary requirements laid down by the authorities of the country of destination, as this certificate may not be sufficient to meet all the requirements of the country of destination. Le présent certificat est valable à partir du trentième jour et jusqu’à la fin du douzième mois après la date de la première vaccination ; dans le cas d’une revaccination au cours de la période de validité, pendant les douze mois qui suivent la date de revaccination. This certificate is valid from the 30th day until the end of the 12th month after the date of the first vaccination; in the case of revaccination within the validity period, for 12 months from the date of revaccination. Le présent certificat doit être imprimé et complété en Français et en Anglais, et si nécessaire, dans la langue du pays d’origine. This certificate must be printed and completed in French and English and, if necessary, the language of the country of origin.

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Annex 8. WHO collaborating centres on rabies, neurovirology, viral zoonoses and zoonoses control WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris, France Head, Dr Hervé Bourhy; e-mail: herve.bourhy@pasteur.fr WHO Collaborating Centre on Research and Management on Zoonoses Control, AFSSALERPAS, Laboratoire d’études sur la rage et la pathologie, des animaux sauvages, Domaine de Pixérécourt, BP 9, 54220 Malzéville, France Head, Dr Florence Cliquet; e-mail: florence.cliquet@anses.fr WHO Collaborating Centre for the Characterization of Rabies and Rabies-related Viruses, Animal Health and Veterinary Laboratories Agency, Weybridge, Surrey KT15 3NB, United Kingdom Head, Dr Anthony Fooks; e-mail: t.fooks@ahvla.gsi.gov.uk WHO Collaborating Centre for Rabies Surveillance and Research, Friedrich-Loeffler Institut, Federal Research Institute for Animal Health, Sudufer 10, 17493 GreifswaldInsel Reims, Germany Head, Dr Thomas Müller; e-mail: thomas.mueller@fli.bund.de WHO Collaborating Centre for Control, Pathogenesis and Epidemiology of Rabies in Carnivores, 106 Pineridge Road, Carp, ON, Canada Head, Dr Christine Fehlner-Gardiner; e-mail: Christine.Fehlner-Gardiner@inspection.gc.ca WHO Collaborating Centre for Neurovirology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19105, USA Head, Dr Bernhard Dietzschold; e-mail: bernhard.dietzschold@jefferson.edu WHO Collaborating Centre for Reference and Research on Rabies, Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA Head, Dr Hildegund Ertl; e-mail: ertl@wistar.upenn.edu WHO Collaborating Centre for Reference and Research on Rabies, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA Head ad interim, Inger Damon, Chief, Poxvirus and Rabies Branch, CDC

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WHO Collaborating Centre for Rabies Epidemiology, Centre for AIDS and Related Diseases, National Centre for Disease Control, 22-Sham Nath, Delhi 110054, India Head, Dr Rattan Lal Ichhpujani; e-mail: ichhpujani@hotmail.com WHO Collaborating Centre for Reference and Research in Rabies, Department of Neurovirology, National Institute of Mental Health and Neurosciences, PO Box 2900, 560029 Bangalore, India Head, Professor S.N. Madhusudana; e-mail: mshampur@hotmail.com WHO Collaborating Centre for Research and Training on Viral Zoonoses, Chulalongkorn University Hospital, Rama 4 Road, Bangkok 10330, Thailand Head, Dr Thiravat Hemachudha; e-mail: fmedthm@gmail.com WHO Collaborating Centre for Research on Rabies Pathogenesis and Prevention, Queen Saovabha Memorial Institute, Thai Red Cross Society, 1871 Rama IV Road, 10330 Bangkok, Thailand Head, Professor Visith Sitprija; e-mail: sitprija@yahoo.com; and Dr Pakmanee Narumol; e-mail: npakmanee@yahoo.com

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982 WHO Expert Consultation on Rabies

W H O

Te c h n i c a l

R e p o r t

S e r i e s

982 Although there is debate about the estimated health burden of rabies, the estimates of direct mortality and the DALYs due to rabies are among the highest of the neglected tropical diseases. Poor surveillance, underreporting in many developing countries, frequent misdiagnosis of rabies, and an absence of coordination among all the sectors involved are likely to lead to underestimation of the scale of the disease. It is clear, however, that rabies disproportionately affects poor rural communities, and particularly children. Most of the expenditure for postexposure prophylaxis is borne by those who can least afford it. As a result of growing dog and human populations, the burden of human deaths from rabies and the economic costs will continue to escalate in the absence of concerted efforts and investment for control. Since the first WHO Expert Consultation on Rabies in 2004, WHO and its network of collaborating centres on rabies, specialized national institutions, members of the WHO Expert Advisory Panel on Rabies and partners such as the Gates Foundation, the Global Alliance for Rabies Control and the Partnership for Rabies Prevention, have been advocating the feasibility of rabies elimination regionally and globally and promoting research into sustainable cost-effective strategies. Those joint efforts have begun to break the cycle of rabies neglect, and rabies is becoming recognized as a priority for investment. This Consultation concluded that human dog-transmitted rabies is readily amenable to control, regional elimination in the medium term and even global elimination in the long term. A resolution on major neglected tropical diseases, including rabies, prepared for submission to the World Health Assembly in May 2013 aims at securing Member States’ commitment to the control, elimination or eradication of these diseases. Endorsement of the resolution would open the door for exciting advances in rabies prevention and control.

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SELECTED WHO PUBLICATIONS OF RELATED INTEREST

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This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the World Health Organization

Contents Introduction 1. The burden of rabies 1.1 1.2 1.3 1.4 Methods for estimating the burden of rabies Estimated burden of rabies in the world Global summary References

1 2 2 3 8 10 13 13 13 14

2. Classification of lyssaviruses 2.1 Distinguishing features of lyssaviruses 2.2 Criteria for differentiating between lyssaviruses 2.3 Present structure of the Lyssavirus genus 2.4 References

15 19 23

23 24 25 27 28 30 31

34 34 35 35 36 36 36

37 37 39 40 41 42 42 42 43

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7. Vaccines for animals 7.1 Vaccine types 7.2 Potency requirements for animal rabies vaccines 7.3 Safety of animal vaccines 7.4 Parenteral rabies vaccination 7.5 References

47 48 49 50 51 51

54 54 54 55 59 59 60 60 61 62

63 64 66 67 69 70 70 72 74

77 77 80 82 83 83 88 88 88

92 94 97 97 97

13.3 Special exemption of guide dogs for people with disabilities and of other service dogs 13.4 References

98 98

14. Global and regional activities on rabies 14.1 WHO global and regional activities 14.2 Examples of activities by partners 14.3 References

98 99 102 107

110 110 110 111 112 113 113 114 114

Concluding remarks Acknowledgements Annex 1 List of participants

117 118 119 125

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128 130 131 134

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Introduction

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The burden of rabies

1.1

Methods for estimating the burden of rabies

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Post-exposure prophylaxis, preventive 261 vaccination of cattle, dogs and cats, oral rabies vaccination Oral rabies vaccination Oral rabies vaccination and surveillance 122 15.5

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1.2.2 Countries in which canine rabies is endemic Latin America and the Caribbean

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Africa

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Table 2 Estimated numbers of deaths from rabies (with 95% confidence intervals) in various areas of the world Africa China India Other Asian countries All Asia All Asia and World Africa

2003

(26)

Multi-centre study (community surveys and hospital records)

2005

(9)

Verbal autopsies

2003

(5)

Probability decision-tree approach

2010

(11,12)

‘Cause-of-death ensemble’ model

2010

(27)

National surveillance data

2010

PRP

Probability decision-tree approach

PRP, Partners for Rabies Prevention

*Excluding Central Asia

The burden of rabies

**Excluding North Africa

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The burden of rabies

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28. Yin C-P. Analysis on factors related to rabies epidemic in China from 2007–2011. Virologica Sinica, 2012, 27(2):132–143. WHO Technical Report Series No. 982, 2013

Classification of lyssaviruses

2. 2.1

Classification of lyssaviruses Distinguishing features of lyssaviruses

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2.3

Present structure of the Lyssavirus genus

Classification of lyssaviruses

3. Boulger LR, Porterfield JS. Isolation of a virus from Nigerian fruit bats. Transactions of the Royal Society of Tropical Medicine and Hygiene, 1958, 52:421–424.

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Table 3 Viruses currently included in the genus Lyssavirus Recognized and proposed species (abbreviation) Rabies virus (RABV) Primary host Geographical range Comments

Carnivora and bats (Chiroptera)

South-eastern Europe United Republic of Tanzania

13 14

Ikoma lyssavirus Not known (IKOV)

Classification of lyssaviruses

1. 2. 3.

4. 5. 6. 7. 8. 9.

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4. Shope RE et al. Two African viruses serologically and morphologically related to rabies virus. Journal of Virology, 1970, 6:690–692.

11. King A, Davis P, Lawrie A. The rabies viruses of bats. Veterinary Microbiology, 1990, 23:165–174. WHO Technical Report Series No. 982, 2013

12. Bourhy H, Kissi B, Tordo N. Molecular diversity of the Lyssavirus genus. Virology, 1993, 194:70–81.

Pathogenesis

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Pathogenesis

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20. Roy A, Hooper DC. Immune evasion by rabies viruses through the maintenance of blood–brain barrier integrity. Journal of Neurovirology, 2008, 14(5):401–411.

Diagnosis

One or more of the following laboratory criteria should be used to confirm a clinical case: ■■ presence of viral antigens; ■■ isolation of virus in cell culture or in laboratory animals;

■■ presence of viral-specific antibodies in the cerebrospinal fluid or the serum of an unvaccinated person; or 23

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Diagnosis

4.3

Biosafety, sampling and specimen transport for laboratory diagnosis

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Diagnosis

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commercially available assays have not been standardized or harmonized for proper use and adequate validation according to international standards (5).

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Diagnosis

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Diagnosis

13. Laothamatas J et al. MR imaging in human rabies. American Journal of Neuroradiology, 2003, 24:1102–1109.

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Diagnosis

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Management of patients before and after death

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5.6 References 1. Hemachudha T et al. Human rabies: neuropathogenesis, diagnosis and management. Lancet Neurology, 2013, 12(5):498–513.

Vaccines and rabies immunoglobulin for humans

5. Maier T et al. Management and outcomes after multiple corneal and solid organ transplantation from a donor infected with rabies virus. Clinical Infectious Diseases, 2010, 50(8):1112–1119.

Vaccines and rabies immunoglobulin for humans

6.1.2 Nerve tissue vaccines Nerve tissue vaccines induce more severe adverse reactions and are less immunogenic than CCEEVs. Since 1984, WHO has recommended discontinuation 37

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Vaccines and rabies immunoglobulin for humans

■■ Chile (2003) ■■ Dominican Republic (2009) ■■ El Salvador (2009) ■■ Mexico (1995) ■■ Nicaragua (2005) ■■ Paraguay (2006)

6.2

WHO prequalification of human rabies vaccines

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6.3

Requirements for human rabies vaccines

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6.4

Routes of vaccine administration

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safe and effective when given by this route should register their product for intradermal use and state in the product insert that their vaccine can be used intradermally.

6.5

Adverse events after active immunization

6.6

Duration of immunity

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6.7

Rabies vaccine and full post-exposure prophylaxis failures

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6.8

Rabies immunoglobulins

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Management of patients before and after death

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20. Sudarshan MK et al. Assessing the relationship between antigenicity and immunogenicity of human rabies vaccines. Results of a meta-analysis. Human Vaccines, 2005, 1:187–190.

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Vaccines for animals

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7.1.1 Vaccines for domestic animals Injectable modified-live virus vaccines

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A canarypox virus expressing the rabies virus glycoprotein has been licensed in the USA as a parenteral vaccine for cats. The commercially available vaccine

Vaccines for animals

combines the rabies–canarypox with feline panleukopenia, feline calicivirosis and feline herpesvirus components. Live replication-competent vaccines for oral use

7.2

Potency requirements for animal rabies vaccines

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Live recombinant vaccines

Vaccines for animals

7.5 References 1. WHO Expert Committee on Rabies. Eighth report. Geneva, World Health Organization, 1992 (WHO Technical Report Series, No. 824).

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Vaccines for animals

14. Matter HC, Fico R. Accessibility of dogs to oral and parenteral vaccination against rabies in Tunisia and Turkey. Geneva, World Health Organization, 1992 (WHO/Rabies/93.206).

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Prevention of human rabies

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Prevention of human rabies

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Prevention of human rabies

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Prevention of human rabies

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Figure 1 Four categories of countries or areas, from those at no risk to those at low, moderate and high risk

National programmes for dog rabies control

9. Schneider MC. et al Rabies transmitted by Vampire bats to humans: an emerging zoonosis in Latin America? Pan American Journal of Public Health, 2009, 25(3):260–269.

National programmes for dog rabies control

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National programmes for dog rabies control

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National programmes for dog rabies control

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National programmes for dog rabies control

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National programmes for dog rabies control

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9.7

Operational research for dog rabies control

National programmes for dog rabies control

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ferent dog population management options and to determine whether and how fertility control could be used as an adjunct to optimize rabies elimination programmes in some contexts.

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Prevention and control of rabies in wild animals

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Prevention and control of rabies in wild animals

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Prevention and control of rabies in wild animals

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10.2.3 Vampire bat rabies

10.3 Rabies in rodents Testing of tens of thousands of wild and synanthropic rodents in areas endemic for rabies across the world has revealed only exceptional instances of dead-

Prevention and control of rabies in wild animals

end spillover of rabies virus infection, indicating that these animals are neither primary hosts nor play a role in the epidemiology and transmission of the disease.

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Prevention and control of rabies in wild animals

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apparent success in some areas of North America, primarily for skunks and raccoons (46,47).

10.7 Other public health measures WHO Technical Report Series No. 982, 2013

10.8 References 1. Weyer J et al. Epidemiology of human rabies in South Africa, 1983–2007. Virus Research, 2011, 155(1):283–290.

2. Sabeta CT et al. Molecular epidemiology of rabies in bat-eared foxes (Otocyon megalotis) in South Africa. Virus Research, 2007, 129(1):1–10.

Prevention and control of rabies in wild animals

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in the Middle East, 23–25 June 2008, Amman, Jordan. Paris, 2008 (www. oie.int/doc/ged/D6490.pdf; accessed 3 December 2012).

24. Banyard AC et al. Bats and lyssaviruses. Advances in Virus Research, 2011, 79:239–289.

30. McElhinney LM et al. Molecular epidemiology of bat lyssaviruses in Europe. Zoonoses and Public Health, 2012 (doi: 10.1111/zph.12003).

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47. Slavinski S et al. Trap–vaccinate–release program to control raccoon rabies, New York, USA. Emerging Infectious Diseases, 2012, 18(7):1170– 1172.

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Rabies surveillance

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information should be shared with the medical and veterinary sectors to facilitate management of animal bites, outbreak investigation and implementation of control measures.

References 1. Making surveillance work [modules 1–4]. Geneva, World Health Organization Department of Vaccines and Biologicals, 2001 (V&B/00.08 to 00.11).

7. Bourhy H et al. The origin and phylogeography of dog rabies virus. Journal of General Virology, 2008, 89:2673–2681.

8. Talbi C et al. Phylodynamics and human-mediated dispersal of a zoonotic virus. PLoS Pathogens, 2010, 6(10):e1001166.

Rabies-free countries or areas

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International movement of animals

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13.4 References 1. Terrestrial animal health code [vol. 2, chapter 8.10: Rabies]. Paris, World Organisation for Animal Health, 2011.

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14.3 References 1. WHO Expert Consultation on Rabies. First report. Geneva, World Health Organization, 2005 (WHO Technical Report Series, No. 931).

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Research

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for domestic animals and humans) and to verify whether commercial rabies biologicals, such as vaccines and antibodies, protect against them.

112

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4. Knobel DL et al. Re-evaluating the burden of rabies in Africa and Asia. Bulletin of the World Health Organization, 2005, 83:360–368.

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Members of the WHO Expert Advisory Panel on Rabies Dr Ahmad Fayaz, Former Head, Rabies Laboratory, Pasteur Institute, Tehran, Islamic Republic of Iran Professor Louis Hendrik Nel, Professor of Virology, Department of Microbiology and Plant Pathology, Faculty of Natural and Agricultural Sciences, University of Pretoria, Hillcrest, South Africa, President of the South Eastern Africa Rabies Group (SEARG) (Chair) Dr Beatriz P. Quiambao, Chief, Clinical Research Division, Research Institute for Tropical Medicine, Philippines. Dr Charles E. Rupprecht, Former Chief, Rabies Section, Centers for Disease Control and Prevention, Atlanta, USA Dr Naseem Salahuddin, Indus Hospital, Korangi, Karachi, Pakistan (Rapporteur) Professor Dr Mysore K. Sudarshan, Dean, Principal and Professor of Community Medicine Kempegowda Institute of Medical Sciences, Bangalore, India. Asia Foundation (RIA) Dr Alexander Wandeler, Former Head, rabies laboratory, Canadian Food Inspection

Other experts Professor Sarah Cleaveland, Consultant, University of Glasgow, Glasgow, Scotland Dr Raffy Deray, National Program Manager, National Rabies Prevention and Control Program, National Center for Disease Prevention and Control, Department of Health, Manila, Philippines Dr Katie Hampson, Research Scientist, Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland Mr Kevin Le Roux, Rabies Project Management, Veterinary Services, Pietermaritzburg, KwaZulu-Natal, South Africa Dr Giovanna Massei, Ecologist, Food and Environment Research Agency, York, England Dr Mathew Maziku, National Project Coordinator, Rabies, WHO Country Office, Dar es Salaam, United Republic of Tanzania Dr Maria P. Rebollo, Scientific Manager, Monitoring and Evaluation, Liverpool School of Tropical Medicine, Centre for Neglected Tropical Diseases, Liverpool, England Dr Graham Smith, Senior Researcher, Food and Environment Research Agency, York, England Dr Mathurin Cyrille Tejiokem, Epidemiologist, Epidemiology and Public Health Laboratory, Centre Pasteur du Cameroun, Yaoundé, Cameroon

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Dr Henry Wilde, Professor of Medicine, Senior Consultant, WHO Collaborating Centre for Zoonoses and Rabies, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand Representatives of other organizations

Representatives of other organizations Intergovernmental organizations World Organisation for Animal Health (OIE) Dr Marta Martinez, Veterinary Epidemiologist Dr Dietrich Rassow, Chargé de mission, WHO Nongovernmental organizations Association for the Prevention and Control of Rabies in India Dr A. Rahman, President, Commonwealth Veterinary Association for Prevention and Control of Rabies in India, Department of Community Medicine, Bangalore, India Dodet Science Dr Betty Dodet, Caluire et Cuire, France Global Alliance for Rabies Control Mr Kim Doyle, Trustee of the Alliance for Rabies Control, Global Alliance for Rabies Control, c/o Balfour and Manson, Edinburgh, Scotland Ms Maylin Meincke, Development Studies, University of Helsinki, Helsinki, Finland Dr Elizabeth Miranda, Asian Coordinator, Global Alliance for Rabies Control, Laguna, Philippines Marwar Trust Mr Federico Spinola, Founder, Partnership for Animals, Geneva, Switzerland PATH Dr Darin Zehrung, Technical Officer, Portfolio Leader Vaccine Delivery Technologies, Seattle, USA World Society for the Protection of Animals Dr Elly Hiby, Scientific Advisor, World Society for the Protection of Animals, Cambridge, England Dr Esmée Russell, Campaign Manager, World Society for the Protection of Animals, London, England

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Observers Dr Michaël Attlan, Director, Traveler Endemic and Emerging Vaccines Franchise, SanofiPasteur, Lyon, France Dr Jac Bergman, Global Marketing Director, Small Animal Vaccines, Global Companion Animal Business Unit, Boxmeer, the Netherlands Dr Rachel Chikwamba, Technical Lead, Rabies Initiatives, Pretoria 0001, South Africa Dr Pradip Desai, Director, Span Diagnostics Ltd, Surat, India Dr Alexandra Giesen, Novartis Vaccines and Diagnostics, Global Medical Affairs, Munich, Germany Dr Reinhard Glueck, CSO, Zydus Cadila Healthcare, Zydus Research Centre, Gujarat, India Dr Françoise Guinet-Morlot, Project Director New Vaccines, Sanofi Pasteur, Marcy l’Etoile, France Dr Gaurav Gupta, Head, Viral Vaccines, Zydus Cadila, Zydus Research Centre, Gujarat, India Dr K. Jager, Intervet, Boxmeer, the Netherlands Dr Philipe Mahl, Rabies Programme Manager, Virbac, Carros, France Dr Joanne Maki, Veterinary Public Health, Global Public Health Director, Athens, Georgia,USA Dr Claudius Malerczyk, Head, Medical Affairs, Middle East and Africa, Novartis Vaccines and Diagnostics, Marburg, Germany Dr Stephanus Francois Marais, Commercialisation Manager, CSIR Biosciences, Pretoria, South Africa Dr Wilfred Marissen, Programme Director, Crucell Holland B.V., Leiden, the Netherlands Dr Anvar Rasuli, Medical Product Leader, Global Medical Affairs, Sanofi Pasteur, 2 Avenue Pont Pasteur, Lyon, France Dr Micha Roumiantzeff, Fondation M. Merieux, 1 rue Dangon, 69004 Lyon, France Dr Carolin Schumacher, Director, Corporate Public Affairs, Merial, Lyon, France Dr Daniela Todorova-Balvay, R&D Manager, Span Diagnostics SARL, Compiegne, France Dr Adriaan Vos, Head, Vaccine Development Technologies, IDT Biologika GmbH, Dessau Rosslau, Germanyy

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WHO secretariat WHO headquarters, Geneva, Switzerland Dr Hiroki Nakatani, Assistant Director-General, HIV/AIDS, Tuberculosis, Malaria and Neglected Tropical Diseases Dr Lorenzo Savioli, Director, Control of Neglected Tropical Diseases, HIV/AIDS, Tuberculosis, Malaria and Neglected Tropical Diseases Dr François Meslin, Team Leader, Neglected Zoonotic Diseases, Control of Neglected Tropical Diseases (NTD), HIV/AIDS, Tuberculosis, Malaria and Neglected Tropical Diseases (HTM), (Organizer and convener) Dr Bernadette Abela-Ridder, Scientist, Foodborne Disease and Epidemiology, Food Safety, Zoonoses and Foodborne Diseases (FOS), Health Security and Environment (HSE) Dr Simone Magnino, Scientist, Foodborne Disease and Epidemiology, Food Safety, Zoonoses and Foodborne Diseases, Health Security and Environment (HSE) Dr Arve Willingham, Special Programme for Research and Training in Tropical Diseases (TDR), Information, Evidence and Research (IER) Dr Martin Friede, Technology Transfer Initiative (TTI), Technology Transfer Initiative (TTi), Health Systems and Innovation (HIS) Ms Erin Sparrow, Project Officer, Technology Transfer Initiative (TTi), Health Systems and Innovation (HIS) Dr Philippe Duclos, Scientist, Immunization, Vaccines and Biologicals (IVB), Family, Women’s and Children’s Health (FWC) Dr Ivana Knezevic, Team Leader, Essential Medicines and Health Products (EMP), Health Systems and Innovation (HIS) Dr Jinho Shin, Scientist, Essential Medicines and Health Products (EMP), Health Systems and Innovation (HIS) Dr David Wood, Coordinator, Essential Medicines and Health Products (EMP), Health Systems and Innovation (HIS) Dr Ana Padilla, Scientist, Quality Assurance and Safety: Medicines, Essential Medicines and Health Products (EMP), Health Systems and Innovation (HIS) Ms Beatrice Wamutitu, Secretary, Neglected Zoonotic Diseases, Department of Control of Neglected Tropical Diseases, HIV/AIDS, Tuberculosis, Malaria and Neglected Tropical Diseases

WHO Regional Office for Africa Dr Landry Bidé, Medical Officer, Neglected Tropical Diseases, Brazzaville, Congo 123

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WHO Regional Office for South-East Asia Dr Gyanendra Gongal, Scientist, Veterinary Public Health, Disease Surveillance and Epidemiology, New Delhi, India

Invited but unable to attend Dr Katinka de Balogh, Senior Officer, Veterinary Public Health, Food and Agriculture Organization of the United Nations, Rome, Italy Dr Philip Binu, Business Development, Zydus Research Centre, Gujarat, India Dr Deborah Briggs, Executive Director, Global Alliance for Rabies Control, c/o Balfour and Manson, Edinburgh, Scotland Dr Yu Hongjie, Director, Division of Infectious Disease Control, Centre for Disease Control, Beijing, China Dr Ivan V. Kuzmin, Rabies Program, Centers for Disease Control and Prevention, Atlanta, Georgia, USA Dr Tiziana Lembo, Institute of Biodiversity, Animal Health and Comparative Medicine College of Medical, Veterinary and Life Sciences, University of Glasgow, Scotland Dr Anastasia Pantelias, Bill & Melinda Gates Foundation, Seattle, Washingthon, USA Dr A. Rowan, Humane Society International, Washington, District of Columbia, USA WHO Technical Report Series No. 982, 2013 124

Dr Louis Taylor, PRP Coordinator, Global Alliance for Rabies Control, Manhattan, Kansas, USA

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III

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General remarks (if any)

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(1) Date de vaccination/Vaccination date

(2) Nom du vaccin/ Name of vaccine

(3) Nom du fabricant/ Name of manufacturer

(4) Numéro de lot/ Batch no.

(5) Date d’expiration/ Expiry date

(6) signature et cachet du vétérinaire officiel/ Signature and stamp of official veterinary surgeon

(7) Valable jusqu’au/ Valid until

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V. Autres vaccinations/Other vaccinations Date Vaccin utilisé/ Type of vaccine Numéro de lot/ Signature et cachet du vétérinaire/ Batch no. Signature and stamp of veterinary surgeon

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WHO Technical Report Series No. 982, 2013

Annexes

WHO Collaborating Centre for Rabies Epidemiology, Division of Zoonosis, National Centre for Disease Control, 22-Sham Nath, Delhi 110054, India Head, Dr Veena Mittal; e-mail: veena_m12@yahoo.com WHO Collaborating Centre for Reference and Research in Rabies, Department of Neurovirology, National Institute of Mental Health and Neurosciences, PO Box 2900, 560029 Bangalore, India Head, Professor S.N. Madhusudana; e-mail: mshampur@hotmail.com WHO Collaborating Centre for Research and Training on Viral Zoonoses, Chulalongkorn University Hospital, Rama 4 Road, Bangkok 10330, Thailand Head, Dr Thiravat Hemachudha; e-mail: fmedthm@gmail.com WHO Collaborating Centre for Research on Rabies Pathogenesis and Prevention, Queen Saovabha Memorial Institute, Thai Red Cross Society, 1871 Rama IV Road, 10330 Bangkok, Thailand Head, Professor Visith Sitprija; e-mail: sitprija@yahoo.com; and Dr Pakmanee Narumol; e-mail: npakmanee@yahoo.com

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世界卫生组织技术报告丛书 982

世界卫生组织狂犬病专家磋商会第二版报告

WHO Library Cataloguing-in-Publication Data: WHO Expert Consultation on Rabies: second report. (WHO technical report series ; no. 982) 1.Rabies – prevention and control. 2.Rabies – diagnosis. 3.Rabies – epidemiology. 4.Rabies vaccines. 5.Rabies virus. 6.National health programs. I.World Health Organization. II.Series. ISBN 978 92 4 520982 9 ISSN 1810-6641 (NLM classification: WC 550)

© 世界卫生组织，2014年版权所有。世界卫生组织出版物可从世卫组织网站（www.who.int）获得，或者自WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland（电话：+41 22 791 3264；传真：+41 22 791 4857；电子邮件：bookorders@who.int）购买。要获得复制许可或翻译世界卫生组织出版物的许可 – 无论是为了出售或非商业性分发，应通过世卫组织网站http://www.who.int/about/licensing/copyright_form/en/index.html）向世界卫生组织出版处提出申请。本出版物采用的名称和陈述的材料并不代表世界卫生组织对任何国家、领地、城市或地区或其当局的合法地位，或关于边界或分界线的规定有任何意见。地图上的虚线表示可能尚未完全达成一致的大致边界线。凡提及某些公司或某些制造商的产品时，并不意味着它们已为世界卫生组织所认可或推荐，或比其它未提及的同类公司或产品更好。除差错和疏忽外，凡专利产品名称均冠以大写字母，以示区别。世界卫生组织已采取一切合理的预防措施来核实本出版物中包含的信息。但是，已出版材料的分发无任何明确或含蓄的保证。解释和使用材料的责任取决于读者。世界卫生组织对于因使用这些材料造成的损失不承担责任。

目

录

导言 ………………………………………… … ………………………………………………… 1 1. 狂犬病的负担 … ……………………………………………………………………………… 1 1.1 估测狂犬病负担的方法……………………………………………………………… 2 1.2 全世界狂犬病负担的估测值………………………………………………………… 2 1.3 全球概述 ………………………… …………………………………… ……………… 6 1.4 参考文献 ………………………… …………………………………… ……………… 9 2. 狂犬病病毒的分类 …… …………………………………………………………………… 11 2.1 狂犬病病毒的特征…………………………………………………………………… 11 2.2 狂犬病病毒属的分类标准 ……………………………………………………… 12 2.3 目前狂犬病病毒属的组成 ……………………………………………………… 13 2.4 参考文献……………………………………………………………………………… 13 3. 发病机理……………………………………………………………………………………… 17 3.1 参考文献……………………………………………………………………………… 18 4. 诊断 ………………………………………………………………………………………… 20 4.1 狂犬病病例的标准定义……………………………………………………………… 20 4.2 临床诊断 ……………………………………………………………………………… 21 4.3 实验室诊断的生物安全、采样和样本运输………………………………………… 22 4.4 狂犬病死后诊断的实验室技术……………………………………………………… 23 4.5 人狂犬病活体诊断技术……………………………………………………………… 25 4.6 应用分子技术确定病毒：流行病学考虑………………………………………………26 4.7 参考文献……………………………………………………………………………… 26 5. 狂犬病人生前和死后的管理……………………………………………………………… 30 5.1 狂犬病存活者及其治疗方案 ………………………………………………………… 30 5.2 狂犬病患者的临床管理……………………………………………………………… 30 5.3 通过器官移植传播 …………………………………………………………………… 30 5.4 对医护人员和病人家属的建议 ……………………………………………………… 31 5.5 死于狂犬病的病人尸体的管理……………………………………………………… 31 5.6 参考文献 ……………………………………………………………………………… 31 6. 人用狂犬病疫苗和免疫球蛋白………………………………………………………………31 6.1 疫苗种类 ……………………………………………………………………………… 32

6.2 人用狂犬病疫苗 WHO 预认证 ……………………………………………………… 33 6.3 人用狂犬病疫苗的要求 ……………………………………………………………… 34 6.4 疫苗用药途径 ………………………………………………………………………… 35 6.5 主动免疫后的不良反应……………………………………………………………… 36 6.6 免疫持续时间 ………………………………………………………………………… 36 6.7 狂犬病疫苗与完全暴露后预防失败 ………………………………………………… 36 6.8 狂犬病免疫球蛋白 …………………………………………………………………… 37 6.9 参考文献 ……………………………………………………………………………… 37 7. 兽用疫苗 ……………………………………………………………………………………… 41 7.1 疫苗种类 ……………………………………………………………………………… 41 7.2 兽用狂犬病疫苗的效力要求 ………………………………………………………… 42 7.3 兽用疫苗的安全性 …………………………………………………………………… 43 7.4 注射用狂犬病疫苗接种 ……………………………………………………………… 44 7.5 参考文献 ……………………………………………………………………………… 44 8. 人类狂犬病的预防……………………………………………………………………………46 8.1 综合考虑 ……………………………………………………………………………… 46 8.2 暴露前预防 …………………………………………………………………………… 47 8.3 暴露后预防 …………………………………………………………………………… 47 8.4 定期加强注射的要求 ………………………………………………………………… 50 8.5 免疫功能受损个体的接种 …………………………………………………………… 51 8.6 狂犬病免疫球蛋白的被动免疫 ……………………………………………………… 51 8.7 禁忌症和注意事项 …………………………………………………………………… 51 8.8 狂犬病流行的国家和地区的旅行者和居民以及暴露前预防适应征……………… 52 8.9 参考文献 ……………………………………………………………………………… 53 9．犬类狂犬病控制的国家项目……………………………………………………………… 54 9.1 犬类大规模注射接种活动 …………………………………………………………… 55 9.2 疫苗接种活动的战略规划和管理…………………………………………………… 56 9.3 犬疫苗接种活动的实施和监控 ……………………………………………………… 57 9.4 增加对犬预防接种的可参与性 ……………………………………………………… 59 9.5 补充措施: 人性化犬群管理…………………………………………………………… 59 9.6 犬狂犬病控制计划的主要组成部分………………………………………………… 60 9.7 犬狂犬病控制的运作研究 …………………………………………………………… 60 9.8 参考文献 ……………………………………………………………………………… 62 10. 野生动物狂犬病的预防和控制…………………………………………………………… 64 10.1 食肉动物狂犬病流行病学和生态学 ………………………………………………… 64

10.2 蝙蝠狂犬病的流行病学和生态学 ………………………………………………… 66 10.3 啮齿动物狂犬病……………………………………………………………………… 68 10.4 特别关注的野生动物物种 …………………………………………………………… 68 10.5 野生食肉动物狂犬病的消除………………………………………………………… 69 10.6 蝙蝠狂犬病控制……………………………………………………………………… 72 10.7 其他公共卫生措施 …………………………………………………………………… 73 10.8 参考文献……………………………………………………………………………… 73 11．狂犬病监测………………………………………………………………………………… 77 参考文献…………………………………………………………………………………… 78 12. 无狂犬病国家和地区 ……………………………………………………………………… 79 参考文献…………………………………………………………………………………… 80 13. 动物的国际间转运………………………………………………………………………… 80 13.1 从有狂犬病的国家或地区跨国运输犬、猫和雪貂 ………………………………… 81 13.2 从有狂犬病的国家或地区跨国运输家畜、动物园动物、研究用和表演以及其他活动用的动物……………………………………………………………………………… 81 13.3 残疾人用引导犬及其他服务犬的特别豁免………………………………………… 81 13.4 参考文献……………………………………………………………………………… 82 14．有关狂犬病的全球性和地区性活动………………………………………………………82 14.1 WHO 全球性和地区性活动 ………………………………………………………… 82 14.2 成员活动范例………………………………………………………………………… 85 14.3 参考文献……………………………………………………………………………… 89 15、研究 ………………………………………………………………………………………… 91 15.1 诊断学 ………………………………………………………………………………… 91 15.2 流行病学 ……………………………………………………………………………… 92 15.3 新病毒分离物的分子、遗传和流行病学特征……………………………………… 92 15.4 生物医学制剂 ………………………………………………………………………… 93 15.5 人类狂犬病预防 ……………………………………………………………………… 94 15.6 病理学 ………………………………………………………………………………… 94 15.7 宿主生态学 …………………………………………………………………………… 95 15.8 参考文献……………………………………………………………………………… 95 结束语 …………………………………………………………………………………………… 98 鸣谢 ……………………………………………………………………………………………… 98 附件 ……………………………………………………………………………………………… 99 附件 1. 参加人员名单 …………………………………………………………………… 99 附件 2. 可能暴露于狂犬病的病例记录表……………………………………………… 104

附件 3. 使用细胞培养或鸡胚细胞生产的现代狂犬病疫苗替代神经组织疫苗的四个步骤 …………………………………………………………………………… 106 附件 4. 狂犬病皮内注射技术及应采用的预防措施 …………………………………… 107 附件 5. 根据暴露类型推荐的暴露后预防方法………………………………………… 109 附件 6. 人狂犬病疫苗接种证书的建议 ………………………………………………… 110 附件 7. 犬、猫、鼬国际狂犬病疫苗接种证书……………………………………………113 附件 8. 世界卫生组织狂犬病、神经病毒学、病毒引起的人畜共患病和人畜共患病控制合作中心 ………………………………………………………………………… 115

世界卫生组织狂犬病专家磋商会

第二版报告

导言世界卫生组织（WHO）狂犬病专家磋商会于 2012 年 9 月 18-20 号在瑞士日内瓦召开。项目经理 Denis Daumerie 博士代表被忽视的热带病控制中心主任 Lorenzo Savioli 博士和总干事向参会者致欢迎词。他指出狂犬病像该中心研究的热带病一样，感染的主要人群其死亡原因并不清楚。这种疾病主要在贫困地区持续发生，尽管早在 1950 年第三次世界卫生大会已经采纳的一项决议明确了人狂犬病的预防首先要控制犬的狂犬病，但这些地区并没有实施通过控制犬狂犬病进而实现预防人狂犬病的措施。狂犬病研究领域已经取得了一些进展，尤其是在人和动物生物制品的生产和使用方面，但是狂犬病仍然被忽视，而且目前没有提出致力于由犬类传播的人狂犬病的新的 WHO 解决方案。Daumerie 博士介绍了被忽视热带疾病控制部门与主要药物制造商在控制和消除热带病方面的成功合作，如麻风、淋巴丝虫病和非洲人类锥虫病，并建议此次磋商要探索有关狂犬病预防和控制方面的这种合作的益处。 Francois-Xavier Meslin 博士，被忽视人兽共患病专家，回顾了 WHO 谴责并抗争视狂犬病为 “被忽视的范畴” 的努力已长达十多年。自从 2004 年第一次 WHO 狂犬病专家磋商会以来， WHO 及其狂犬病合作中心网络、国家专业机构、 WHO 狂犬病专家咨询小组成员和包括比尔和梅林达盖茨基金会、全球狂犬病控制联盟与狂犬病预防合作伙伴一直倡导区域性和全球性消除狂犬病的可行性，并且促进相关策略研究。这些共同的努力已经开始打破狂犬病被忽视的怪圈，狂犬病正逐渐转变为一个优先投入的领域。 Louis Nel 博士被任命为主席， Naseem Salahuddin 博士被任命为此次磋商会的报告人。与会者名单见附件 1。本报告中的信息为狂犬病预防和控制的最新信息，应取代 2005 年出版的首次 WHO 狂犬病专家磋商会报告的信息。

1.狂犬病的负担疾病负担的信息广泛应用于确定公共卫生工作的重点，分配有限的资源于疾病预防与控制以及评估干预措施的影响和成本效益（1）。标准化衡量指标，如伤残调整寿命年（DALY），已经广泛应用于评价地区和全球范围的疾病负担，并且已经成为政策制定者做决策时的基本工具（2）。狂犬病的主要负担归因于犬介导的传播，因此这一章集中在犬介导的狂犬病并简单介绍因其他宿主种类所产生的负担（3）。当基础数据质量欠佳时疾病负担的估测可能会有争议，尽管如此，所获得的信息依然可以作为更准确评估的有帮助的起点，成为更好的可应用数据。 - 1 -

一些因素造成了世界许多地区狂犬病死亡病例的严重漏报。因此产生了估测狂犬病死亡率的方法，这些方法能够解释有犬狂犬病地方性流行的国家的数据报告质量。特别是设计了基于概率决策树的预测方法用于确定人被可疑疯犬咬伤后发生临床狂犬病的可能性。这种方法最初在坦桑尼亚联合共和国用来估测人狂犬病死亡数（4），并由此产生了用于非洲和亚洲狂犬病负担的校正估测（5）。最近，这种方法经过调整后用来估测亚洲特定国家（如不丹（6）和柬埔寨（7））的狂犬病死亡率。相关参数和验证此估测方法的实践研究包括社区调查（8）、大规模口头追溯调查（9）以及主动监测和追踪接触者（10）。 DALYs 包含过早死亡和残疾两项内容（2）。计算狂犬病的 DALYs 时最关键的要素是过早死亡（5），因为狂犬病持续时间短，残疾在狂犬病负担中所占比例较小。然而，注射神经组织疫苗后可能发生残疾，目前仍有少数国家在使用此类疫苗。这些疫苗有严重的副作用，这种副作用因使用疫苗的类型不同可持续 4 至 7 个月，估测发生率约为 0.3-0.8‰ （5）。疾病的经济负担是依据直接和间接两方面费用特别测算出来的。对于狂犬病来讲，暴露后预防的直接费用取决于疫苗、接种的方案和途径以及所使用狂犬病免疫球蛋白的类型；间接费用包括去诊所（或者陪同被咬伤者去诊所）以及与此相关的收入损失。这种费用的数量对狂犬病尤其重要，因为缺少暴露后预防会直接导致人死亡。进一步的经济构成是生产力的损失，可以用该国的国内生产总值乘以损失的寿命年，并采用 3%的贴现率进行贴现得到。至今，在狂犬病负担的研究中还没有考虑生产力的损失。在宿主动物中狂犬病的预防、控制和消除（包括监测）的费用以及动物生产部门的损失也应该考虑到疾病负担中。狂犬病负担还应包括它对情绪和心理的影响，特别是在被狂犬病动物咬伤后无法获得暴露后预防或者所采用的预防措施不可靠所产生的创伤以及长时间无法确认是否感染而带来的心理影响。狂犬病预防合作伙伴汇集成一个工作团队来核对和查阅最新数据并且运用概率决策树方法评估犬狂犬病的全球负担。作为本研究的一部分，健康度量和评估机构又运用 “死亡原因集成” 模型对全球狂犬病负担进行了估计（11,12）。就这些研究的初步结果进行了讨论，却发现这两项评估均存在高度的不确定性，其原因是缺少准确的数据。因此需要现场数据来验证这些评估以解决这一持久的问题。

接下来的部分，根据流行病学的相似性和地理上的接近程度将不同国家的狂犬病负担信息进行了分组。每一个地区都以当地所提供的最准确的数据为准，尽可能少地采用外推的地区性估计值。 - 2 -

世界卫生组织狂犬病专家磋商会

第二版报告

西欧、加拿大、美国、日本、马来西亚和少数拉丁美洲国家已经消除了犬狂犬病，而澳大利亚消除了食肉动物狂犬病，许多太平洋岛屿国家一直都没有狂犬病及其相关病毒。在这些地区，狂犬病人死亡病例仅限于去犬狂犬病流行地区生活或旅游的暴露人群。在欧洲、北美洲和日本，每年大约报告两例输入性狂犬病死亡病例（13,14）。在 1990-2010 年间，输入病例中三分之一来源于南亚和东南亚（主要是印度和菲律宾），另外三分之一来自非洲，大约 20%来自拉丁美洲和加勒比海以及超过 10%来自东欧和中亚。从海外返回的旅行者的暴露后预防和暴露前预防的费用通常很高。在其他无狂犬病地区的暴露后预防的费用会因发生输入性狂犬病动物疫情后而升高，甚至比那些经常有狂犬病动物从地方性流行的国家非法输入的地区更高，从而给卫生服务造成了相当大的负担（15）。在与犬狂犬病流行地区接壤的国家，需要边界防护和强化监测来维持无狂犬病状态。所有无狂犬病国家均需要检疫程序和立法。在许多有野生动物狂犬病和蝙蝠狂犬病病毒流行的国家必须考虑预防的费用。每年需要花费数百万美元用于投放口服狂犬病疫苗来消除野生动物狂犬病，这些费用会随着疫苗接种场所和策略的不同而显著变化（16）。例如，在美国由于野生动物狂犬病的缘故每年发生 1-8 个人狂犬病的死亡（17），根据疾病预防控制中心的数据，每年大约有 3 亿美元用于狂犬病的预防。目前一些州正在试图消除浣熊狂犬病以减少暴露后预防的需求。自从狐狸狂犬病从西欧消除以后，口服疫苗的费用则显著减少（表 1），但其他目前正致力于消除狐狸狂犬病的欧洲国家则承担着较高的费用。最近狂犬病侵入了意大利，虽然形势得到了控制，但仍然需要大量的财政支持，并且其他方面的费用也会上升，并且给一些没有狂犬病的国家比如希腊带来了再次出现疫情的威胁。在欧盟的整个东部边界建立一道预防警戒线来防止这样的侵入所需的费用估计每年超过 650 万美元（21）。

国家（参考文献）（18）法国德国（19）

项目时间 1988-1993

项目包括费用类别暴露后预防，牛、犬和猫的预防性疫苗接种口服狂犬病疫苗接种

项目费用（百万，美元）

261 122 15.5

1983-2008 口服狂犬病疫苗接种

（20） 2005-2010 口服狂犬病疫苗接种和监测爱沙尼亚

在过去的二十年中犬狂犬病控制项目在这些国家取得了很大的成功。随着犬狂犬病数量的下降，官方报告的由犬传播的人狂犬病病例数从 1990 年的 250 例下降到 2010 年的 10 - 3 -

例以下（22）。然而，在犬狂犬病持续流行的中心地区，官方的报告可能低于实际的流行，特别是在玻利维亚、古巴、多米尼加共和国、危地马拉、萨尔瓦多、海地、洪都拉斯、巴西的部分地区、墨西哥和秘鲁。在这些国家，狂犬病引起人死亡的事件仍然时有发生或者面临发生的危险。概率决策树模型初步估计显示在美洲因犬狂犬病引起的人死亡数每年可能达到 200 例，并且大部分发生在海地。尽管在美洲逐步淘汰神经组织疫苗方面取得了一些进展，但在阿根廷、玻利维亚、洪都拉斯、秘鲁和委内瑞拉玻利瓦尔共和国仍然在广泛使用神经组织疫苗，由此引发的副反应和导致的残疾仍然是一个问题。在这些地区每年狂犬病的公共卫生负担大概超出了 15000DALYs，其中大概有 100DALYs 可能是由神经组织疫苗引发的副反应造成的；然而，还需要合适的副反应报告系统准确测量这个数字。泛美卫生组织设定了到 2015 年消除美洲犬狂犬病的目标。为了达到这个目标，估计每年需要超过两千万美元的预算（23）；然而，目前每年大约还有 4 百万美元的资金缺口（24）。总年度预算中大约有 75%用于犬的疫苗接种， 5-10%用于暴露后预防相关的花费。该预算不包括人们接受暴露后预防产生的费用（包括消耗的时间、收入损失和副作用），也不包括人或牲畜中发生的蝙蝠相关狂犬病所产生的费用。

亚洲死于狂犬病的病例数居全球首位， 2003 年估计每年因地方性犬狂犬病导致的人死亡数超过了 30000 （95%的置信区间（CI）， 8100-61400）（5）。自 2003 年以来，随着许多地区狂犬病预防和控制状况的改善，尤其是在暴露后预防措施提供方面，亚洲多个地区的流行病学状况发生了改变。然而，仍有一些地区会有紧急状况发生。神经组织疫苗在亚洲几乎已完全被淘汰，除了蒙古、缅甸和巴基斯坦仍在使用这种疫苗。使用这些疫苗引发的副反应导致的 DALYs 大概从 40000 （5）下降到了 2010 年的 10000。 2011 年底，孟加拉共和国淘汰了神经组织疫苗，缅甸和巴基斯坦也正在计划停止生产和使用该疫苗。广泛应用暴露后预防可能已经减少了很多地区的死亡例数，包括印度，但暴露后预防的增加花费昂贵，因为犬的狂犬病控制项目没有受到同样的重视，暴露于狂犬病的风险依然存在并且可能会增加。暴露后预防相关的费用在亚洲要高于任何其他的地区，估计约为 15 亿美元。包括斯里兰卡和泰国在内的两个极端例子，在这两个国家每年用于暴露后预防的直接费用超过 1000 万美元（25）。估计 2010 年在亚洲，中亚除外，死于狂犬病的人数在 15900 （ “ 死亡原因集成 ” 模型方法）到 34500 （概率决策树方法）之间，由此造成该地区约 120 万 DALYs 损失。由于置信区间 - 4 -

世界卫生组织狂犬病专家磋商会

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重叠，这两种估计方法均不准确，还需要现场数据去验证模型的结果。此类研究中一些较好的实例，在孟加拉国农村地区人狂犬病死亡率估计为 1.1-1.8/10 万人（8），不丹处于危险的人群为 2.5-7.5/100 万（6）而柬埔寨则为 2.8-11.5/10 万（7）。印度报道为全球狂犬病最高发病率。2003 年一个多中心研究显示每年有 20565 人死于狂犬病（26），在 2005 年一个大规模的口头追溯研究，对于后一种方法未捕获的非典型病例没有进行校正的情况下，保守估计为 12700 人死于狂犬病（9）。大多数病例报告自农村地区（9， 26），在这些地区没有实施大规模犬的疫苗接种项目并且犬狂犬病发生率推测仍然很高。虽然暴露后预防的可及性得到了改善，但仍然不清楚有多少农村地区从中获益；此外，大部分死亡发生在没有接受治疗的人群。因此印度死于狂犬病的人数仍然不能确定。中国狂犬病负担的估测也不能确定。监测记录显示狂犬病的发病率自 2007 年以来已经降低，官方记录了 3300 多例狂犬病疑似（临床诊断）病例的死亡（27）。这些记录可能仍然低于狂犬病的实际发生率（27），所以仍然急需现场调查提高数据的准确性。虽然这些估计的数据具有不确定性，狂犬病显然是亚洲的一个重要问题，主要影响农村贫穷人群。在许多国家，官方记录大大低于问题的严重程度（6,7,9），因此鼓励重新评估。印度已经开始计划该项目。

2003 年在非洲因地方性犬狂犬病导致的人死亡数大约为 23700 （95%CI， 6900-45900）（5）。然而，因为缺乏质量较好的数据，非洲的狂犬病负担一直无法确定。在过去的十年中，该地区很少实施大规模犬疫苗接种项目，狂犬病仍然一直大范围流行。最近的调查显示在撒哈拉以南的非洲暴露后预防的可及性非常有限。深入研究发现，因为大多数死亡病例发生在社区而非医院（4,10），而且在医院就诊的病例常常被误诊为脑炎，因此官方报告可能低估狂犬病发生率 100 多倍（29）。经概率决策树方法校正 2010 年非洲狂犬病负担约有 23800 例死亡病例（95%CI， 21000-28000）， 609000DALYs （95%CI， 522000-707000）与早期的估计是一致的（5）。健康度量和评价机构的研究发现， 2010 年大约有 9500 人死于狂犬病（11），而 DALYs 则与之前的报告相似（750000； 95%CI， 169000-2733000）。由于缺乏验证，这些数据应该谨慎使用，并且应在该地区进行进一步的调查。神经组织疫苗在埃塞俄比亚仍然广泛使用，因此每年导致大约 1000DALYs。阿尔及利亚仍然在生产神经组织疫苗，而北非和非洲之角的其他国家的状况尚不清楚。 - 5 -

中东或中亚的狂犬病信息很少，之前没有调查过这些地区的狂犬病负担。采用概率决策树模型基于文献和人口数据，初步估计中东有 350 人死亡（95%CI， 270-450）和 13100DALYs （95%CI， 11100-15900），中亚有 1900 人死亡（95%CI， 1600-2350）和 55200DALYs （95%CI， 47500-66600）。

在拉丁美洲和加勒比海，大多数受吸血蝙蝠狂犬病病毒感染的病例被漏报。在 1985 年，估计每年牛的死亡总数为 100000，估计每年的价值为 3 千万美元。然而有证据表明蝙蝠狂犬病的发生率已经提高，这可能会导致更多的人类病例和牲畜损失（30）。

全球每年狂犬病死亡人数在 2010 年估计为 26400 （95%CI， 15200-45200）（ “死亡原因集成” 模型方法）至 61000 （95%CI， 37000-86000）（概率决策树方法）之间（表 2）。死亡病例的绝大多数（84%）发生在农村地区。估计总共有 190 万（95%CI， 130-260 万） DALYs。大约 12600DALYs 是由神经组织疫苗产生的副反应引起。每年狂犬病的花费估计达到 60 亿美元（95%CI， 46-73 亿美元），其中约有 20 亿美元（~40%）是由于过早死亡引起的生产力损失，另有 16 亿美元直接花费于暴露后预防。虽然对于被忽视的热带病负担的估测仍存在较大争议，但由于狂犬病引起的直接死亡率的估测仍处于最高之中（可能是最高的），狂犬病导致的 DALYs 也很高（31）。挽救生命的预防花费无论对国家经济或对贫困家庭都是沉重的负担，像中国（例如： 2010 年报告有 1 千万病例进行了暴露后预防治疗）和印度自 2004 年以来每年都能获得越来越多的关于暴露后预防数量增加的数据，这些数据表明即使大部分患者并非来源于狂犬病动物，但人群感染狂犬病的风险更高了。被疑似患有狂犬病的犬咬伤之后所带来的创伤和恐惧的心理影响很难用金钱来衡量，但是据估计在非洲导致 32000DALYs，亚洲为 140000DALYs （32）。在很多具有犬狂犬病地方性流行的国家，暴露后预防的可及性、质量和支付能力的不确定性加重了这些影响。监测能力不足、许多发展中国家的漏报、频繁发生的狂犬病误诊（29）以及缺乏所有相关部门之间的协调等均会导致这种疾病负担程度的低估。应该鼓励特定国家的疾病负担研究和改进监测（见 11 节）从而获得更多可靠的全球狂犬病负担估测数据。即便如此，狂犬病不同程度影响着贫困的农村社区，尤其是儿童，是显而易见的。暴露后 - 6 -

世界卫生组织狂犬病专家磋商会

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预防的大部分费用是由这些支付能力最差的人负担的。例如，在印度，病人大约需要支付狂犬病经济负担的一半。之前的估测显示，暴露后预防的全程治疗费用在亚洲占人均国民总收入的 3.87%，在非洲占 5.80% （相当于一个非洲公民平均 51 天的工资，一个亚洲公民平均 31 天的工资）。坦桑尼亚联合共和国最近的一个现场调研发现，即使这样，这些估测值仍然显著低于高危人群的实际花费。结果，许多病人没有完成全程治疗并常采用未被推荐的治疗方法。狂犬病每年导致的牲畜损失也是巨大的：大约 1230 万美元（90%CI， 1100 万 -1370 万），不同程度地影响着靠牲畜谋生的农村贫穷人群。随着犬数量和人口数量的持续增长，如果缺乏控制狂犬病的一致努力和投入，人狂犬病死亡的负担和经济费用将会持续增加。狂犬病是完全可以预防的。如果国家致力于减少人死亡数并改进暴露后预防的可及性，投入将会增加；然而，通过大规模的犬疫苗免疫实现了犬狂犬病的控制和最终消除，暴露后预防的需求和费用都会降低。国家疫苗接种项目需要一致、持久保障，这样会产生广泛的健康效益，尤其对于世界最贫穷的地区。

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1. Murray CJL et al. Summary measures of population health: concepts, ethics,measurements, and applications. Geneva, World Health Organization,2002. 2. Stein C et al. The global burden of disease assessments-who is responsible PLoS Neglected Tropical Diseases, 2007, 1(3):e161. 3. Essential rabies maps. Geneva, World Health Organization bies_maps/en/; accessed March 2013). (http://www. who.int/rabies/ra-

4. Cleaveland S et al. Estimating human rabies mortality in the United Republic of Tanzania from dog bite injuries. Bulletin of the World Health Organization, 2002, 80(4):304-310. 5. Knobel DL et al. Re-evaluating the burden of rabies in Africa and Asia. Bulletin of the World Health Organization, 2005, 83(5):360-368. 6. Tenzin et al. Dog bites in humans and estimating human rabies mortality in rabies endemic areas of Bhutan. PLoS Neglected Tropical Diseases,2011, 5(11):e1391. 7. Ly S et al. Rabies situation in Cambodia. PLoS Neglected Tropical Diseases,2009, 3(9):e511. 8. Hossain M et al. Human rabies in rural Bangladesh. Epidemiology and Infection, 2012, 140(11): 1964-1971. 9. Suraweera W et al. Deaths from symptomatically identifiable furious rabies in India: a nationally representative mortality survey. PLoS Neglected Tropical Diseases, 2012, 6(10):e1847. 10. Hampson K et al. Rabies exposures, post-exposure prophylaxis and deaths in a region of endemic canine rabies. PLoS Neglected Tropical Diseases, 2008, 2(11):e339. 11. Lozano R et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet, 2012, 380 (9859):2095-2128. 12. Murray CJL et al. Disability-adjusted life years (DALYs) for 291 diseases and injuries in 21 regions, 1990-2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet, 380 (9859):2197-2223.

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13. Gautret P, Parola P. Rabies vaccination for international travelers. Vaccine,2012, 30(2):126-133. 14. Malerczyk C, DeTora L, Gniel D. Imported human rabies cases in Europe, the United States, and Japan, 1990 to 2010. Journal of Travel Medicine, 2011, 18:402-407. 15. Lardon Zl et al. Imported episodic rabies increases patient demand for and physician delivery of antirabies prophylaxis. PLoS Neglected Tropical Diseases, 2010, 4(6):e723. 16. Sterner RT et al. Tactics and economics of wildlife oral rabies vaccination, Canada and the United States. Emerging Infectious Diseases, 2009,15(8):1176-1184. 17. Blanton JD et al. Rabies surveillance in the United States during 2010. Journal of the American Veterinary Medicine Association, 2011,239(6):773-783. 18. Aubert MF. Costs and benefits of rabies control in wildlife in France.Revue Scientifique et Technique (International Office of Epizootics), 1999,18(2):533-543. 19. Müller T et al. Elimination of terrestrial rabies in Germany using oral vaccination of foxes. Berliner und Munchener tierarztliche Wochenschrift, 2012, 125(5-6):178-190. 20. Cliquet F et al. Eliminating rabies in Estonia. PLoS Neglected Tropical Diseases, 2012, 6 (2): e1535. 21. Demetriou P, Moynagh J. The European Union strategy for external cooperation with neighbouring countries on rabies control. Rabies Bulletin Europe, 2011, 35(1):5-7. 22. Sistema de Información Epidemiológica. Washington DC, Pan American Health Organization and World Health Organization. (http://siepi.panaftosa.org.br; accessed March 2013). 23. Elimination of neglected diseases and other poverty-related infections. Pan American Health Organization and World Health Organization. 49th Directing Council. 61st session of the Regional Committee. Washington DC, 2009 [resolution CD49.R19]. (http://new.paho.org/hq/dmdocuments/2009/CD49.R19%20(Eng.).pdf; accessed March 2013). 24. Interagency meeting on planning the prevention and control of neglected zoonotic diseases, Geneva, 5-6 July 2011. Geneva, World Health Organization, 2011 (WHO/HTM/NTD/NZD/2011; also available at whqlibdoc.who.int/publications/2011/9789241502931_eng.pdf;accessed March 2013). 25. Strategic framework for elimination of human rabies transmitted by dogs in the South-East Asia - 10 -

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Region. New Delhi, WHO Regional Office for South-East Asia, 2012 (http://www.searo.who.int/topics/rabies/en/;accessed March 2013). 26. Sudarshan MK et al. Assessing the burden of human rabies in India: results of a national multi-center epidemiological survey. International Journal of Infectious Diseases, 2007, 11(1):29-35. 27. Yu J et al. The spatial and temporal dynamics of rabies in China. PLoS Neglected Tropical Diseases, 2012, 6(5):e1640. 28. Yin C-P. Analysis on factors related to rabies epidemic in China from 2007-2011. Virologica Sinica, 2012, 27(2):132-143. 29. Mallewa M et al. Rabies encephalitis in malaria-endemic area, Malawi,Africa. Emerging Infectious Diseases, 2007, 13(1):136-139. 30. Streicker DG et al. Ecological and anthropogenic drivers of rabies exposure in vampire bats: implications for transmission and control.Proceedings of the Royal Society B. Biological Sciences, 2012,279(1742):3384-3392. 31. Mathers CD, Ezzati M, Lopez AD. Measuring the burden of neglected tropical diseases: the Global Burden of Disease Framework. PLoS Neglected Tropical Diseases, 2007, 1(2):e114. 32. WHO Expert Consultation on Rabies. First report. Geneva, World Health Organization, 2005 (WHO Technical Report Series, No. 931).

2．狂犬病病毒的分类

狂犬病是一种由狂犬病病毒感染引起的急性脑炎或脑膜脑炎。狂犬病脑炎的病原体属于单股负链病毒（Mononegavirales）目、弹状病毒（Rhabdoviridae）科、狂犬病病毒（Lyssavirus）属。狂犬病病毒有一个长 12kb 不分节段负链 RNA 基因组，编码 5 种病毒蛋白（3’ 到 5’ ）：核蛋白（N）、磷蛋白（P）、基质蛋白（M）、糖蛋白（G）和 RNA 依赖的 RNA 聚合酶（或大蛋白， L）。狂犬病病毒颗粒为子弹形状，长 100-300nm，直径 75nm。狂犬病病毒由 2 个结构和功能单位组成：一个内部螺旋状排列的核壳体和一个外部包膜。核壳体由包含基因组 RNA 及紧密盘绕的 N 蛋白与 L 和 P 蛋白一起构成的核蛋白复合体组成。核壳体负责转录和翻译： N-RNA 模板由 L 蛋白及其辅助因子 P 蛋白进行加工， L 蛋白包含大部分 RNA 聚合酶活性。脂质膜在出芽过程中从宿主细胞质膜获得。圆形糖蛋白纤突（5-10nm 长，直径约 3nm）由三个糖基 - 11 -

化的外部功能区组成，突起于病毒膜，连接病毒和宿主细胞受体。M 蛋白形成的低聚物结合在核衣壳的外侧，保持病毒结构的硬度并提供给病毒糖蛋白及其囊膜一个结合平台（1,2）。

直到二十世纪五十年代，狂犬病病毒一直被认为是唯一的。通过对来自尼日利亚的血清学相关病毒的鉴定—狐蝠科蝙蝠的 Lagos 蝙蝠病毒（3）和鼩鼱分离到的 Mokola 病毒（4） — —— 发现狂犬病病毒群的结构更加复杂，由此出现了狂犬病相关病毒和狂犬病血清群的术语（4）。另外一种血清学相关的病毒， Duvenhage 病毒，是 1970 年在南非被食虫蝙蝠咬伤后死于狂犬病的一个病人身上分离到的（5），代表第四种血清型。从 1950 年以来，在欧洲规律性地从蝙蝠分离到的病毒与 Duvenhage 病毒血清学相关并且最初归类为 Duvenhage 血清型（6,7）。此后，单克隆抗体的应用使狂犬病血清型的分类可以细化（8）。欧洲蝙蝠狂犬病病毒不仅与非洲 Duvenhage 病毒相互区别（9），并进一步被分成两个独特的血清型（10），暂时称为 “生物型” （11）。这种区分随后得到基因测序和种系进化分析的支持（12,13）。狂犬病相关病毒多样性的广泛种系进化研究产生了新的专业术语“基因型” ，自此基因型广泛应用于科学文献中（12）。新基因型得到鉴定，有关基因型鉴别的定量标准也随之产生（12,14-18）。为了适应越来越多的狂犬病相关病毒，国际病毒分类委员会主持设立了狂犬病病毒属。属的名称来自于希腊神话： Lyssa(Λυσσα)是一个女神，或者疯狂和愤怒的幽灵。现存的基因型作为狂犬病病毒分类的基础，限定于符合国际委员会的正式规则，应用于更复杂的诸如病毒种类的本质。狂犬病病毒种类的界定标准包括（19）：遗传距离，完整 N 基因核苷酸序列同源性达到 80-82% 的临界值，与其他基因相比较可以提供更好的定量结论，或者是 N+P+M+G+L 基因的联合编码区核苷酸序列有 80-81%的同源性。一般来说，所有属于同一种类的分离物均较阈值有更高的同源性，除了目前归在 Lagos 蝙蝠病毒种的病毒以外。因此，一些作者建议将 Lagos 蝙蝠病毒进一步分成几种基因型（20,21）。然而，由于缺少其他足够的界定特征， Lagos 蝙蝠病毒仍然没有分成为几个种类，尽管这些代表株在大多数种系进化重建的过程中分化成为一个单系簇。用各种进化模型获得的拓扑结构和种系进化树的一致性。用核衣壳蛋白单克隆抗体反应的抗原模式（来源于血清学交叉反应和多克隆抗血清的狂犬病病毒血清型定义）。 - 12 -

世界卫生组织狂犬病专家磋商会

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在可应用的情况下，额外的特性，例如生态特性、宿主、地理范围和病理特征等。

目前，国际病毒分类委员会明确了 12 种狂犬病病毒（表 3）。根据遗传距离和血清学交叉反应特性，该病毒属分为两个遗传系谱。系谱Ⅰ包括狂犬病病毒、欧洲蝙蝠狂犬病病毒 1 型和 2 型、 Duvenhage 病毒、澳大利亚蝙蝠狂犬病病毒、 Aravan 病毒、 Khujand 病毒和 Irkut 病毒。系谱Ⅱ包括 Lagos 蝙蝠病毒、 Mokola 病毒和 Shimoni 蝙蝠病毒。其余基因种类，如 West Caucasian 蝙蝠病毒，不能归入这两类系谱中的任何一个，有专家建议可以考虑将其做为独立的系谱Ⅲ的代表。该病毒种类进一步可能的延伸，一种新的 Bokeloh 蝙蝠狂犬病病毒最近从法国和德国的食虫蝙蝠（Myotis nattereri）中分离到。这种病毒在种系进化方面与欧洲蝙蝠狂犬病病毒 2 型和 Khujand 病毒相关（17， 22）。另外一种不同的狂犬病病毒，与 West Caucasian 蝙蝠病毒（有可能是所提议的系谱Ⅲ 中的一员）种系进化相关，暂时命名为 Ikoma 狂犬病病毒，在坦桑尼亚共和国的非洲灵猫身上发现（18）。蝙蝠是目前已经认识到的 14 种狂犬病病毒中的 12 种病毒的宿主和传播媒介，而 Mokola 病毒和 Ikoma 狂犬病病毒的宿主仍然不能确定。狂犬病病毒在核壳体水平上表现出广泛的抗原交叉反应，其主要原因是 N 蛋白序列的保守性。因此，可以用相似的试剂进行免疫荧光诊断。G 蛋白的外功能区（携带主要抗原位点）更容易变异，同一系谱（胞外区氨基酸同源性， >74%）的狂犬病病毒之间存在交叉中和作用，而不同系谱（胞外区氨基酸同源性， <62%）的病毒之间则没有此作用。实验证明显示目前应用的疫苗株均属于系谱Ⅰ，对系谱Ⅱ的狂犬病病毒和 West Caucasian 蝙蝠病毒的感染起不到保护作用，对 Ikoma 狂犬病病毒的感染可能也同样缺乏保护作用。

1. Graham SC et al. Rhabdovirus matrix protein structures reveal a novel mode of self-association. PLoS Pathogens, 2008, 4:e1000251. 2. Ge P et al. Cryo-EM model of the bullet-shaped vesicular stomatitis virus. Science, 2010, 327: 689-693. - 13 -

3. Boulger LR, Porterfield JS. Isolation of a virus from Nigerian fruit bats.Transactions of the Royal Society of Tropical Medicine and Hygiene, 1958,52:421-424. 4. Shope RE et al. Two African viruses serologically and morphologically related to rabies virus. Journal of Virology, 1970, 6:690-692. 5. Meredith CD, Rossouw AP, van Praag Koch H. An unusual case of human rabies thought to be of chiropteran origin. South African Medical Journal, 1971, 45:767-769. 6. Schneider LG. Antigenic variants of rabies virus. Comparative Immunology, Microbiology and Infectious Diseases, 1982, 5:101-107. 7. Schneider LG, Barnard BJH, Schneider HP. Application of monoclonal antibodies for epidemiological investigations and oral vaccination studies: I. African viruses. In: Kuwert E et al., eds, Rabies in the tropics.Berlin, Springer-Verlag, 1985:49-53. 8. Wiktor TJ, Koprowski H. Monoclonal antibodies against rabies virus produced by somatic cell hybridization: detection of antigenic variants. Proceedings of the National Academy of Sciences of the United States of America, 1978, 75:3938-3942. 9. Dietzschold B et al. Antigenic diversity of the glycoprotein and nucleocapsid proteins of rabies and rabies-related viruses: implications for epidemiology and control of rabies. Reviews of Infectious Diseases,1988, 10(S4):785-798 10. Bourhy H et al. Antigenic and molecular characterization of bat rabies virus in Europe. Journal of Clinical Microbiology, 1992, 30:2419-2426. 11. King A, Davis P, Lawrie A. The rabies viruses of bats. Veterinary Microbiology, 1990, 23:165174. 12. Bourhy H, Kissi B, Tordo N. Molecular diversity of the Lyssavirus genus.Virology, 1993, 194: 70-81. 13. Davis PL et al. Phylogeography, population dynamics, and molecular evolution of European bat lyssaviruses. Journal of Virology, 2005,79:10487-10497. 14. Fraser GC et al. Encephalitis caused by a lyssavirus in fruit bats in Australia. Emerging Infectious Diseases, 1996, 2:327-331.

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世界卫生组织狂犬病专家磋商会

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已经认识到的和提议的种类（简称）狂犬病病毒（RABV）

初始宿主

地理范围陆地哺乳动物分布在世界上除澳大利亚、南极洲和几个岛屿之外的地域；仅在新大陆的蝙蝠

注释

食肉目和蝙蝠（翼手目）

狐蝠科蝙蝠（至少有四种狐蝠澳大利亚蝙蝠狂犬澳大利亚（可能包括几个临近属蝙蝠）和食虫蝙蝠（囊喉墓病病毒（ABLV）的岛屿）蝠属）欧洲蝙蝠狂犬病病食虫蝙蝠（主要是 Eptesicus 欧洲大部，从西班牙到乌克兰毒， 1型（EBL1） serotinus）欧洲蝙蝠狂犬病病食虫蝙蝠（主要是 Myotis 毒， 2型（EBL2） daubentonii and M. dasycneme） Khujand 病毒（KHUV） Aravan 病毒（ARAV）食虫蝙蝠须鼠耳蝠食虫蝙蝠须鼠耳蝠中亚中亚法国，德国东亚撒哈拉以南的非洲欧洲西北部

5 6 7 8 9

Bokeloh 蝙蝠狂犬病食虫蝙蝠纳氏鼠耳蝠病（BBLV） Irkut 病毒（IRKV） Duvenhage 病毒（DUVV） Lagos 蝙蝠病毒（LBV）食虫蝙蝠白腹管鼻蝠食虫蝙蝠

几个属（比如 Eidolon helvum, Rousettus aegyptiacus, 撒哈拉以南的非洲 Epomophorusspp）的狐蝠科蝙蝠撒哈拉以南的非洲肯尼亚欧洲东南部坦桑尼亚联合共和国

Mokola 病毒（ MOKV ）未知 Shimoni 蝙蝠病毒食虫蝙蝠康氏蝠属（SHIBV） West Caucasian 蝙蝠长翼蝠属的食虫蝙蝠病毒（WCBV） Ikoma 狂犬病病毒（IKOV）未知

11 12 13 14 - 15 -

1.导致世界上绝大多数人狂犬病病例。所有目前使用的人用和兽用狂犬病疫苗株均源自该种病毒。 2.由于有限的监测，在食虫蝙蝠中的宿主范围有可能会更大。已有两例人类病例的记录。 3. 仅在东欧和亚洲地区有限的监测，按照贮存宿主种类的范围，实际分布范围可能更广。野生动物和伴侣动物中的溢出感染以及极少数量人类的病例已有文献记载。 4.已有两个人类病例的文献记录。 5.已知来源于一单独的分离株。在东欧和亚洲地区实施有限的监测，实际分布范围可能更广。尚无人类病例记录。 6.已知来自两个分离株。在东欧和亚洲地区实施有限的监测，实际分布范围可能更广。尚无人类病例记录。 7.已知来自一单独的分离株。不具有物种形态且没有列在现有国际病毒分类委员会文件列表中。尚无人类病例记录。 8.发现了两个分离株，分别分离自蝙蝠和人类。 9.已知有四个分离株，其中三个分离自被蝙蝠咬伤的病人，另一个分离自蝙蝠，推测为长翼蝙蝠属。 10.由几个遗传距离较远的种系组成。未来，可能细分为两个或三个独立的种类。在野生动物和伴侣动物中有溢出感染的报告。迄今尚无人类病例文献记录。 11.两次分离自鼩鼱，一次分离自啮齿类动物。其他分离株大多来自伴侣动物，如猫，是溢出感染的结果。已有两例人类病例的报告。 12.发现于一个单独的分离株。血清学调查提示 H. commersoni 是可能的贮存宿主。没有人类病例文献记录。 13.发现于一个单独的分离株，然而，血清学调查提示西高加索蝙蝠病毒（或另一种血清学相关病毒）存在于非洲（肯尼亚）长翼蝙蝠属。尚无人类病例文献记录。 14.已知从一个非洲麝猫（非洲灵猫）的单独分离株。自然宿主尚不清楚。与西高加索蝙蝠病毒系谱相关，在非洲麝猫中的个例有可能是蝙蝠来源的溢出感染导致的结果。尚无人类病例文献记录。 15. Kuzmin IV et al. Bat lyssaviruses (Aravan and Khujand) from Central Asia: phylogenetic relationships according to N, P and G gene sequences.Virus Research, 2003, 97:65-79. 16. Kuzmin IV et al. Phylogenetic relationships of Irkut and West Caucasian bat viruses within the Lyssavirus genus and suggested quantitative criteria based on the N gene sequence for lyssavirus genotype definition. Virus Research, 2005, 111:28-43. 17. Freuling C et al. Novel lyssavirus in a Natterer’ s bat (Myotis nattereri),Germany. Emerging Infectious Diseases, 2011, 17:1519-1522. - 16 -

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18. Marston DA et al. Ikoma lyssavirus: identification of a highly divergent novel lyssavirus in an African civet (Civettictis civetta). Emerging Infectious Diseases, 2012, 18:664-667. 19. Dietzgen RG et al. Family Rhabdoviridae. In: King AMQ et al., eds. Virus taxonomy: ninth report of the International Committee on Taxonomy of Viruses. Oxford, Elsevier, 2011:686-714. 20. Delmas O et al. Genomic diversity and evolution of the lyssaviruses.PLoS One, 2008, 3:e20-57. 21. Markotter W et al. Phylogeny of Lagos bat virus: challenges for lyssavirus taxonomy. Virus Research, 2008, 135:10-21. 22. Picard-Meyer E et al. Découverte d’ une chauve-souris de Natterer infectée par un lyssavirus Bokeloh en Moselle en 2012. Bulletin Epidémiologique Santé animale, Alimentation, 2012, 55:25 (http://www.anses.fr/bulletin-epidemiologique/).

3. 发病机理狂犬病病毒通过伤口或与粘膜表面直接接触而进入体内，但病毒不能穿过没有损伤的皮肤。狂犬病病毒在咬伤部位的肌肉组织中复制，然后通过运动神经元的终板和轴突到达中枢神经系统(1-5)。病毒粒子以运输小泡为载体，快速地沿运动轴突以独有的逆向运输方式到达中枢神经系统，而不被感觉或交感神经末梢所摄取(1-3,5)。病毒也可以通过较深的伤口直接进入周围神经的运动神经元轴突(1,3,4)。在一些蝙蝠变异株中，由于皮肤嗜性病毒繁殖也可以发生在感觉神经(3,7,8)。根据侵入的病毒量、受伤部位运动神经元终板的密度以及病毒接近中枢神经系统的距离，病毒潜伏期从 5 天到几年不等（一般为 2-3 个月，很少超过 1 年） (3-5)。肌肉特异性小 RNA 可能通过抑制病毒在肌肉中的转录和复制影响潜伏期(9,10)。估测的病毒移动速度取决于病毒是向心性的逆向轴突运输或者离心扩散。在向心性逆向轴突运输中，移动较快，约为 5-100mm/ 每天，甚至更快，其原因是处于不同距离的，例如 10μm 至 2cm，具有相同突触排列的神经元群会同时被感染（1,5）。与之相反，离心性扩散的速度会慢，可能是缘于病毒为被动扩散而非主动扩散（1-3,5）。第一阶段的向心移动会导致病毒在中枢神经系统内广泛的跨神经元穿行并且通过最初感染的运动神经元与脊髓中间神经元之间的中央连接感染背根神经节（1-3,5）。病毒然后从中枢神经系统离心性移动缓慢通过在运动轴突的顺向轴浆流动进入腹侧根和神经以及在感染的背根神经节周围的感觉轴突，从而通过这些感觉神经感染肌梭、皮肤、毛囊以及其他非神经组织，如唾液腺、心肌、肺和腹部内脏器官（3-5）。到临床发病时，病毒已经广泛分布于中枢神经系统以及神经以外的器官中（11）。 - 17 -

临床初始特异性症状是在咬伤的部位有神经性疼痛。这种情况是由于病毒在背根神经节复制和细胞免疫引发的炎症造成的（12）。人狂犬病症状可以表现为狂躁型或麻痹型，并且两者均不能与狂犬病病毒在中枢神经系统内的解剖定位相关联（12,14）。最主要的临床体征可能是由不同的位点特异性反应引起（14）。神经功能损伤也解释了昏迷症状的发生。电生理学方法研究病理机制发现麻痹型狂犬病的虚弱无力与外周神经轴突病变或者脑白质变性有关。病毒优先侵入运动神经元途径解释了为什么狂躁型狂犬病前角细胞功能障碍引起的亚临床症状要先于感觉丧失（7,12），并且最初发生在与咬伤部位相对应的躯体部分，渐渐扩展到其他部位（3， 5,12）。同样的考虑也可以解释麻痹型狂犬病人的前兆性症状和体征（3,5）。也有可能是麻痹型狂犬病人（意识清醒的时候）脑中存在的病毒要少于狂躁型狂犬病患者。犬类麻痹型狂犬病的弥散张量成像显示脑干部位神经束的完整性受损，影响了病毒向前脑的传播（5,15,16）。病毒免疫逃避策略加之血脑屏障的完整性阻碍了中枢神经系统中病毒的清除（4,16-21）。在狂犬病感染的病人中没有因免疫抑制或加强而死亡的证据（15,16）。现在对于具有非典型临床症状或者神经影像学特征的狂犬病人的了解越来越多（4,22-26）。然而这些是因为非典型的病毒变异株，还是宿主免疫反应或是大剂量病毒接种（例如在狂犬病感染者作为供体的器官移植病例中）造成的尚不清楚。如果没有重症监护，病人在出现临床症状 2 周内发生死亡（5,7）。

1. Ugolini G. Use of rabies virus as a transneuronal tracer of neuronal connections: implications for the understanding of rabies pathogenesis. Developments in Biologicals (Basel), 2008, 131:493-506. 2. Ugolini G. Advances in viral transneuronal tracing. Journal of Neuroscience Methods, 2010, 194:2-20. 3. Ugolini G. Rabies virus as a transneuronal tracer of neuronal connections. Advances in Virus Research, 2011, 79:165-202. 4. Hemachudha T, Laothamatas J, Rupprecht CE. Human rabies: a disease of complex neuropathogenetic mechanisms and diagnostic challenges. Lancet Neurology, 2002, 1(2):101-109. 5. Hemachudha T et al. Human rabies: neuropathogenesis, diagnosis and management. Lancet Neurology, 2013, 12(5):498-513. 6. Klingen Y, Conzelmann KK, Finke S. Double-labeled rabies virus: live tracking of enveloped virus transport. Journal of Virology, 2008,82(1):237-245. 7. Hemachudha T et al. Pathophysiology of human paralytic rabies. Journal of Neurovirology, 2005, 11(1):93-100. - 18 -

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8. Morimoto K et al. Characterization of a unique variant of bat rabies virus responsible for newly emerging human cases in North America. Proceedings of the National Academy of Sciences of the United States of America, 1996, 93 (11): 5653- 5658. 9. Israsena N et al. Inhibition of rabies virus replication by multiple artificial microRNAs. Antiviral Research, 2009, 84(1):76-83. 10. Israsena N, Mahavihakanont A, Hemachudha T. Rabies virus infection and microRNAs. Advances in Virus Research, 2011, 79:329-344. 11. Hemachudha T et al. Rabies. Current Neurology and Neuroscience Reports, 2006, 6 (6):460468. 12. Mitrabhakdi E et al. Difference in neuropathogenetic mechanisms in human furious and paralytic rabies. Journal of Neurological Science, 2005,238(1-2):3-10. 13. Dumrongphol H et al. Alteration of muscarinic acetylcholine receptors in rabies viral-infected dog brains. Journal of Neurological Science, 1996,137(1):1-6. 14. Thanomsridetchai N et al. Comprehensive proteome analysis of hippocampus, brainstem, and spinal cord from paralytic and furious dogs naturally infected with rabies. Journal of Proteome Research, 2011,10(11):4911-4924. 15. Laothamatas J et al. Furious and paralytic rabies of canine origin:neuroimaging with virological and cytokine studies. Journal of Neurovirology, 2008, 14(2):119-129. 16. Laothamatas J, Sungkarat W, Hemachudha T. Neuroimaging in rabies.Advances in Virus Research, 2011, 79:309-327. 17. Lafon M. Evasive strategies in rabies virus infection. Advances in Virus Research, 2011, 79:33-53. 18. Laothamatas J et al. MR imaging in human rabies. American Journal of Neuroradiology, 2003, 24 (6):1102-1109. 19. Roy A et al. Failure to open the blood brain barrier and deliver immune effectors to central nervous system tissues leads to the lethal outcome of silver-haired bat rabies virus infection. Journal of Virology, 2007,81(3):1110-1118. - 19 -

20. Roy A, Hooper DC. Immune evasion by rabies viruses through the maintenance of blood-brain barrier integrity. Journal of Neurovirology, 2008, 14(5):401-411. 21. Kasempimolporn S et al. Human immune response to rabies nucleocapsid and glycoprotein antigens. Clinical and Experimental Immunology, 1991,84(2):195-199. 22. Hemachudha T, Phuapradit P. Rabies. Current Opinions in Neurology,1997, 10(3):260-267. 23. Burton EC et al. Rabies encephalomyelitis: clinical, neuroradiological,and pathological findings in 4 transplant recipients. Archives of Neurology,2005, 62(6):873-882. 24. Maier T et al. Management and outcomes after multiple corneal and solid organ transplantations from a donor infected with rabies virus. Clinical Infectious Diseases, 2010, 50(8):1112-1119. 25. Shantavasinkul P et al. Failure of rabies postexposure prophylaxis in patients presenting with unusual manifestations. Clinical Infectious Diseases, 2010, 50(1):77-79. 26. Human rabies-Minnesota, 2007. Morbidity and Mortality Weekly Report, 2008, 57(17):460-462.

4.诊断狂犬病是由狂犬病病毒引起的一种急性、进行性脑炎。脑炎的临床诊断具有挑战性，在可能的情况下，所有可能或者疑似的临床狂犬病病例都要用实验室方法进行确诊。在过去的十年间，用于临床病例确认的实验室诊断技术取得了显著的进步。每个国家都应该有一个具有应用推荐的现代技术进行初步狂犬病诊断和病例确认能力的国家参比实验室（1-7）。缺乏专业知识的地区可以向 WHO 合作中心（8）（附件 8）以及世界动物卫生组织（OIE）的参比中心寻求培训以及可参照的诊断能力（9）。

所有国家均应使用基于对人类和动物狂犬病可疑病例实验室监测基础上的狂犬病标准病例定义。根据 WHO 所推荐的针对传染病的监测、预防、控制的标准和策略，狂犬病的临床病例定义如下：病例具有急性神经性综合征（如脑炎），主要表现为机能亢奋（如狂躁型狂犬病）或者麻痹综合征（如麻痹型狂犬病），如果没有得到重症监护治疗，病人通常会在首发症状出现后 7-11 天内进行性发展为昏迷和死亡，通常是由于心脏或呼吸衰竭所致。 - 20 -

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在进行临床病例确认时应使用下列实验室标准中的 1 种或几种： ■ 存在病毒抗原； ■ 细胞培养方法或实验动物接种中分离到病毒； ■ 未接种疫苗者的脑脊液或血清中存在病毒特异性抗体； ■ 通过分子生物学方法在尸检或活检样本（如脑活检样本、皮肤、唾液、浓缩尿）中检测到病毒核酸。狂犬病病例基本分类如下： ■ 疑似病例：符合临床病例定义的病例； ■ 可能病例：疑似病例，同时具有与疑似狂犬病动物接触的可靠病史； ■ 确认病例：实验室确认的疑似病例或可能病例。有些情况下，可以缺少临床疑似脑炎症状或动物暴露史，但适当的实验室诊断检测仍然是病例确认所需要的。可能的狂犬病暴露记录表格见附件 2。

如果表现为急性进行性脑脊髓炎，并且在所有传染病中病死率最高，又具有文件证明是暴露于实验室确诊的狂犬病动物后出现相关症状的病人，推定狂犬病的诊断并不难。如果有明确暴露史，对于出现特征性恐水或恐风症状的患者，应高度怀疑为狂犬病。如果没有明确暴露史和典型临床症状，单从临床表现诊断狂犬病是困难的并且通常也是不可靠的。例如，某些患者可以表现为麻痹或与格林 - 巴利综合征相似的症状，或者出现其他非典型特征（10）。非典型或者非经典的狂犬病越来越多地被认识，这种情况可能是造成狂犬病漏报的原因。现今已有关于非典型狂犬病病例，特别是与蝙蝠或者其它野生动物暴露有关的病例，的详细临床信息报告（11,12）。人病例报告可以从众多同行评议类出版物、国家和国际的报告以及电子来源信息，例如美国疾病预防控制中心（美国 CDC）网站，查阅获得。脑部受累的典型症状包括对触觉、听觉、视觉或嗅觉的刺激有痉挛反应（如怕风和恐水），交替出现周期性清醒、烦躁、错觉和自主神经功能障碍（10）。痉挛会发生在主要表现为兴奋的狂犬病患者中。自发性的呼吸痉挛会持续发生直至死亡，这些表现常常可以帮助诊断。麻痹型狂犬病很少出现兴奋，这样的病人中仅有 50%会出现恐惧性痉挛。麻痹型狂犬病的早期阶段，典型体征包括在叩诊部位的肌水肿，通常在胸部、三角肌和大腿，毛发竖立和肌束震颤。 - 21 -

对可能感染狂犬病的患者在采取适当预防措施情况下进行磁共振成像检查可能有助于诊断（10,13）。无论临床类型如何，当脑干、海马、下丘脑、深层和皮层下白质以及深层和皮质灰质的 T2 成像出现模糊、微弱的异常高信号时提示狂犬病。疾病晚期，当患者进入昏迷状态时，钆增强可以清楚地显示。当比较意识状态时，这些特征可以用来将狂犬病与其它病毒性脑炎相区别，不仅仅从位置上、还从 T2 成像的表现以及对比增强等方面。脑部 CT 几乎没有诊断价值。狂犬病应该包括在所有不明原因的急性、进行性病毒性脑炎病人的鉴别诊断中，即便是在狂犬病罕见的地区，因为狂犬病可以在当地的野生动物中发生，如蝙蝠，因此有可能在去地方性动物流行的地旅行时感染；而且人类及动物的输入性狂犬病病例持续存在（2,12）。此外，在没有足够的流行病学调查和实验室确认的情况下，狂犬病可能被误诊并且死亡被归于另一原因（如脑型疟疾）（2,4,14）。鉴于已有通过接受实体器官移植感染狂犬病病毒的报告，所有具有脑炎表现的可能捐献器官者均应采集生前或死后样本通过敏感、特异的实验方法进行筛查和检测以确定其是否具有传染风险（2,4,6）。

狂犬病是目前已经认识到的所有传染病中病死率最高的。因此，安全性对于从事狂犬病病毒的工作至关重要。一般来说，生物安全 II 级实验室的安全操作适用于动物处理、尸体解剖、样本采集、准备、处理等常规的实验室活动（5-7）。除了符合基本设施要求之外，预防措施还包括个人防护设备（如服装、手套、护目镜）和疫苗接种。在某些情况下，如生产大量浓缩病毒、进行可能产生气溶胶的操作（例如组织悬液匀化）、操作尚不清楚当前的预防措施是否具有保护效果的新分离狂犬病病毒时，可考虑采用生物安全 III 级。所有用于操作传染性病原的国家安全指南均应严格遵守。

分泌物、体液（唾液、脊髓液、泪液等）和组织（皮肤活体组织样本及后颈部毛囊）可用于存活狂犬病病人的诊断（1,2,5,6,15,16）。三份唾液样本取样需要间隔 3-6 小时，皮肤和毛囊是最敏感的样本。样本最好在 -20℃或 -20℃以下保存。血清应在冷冻前从采集的血液样本中分离并保存在 -20℃或 -20℃以下。

脑组织对于人和动物两者的死亡后诊断都是最佳样本（4,5,7）。如果脑组织切片检查无法进行，如在现场研究时，可通过眼眶或枕骨大孔途径采集脑组织样本（1）。脑组织保存在甘 - 22 -

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油中（+4℃或– 20℃）或涂片干燥后用含灭活剂的滤纸包裹（+30℃）可以作为感染性物质被安全稳定地转运，但是在装载前必须保证安全、有效的病毒灭活（1,17）。其他样本，如皮肤和颈背部毛囊，对于死后诊断也具有高度敏感性（5,6,10）。

用于狂犬病诊断的样本应按照国家和国际条例进行运送以避免暴露危险。相关包装分类信息可查阅国际航空运输协会网站（19），包装说明可查阅 WHO 有关感染性物质运输的建议（20）。用于诊断的样本应冷冻或冷藏，如果是在外界环境下进行运送，则应保存在 50% 甘油 - 生理盐水液中。用于诊断样本的来源和储存条件直接影响实验室操作的结果。狂犬病可以通过几种不同组织来源的新鲜（未固定）样本进行诊断，但样本最好冷藏或冷冻保存。如果样本保存在 50%甘油 - 生理盐溶液中，检测之前应彻底洗净，并且不推荐冷冻和长时间储存。与新鲜或冷冻组织的处理不同，在对保存在甘油生理盐溶液中的样本进行直接荧光抗体检测之前不建议丙酮固定。样本的选择和处理取决于要进行的检测试验和疾病的阶段（1,6）。如使用恰当的组织和检测方法，化学固定标本进行病毒抗原检测同样可以敏感性和特异性俱佳（21）。然而，福尔马林固定的脑组织不适于常规诊断，因为会延迟检测结果。如果收到用福尔马林固定的样本，在将其包埋入石蜡油之前要固定 7-14 天。湿的组织样本应由福尔马林转入无水乙醇用于随后的分子诊断和抗原检测。在固定的脑组织中通过有效的免疫组化法可以检测到神经元内典型的胞浆内包涵体（22）。

只有适当的实验室检测才能确诊狂犬病。基本检测技术可以参照 WHO 出版的《狂犬病实验室技术》（5）和 OIE 陆生动物诊断检测和疫苗的指南（7）。在使用任何实验室技术时均强烈建议执行常规的质量管理（6）。

直接荧光抗体技术是诊断动物和人类狂犬病的一种快速、敏感、特异的方法（5-7,23,24），也是狂犬病诊断的金标准。然而，该技术的准确性依赖于检测者的经验、抗狂犬病抗体结合物的质量以及基本设备，包括荧光显微镜。该检测是基于脑组织印片或涂片与异硫氰酸荧光素标记的抗狂犬病多克隆球蛋白或可广泛交叉反应的单克隆抗体结合物反应后的显微镜检测。诊断性结合物应是高质量的，并且需要确定病毒特异性抗原最佳显现和检测的适宜工作浓度。 - 23 -

脑干或小脑样本的组织印片（或涂片）因其检测的高敏感性而优先推荐。（6）。也可以使用海马（Ammon 角）但对于确诊并非必须。检测狂犬病病毒抗原的其他方法，如酶联免疫吸附试验（ELISAs）和直接快速免疫组化检测，在一些实验室也能得出一致的可重复的结果（6,25-28）。直接快速免疫组化检测的广泛评估表明其敏感性和特异性至少能够与直接荧光抗体试验，狂犬病诊断的传统标准，相媲美。这个检测可以使用光学显微镜进行快速的现场检测，并且如果其检测试剂可以商业化应用，该检测方法有助于进行分散的流行病学调查。本次会议推荐进一步发展直接快速免疫组化检测作为除直接荧光抗体检测以外的一种替代方法以改善分散的基于实验室的监测。在野外条件下进行的狂犬病病毒抗原快速检测的横向流动测试得到了发展（29-31），然而，这一商业化应用的检测方法尚未根据国际标准进行标准化或一致化的适当使用和充分验证（5）。

只有进行病毒分离才可以确认病毒抗原检测结果以及进一步的病毒扩增和明确特性（5）。病毒可以通过细胞培养分离，如神经瘤细胞，或通过小鼠颅内接种分离。只要有可能，动物接种分离病毒应该由其他可以替代的方法来取代。鼠神经瘤细胞（例如 NA1300）用于狂犬病病毒的现场分离要比其他测试过的细胞系更为敏感（5,6）。病毒通过神经瘤细胞培养分离至少与动物接种分离有相同的效率，特别是病毒量少的时候。细胞培养分离病毒还可以减少诊断所需要的时间，将小鼠接种试验所需的 10-21 天减少至仅 1-2 天。然而，如果条件并不适合，例如腐败的脑组织，也有可能得到假阴性结果。在细胞培养设备或分子方法不能应用时，可以使用动物接种进行分离。如果需要尽快得到结果，乳鼠（<3 日龄）比断乳期或成年的小鼠更好，因为乳鼠比年长的动物更加敏感。接种病毒后 14-21 天（或更长时间）或出现临床症状时的小鼠实施安乐死后再对其脑组织进行荧光抗体检测有可能缩短观察期。

分子生物学方法，如逆转录聚合酶链反应（PT-PCR）和其他扩增技术，在许多国家发挥着越来越重要的作用，但如果能得到脑组织则在目前不推荐用这些方法进行狂犬病的常规死后诊断，而应该使用直接荧光抗体检测（5）。然而，有严格质量控制程序以及具有应用这些技术的经验和专业知识的实验室进行流行病学调查时，可以使用这些分子技术；分子技术还可以用于人类狂犬病的生前诊断。强烈推荐应用强阳性对照或检测过程中的质量控制。 - 24 -

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许多实验室方法可以用于患者活着时确定该临床病例是否为狂犬病（2,32）。然而应用活体技术诊断动物狂犬病却困难重重。诊断狂犬病技术的敏感性根据疾病阶段、免疫状况、间歇分泌病毒以及技术人员培训情况的不同而差异很大。经过验证的阳性结果表明有狂犬病，但阴性结果不一定能排除感染的可能。不推荐单独使用脑活检标本来诊断狂犬病，但在可以获得的情况下仍可以帮助诊断（6,10）。对怀疑患狂犬病的病人进行狂犬病诊断时从多方面分析会更有价值，包括致病病原及潜在感染源的特异性特征，特别是在缺乏动物暴露史的情况下；在公共健康调查中确定有可能暴露于同一动物的其他人群；采取适当控制感染的措施来预防因接触病人而造成的暴露；对暴露于病人感染性分泌物的人群给予暴露后预防；病例终了以及安抚病人家庭成员；斟酌实验性治疗方案；如果接受治疗的话则监测病毒载荷及病人反应；较少创伤的技术用于记载人类疾病负担，减少尸检；如果检测为阴性则提示其他感染性病原体。

直接荧光抗体技术在狂犬病临床病例的皮肤活体组织标本或毛囊中可以检测到病毒抗原（33）。检测结果与病人的抗体状态无关，并且在病程早期样本检测也可能呈阳性。皮肤标本通常取自颈后部，内有包含周围神经的毛囊。需要检测多个截面以确定围绕毛囊的基部是否含有病毒抗原。样本的质量极其重要，例如毛囊的缺失会降低检测的敏感性。由于需要有恒冷切片机来制备冰冻皮肤组织切片，所以此项技术并非适用于所有环境，可以用检测病毒 RNA 来替代（6,12,18,34）。角膜印片的荧光抗体检测在大多数临床环境下是不可靠的，因此为避免角膜划痕的风险不推荐作为常规检测，尤其是对于脑炎而非狂犬病的患者。免疫色谱方法已发展用于直接检测动物唾液或脑组织中的狂犬病抗原（29-31），但该方法仍需要标准化和严格质量控制。

在未免疫患者的血清或脑脊液中的中和抗体可以通过病毒中和试验来测定，包括快速荧光灶抑制试验和荧光抗体病毒中和试验（27,35-37）。通常临床症状出现后平均 7-8 天，血清中会出现病毒中和抗体。脑脊液中很少发现病毒抗体，这取决于疾病的临床阶段。用 ELISA 法测定的抗狂犬病糖蛋白抗体滴度与用病毒中和试验测定的结果有很好的相关性，并且 ELISA 法更易于常规操作（27,35）。快速检测其他病毒抗原（如核蛋白）的抗体（免疫球蛋白 G 和 M ）也可能是有用的，因为这些抗体可能比中和抗体出现的早（27）。

分子检测方法用于诊断是高度敏感的（1-3， 5， 10， 11， 14-18， 24， 32， 38-42），然而就像所 - 25 -

有的实验室方法一样，分子检测方法需要标准化和严格的质量控制。狂犬病病毒 RNA 不仅能在脑组织中检测和扩增到，也可以从其他体液和组织样本（如唾液、脑脊髓液、眼泪、皮肤、浓缩的尿液和毛囊）中检出。由于病毒间歇性排出，应该对连续的样本，如唾液和尿液，进行检测。

最好采用脑、唾液或其他容易检出病毒的生物样本进行病毒分离（1-7）。分离成功率取决于病人的免疫状态（抗体阴性的病人会获得更多阳性结果）、病毒排出的间歇以及细胞培养中连续传代的次数。液体样本或者拭子采集后应冷冻保存，拭子内容物应放入采集液中。采集液中绝不能加入防腐剂。即使在病程晚期，这些样本中也可能没有感染性病毒。

应用分子技术已经对数以千计的来自于人类、家养和野生动物的狂犬病病毒分离物进行了比较，引导了狂犬病病毒的基础鉴定和分类并揭示了从特定地理区域或物种分离到的病毒具有独特的基因序列。在大多数情况下，这些差异可以用来确定主要的宿主动物（如蝙蝠、犬、狐狸）并且在缺乏明确暴露史的情况下推断传染来源（1-3,5,10-12,14-17,29,32,33,38-42）。

1. Barrat J et al. Rabies diagnosis. Developments in Biologics (Basel), 2006,125:71-77. 2. Dacheux L et al. More accurate insight into the incidence of human rabies in developing countries through validated laboratory techniques. PLoS Neglected Tropical Diseases, 2010, 4:e765. 3. Dürr S et al. Rabies diagnosis for developing countries. PLoS Neglected Tropical Diseases, 2008, 2(3):e206. 4. Fooks AR et al. Emerging technologies for the detection of rabies virus: challenges and hopes in the 21st century. PLoS Neglected Tropical Diseases, 2009, 3(9):e530. 5. Meslin FX et al., eds. Laboratory techniques in rabies, 4th ed. Geneva, World Health Organization, 1996. 6. Orciari LA, Rupprecht CE. Rabies. In: Versalovic J et al., eds. Manual of clinical microbiology, 10th ed. Washington DC, ASM Press, 2011:1470-1478. 7. Manual of diagnostic tests and vaccines for terrestrial animals, 6th ed. Paris, World Organisation - 26 -

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for Animal Health, 2011 (http://www.oie.int/international-standard-setting/terrestrial-manual). 8. WHO collaborating centres database and portal. Geneva, World Health Organization (http://apps. who.int/whocc/). 9. Reference experts and laboratories. Paris, World Organisation for Animal Health (http://www.oie. int/our-scientific-expertise/referenceslaboratories/list-of-laboratories/). 10. Rupprecht CE, Hemachudha T. Rabies. In: Scheld M, Whitley RJ, Marra C, eds. Infections of the central nervous system. Philadelphia, Lippincott,Williams & Wilkins, 2004:243-259. 11. Feder HM et al. Rabies: still a uniformly fatal disease? Historical occurrence, epidemiological trends, and paradigm shifts. Current Infectious Disease Reports, 2012, 14:408-422. 12. Petersen BW, Rupprecht CE. Human rabies epidemiology and diagnosis.In: Tkachev S, ed. Non-flavivirus encephalitis. Rijeka, InTech, 2011. 13. Laothamatas J et al. MR imaging in human rabies. American Journal of Neuroradiology, 2003, 24:1102-1109. 14. Mallawa M et al. Rabies encephalitis in a malaria-endemic area of Malawi, Africa. Emerging Infectious Diseases, 2007, 13:136-139. 15. Madhusudana SN, Sukumaran SM. Antemortem diagnosis and prevention of human rabies. Annals of Indian Academy of Neurology, 2008, 11(1):3-12. 16. Wacharapluesadee S, Hemachudha T. Ante- and post-mortem diagnosis of rabies using nucleic acid-amplification tests. Expert Review of Molecular Diagnosis, 2010, 10(2):207-218. 17. Picard-Meyer E et al. Use of filter paper (FTA) technology for sampling, recovery and molecular characterisation of rabies viruses. Journal of Virological Methods, 2007, 140(1-2):174-182. 18. Dacheux L et al. A reliable diagnosis of human rabies based on analysis of skin biopsy specimens. Clinical Infectious Diseases, 2008, 47(11):1410-1417. 19. Shipping guidelines for hazardous goods. Montreal, Quebec, International Air Transport Association (www.iata.org/whatwedo/cargo/dangerous_goods/pages/infectious_substances.aspx). 20. Guidance on the regulations for transport of infectious substances 2007-2008. Geneva, World Health Organization. (www.who.int/csr/resources/publications/biosafety/WHO_CDS_EPR_2007_2cc. pdf). - 27 -

21. Coertse J et al. A case study of rabies diagnosis from formalin-fixed brain material. Journal of the South African Veterinary Association, 2011,82(4):250-253. 22. Stein LT et al. Immunohistochemical study of rabies virus within the central nervous system of domestic and wildlife species. Veterinary Pathology, 2010, 47(4):630-633. 23. Robardet E et al. International interlaboratory trials on rabies diagnosis: an overview of results and variation in reference diagnosis techniques(fluorescent antibody test, rabies tissue culture infection test, mouse inoculation test) and molecular biology techniques. Journal of Virological Methods, 2011, 177:15-25. 24. Rudd RJ et al. A need for standardized rabies-virus diagnostic procedures: effect of cover-glass mountant on the reliability of antigen detection by the fluorescent antibody test. Virus Research, 2005, 111(1):83-88. 25. Lembo T et al. Evaluation of a direct, rapid immunohistochemical test for rabies diagnosis. Emerging Infectious Diseases, 2006, 12(2):310-313. 26. Madhusudana SN et al. Evaluation of a direct rapid immunohistochemical test (dRIT) for rapid diagnosis of rabies in animals and humans.Virologica Sinica, 2012, 27(5):299-302. 27. Welch RJ et al. An evaluation of two commercially available ELISAs and one in-house reference laboratory ELISA for the determination of human anti-rabies virus antibodies. Journal of Medical Microbiology, 2009,58(6):806-810. 28. Xu G et al. WELYSSA: a simple tool using mouse monoclonal antibodies for the detection of lyssavirus nucleocapsid in rabies suspected specimens.Developments in Biologics (Basel), 2008, 131:555-561. 29. Kasempimolporn S et al. Evaluation of a rapid immunochromatographic test strip for detection of rabies virus in dog saliva samples. Journal of Veterinary Diagnostic Investigation, 2011, 23(6):1197-1201. 30. Markotter W et al. Evaluation of a rapid immunodiagnostic test kit for detection of African lyssaviruses from brain material. Onderstepoort Journal of Veterinary Research, 2009, 76(2):257-262. 31. Servat A et al Evaluation of a rapid immunochromatographic diagnostic test for the detection of rabies from brain material of European mammals.Biologicals, 2012, 40(1):61-66. 32. Hemachudha T, Wacharapluesadee S. Ante-mortem diagnosis of human rabies. Clinical Infectious Diseases, 2004, 39:1085-1086. 33. Crepin P et al. Intravitam diagnosis of human rabies by PCR using saliva and cerebrospinal fluid. - 28 -

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Journal of Clinical Microbiology, 1998, 36(4):1117-1121. 34. Macedo CI et al. Diagnosis of human rabies cases by polymerase chain reaction of neck-skin samples. Brazilian Journal of Infectious Diseases, 2006, 10(5):341-345. 35. Feyssaguet M et al. Multicenter comparative study of a new ELISA, Platelia Rabies II, for the detection and titration of anti-rabies glycoprotein antibodies and comparison with the rapid fluorescent focus inhibition test (RFFIT) on human samples from vaccinated and non-vaccinated people. Vaccine, 2007, 25(12):2244-2251. 36. Nishizono A et al. Evaluation of an improved rapid neutralizing antibody detection test (RAPINA) for qualitative and semiquantitative detection of rabies neutralizing antibody in humans and dogs. Vaccine, 2012, 30(26):3891-3896. 37. Wright E et al. A robust lentiviral pseudotype neutralisation assay for in-field serosurveillance of rabies and lyssaviruses in Africa. Vaccine,2009, 27(51):7178-7186. 38. Hughes GJ et al. Evaluation of a TaqMan PCR assay to detect rabies virus RNA: influence of sequence variation and application to quantification of viral loads. Journal of Clinical Microbiology, 2004, 42:299-306. 39. Wacharapluesadee S et al. Development of a TaqMan real-time RTPCR assay for the detection of rabies virus. Journal of Virological Methods,2008, 151:317-320. 40. Wacharapluesadee S et al. Comparative detection of rabies RNA by NASBA, real-time PCR and conventional PCR. Journal of Virological Methods, 2011, 175(2):278-282. 41. Wacharapluesadee S et al. Detection of rabies viral RNA by TaqMan realtime RT-PCR using non-neural specimens from dogs infected with rabies virus. Journal of Virological Methods, 2012, 184(1 2):109-112. 42. Wakeley PR et al. Development of a real-time, TaqMan reverse transcription-PCR assay for detection and differentiation of lyssavirus genotypes 1, 5, and 6. Journal of Clinical Microbiology, 2005, 43:2786-2792.

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5. 狂犬病人生前和死后的管理

虽然狂犬病被认为是一种致命性疾病，但是在过去的几十年里也有存活者的记录，特别是与蝙蝠变异株相关的病例（1）。2004 年在美国通过密尔沃基（Milwaukee）疗法成功治愈一名青少年之后，美国和南美一些国家一直尝试治疗狂犬病病人（3） ,虽然极少获得成功。为狂犬病患者选择可能的治疗方法时，应切记以下几点（1）。 ■ 狂犬病在动物中并非总是致命的，但病人极少存活。 ■ 目前尚无法预测哪些病人有可能康复。 ■ 无论是否接受治疗，所有幸存者都有严重的早期免疫反应。 ■ 鼓励进行鉴别治疗方案、免疫调节程序和新药疗法，包括抗病毒药物的研究。 ■ 用于病人的治疗必须证明是安全的并且不会对病人造成进一步的伤害。

狂犬病患者意识清醒，通常了解所患疾病的性质并且常常表现为极度地激动不安，特别是显著兴奋的时候。进一步，由于已经认识到的通过接触传播病毒的风险常常将病人隔离。确诊为狂犬病的病人应在合适的医疗机构内得到足够的镇静和护理，最好在单人房间，并有适当的身心支持。反复静脉注射吗啡或苯并二氮艹卓类镇静剂能有效缓解困扰狂躁型狂犬病病人的严重烦躁、焦虑和恐惧性痉挛（1）。一旦诊断为狂躁型狂犬病，就应避免侵入性操作，病人应安置在独立、安静、避风的环境接受治疗。鉴于绝大多数病例不可避免的死亡，一旦诊断确立，治疗应以减轻病痛为主，可使用大剂量镇静剂（巴比妥酸盐类，吗啡）并且避免插管或生命支持疗法（1）。

狂犬病病毒在疾病的晚期会存在于许多组织中。因为已有多例因器官和组织移植感染狂犬病的记录，所以对来自于因神经系统症状和体征而死亡病例的器官，进行移植前需要格外注意（4,5）。应权衡进行常见或高致死性传染病的检测以保证紧急的活体器官移植需求。目前只有罕见的疾病尚无可应用的技术进行识别。在发展中国家较为普遍的角膜移植应谨慎进行。 - 30 -

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对于已经诊断为狂犬病的病人的治疗常常会在医护人员和媒体及公众之间引起很大的焦虑。如果采取常规的预防措施，尤其是在气管插管和吸引痰液治疗时，与大多数细菌或病毒性感染相比，人狂犬病并不会对医务人员造成大的风险。在经过仔细评估认为有风险时，应为医护人员提供暴露后预防，如同所有传染病所建议的一样应提醒医护人员坚持防护至关重要。在有可能接受狂犬病病人的医院应考虑为可能涉及狂犬病病人管理的医护人员进行暴露前疫苗免疫。有时需要对病人的伴侣进行免疫，因为在疾病早期的密切接触或性接触会有感染的风险。

怀疑死于狂犬病的病人尸体应该标记为有传染性。如果采取了常规预防措施，传播至他人的风险并不大。血液中不含有病毒，但病毒存在于多种组织和体液中，如中枢神经系统和唾液腺（1）。如果需要进行防腐处理或尸体解剖，应该在具备适当的预防措施和个人防护设备条件下小心进行。组织和体液应该与其他传染性疾病一样使用同样的方法进行处理。死者遗体应该掩埋或火化，具体做法取决于病人的宗教信仰。

1. Hemachudha T et al. Human rabies: neuropathogenesis, diagnosis and management. Lancet Neurology, 2013, 12(5):498-513. 2. Willoughby RE et al. Survival after treatment of rabies with induction of coma. New England Journal of Medicine, 2005, 352:2508-2514. 3. Jackson AC. Therapy of human rabies. Advances in Virus Research, 2011,79:365-375. 4. Srinivasan A et al. Transmission of rabies from an organ donor to four transplant recipients. New England Journal of Medicine, 2005, 352:1103-1111.

6. 人用狂犬病疫苗和免疫球蛋白自四十余年前发展至今，经过浓缩、纯化的细胞培养和鸡胚狂犬病疫苗（共称为 - 31 -

CCEEVs）已被证明在预防狂犬病上是安全和有效的。这些疫苗用于暴露前与暴露后的预防，全球已有数百万人接受了 CCEEVs 注射（1）。暴露后及时注射 CCEEVs，结合恰当的伤口处理以及注射狂犬病免疫球蛋白，对于预防狂犬病（甚至在高风险暴露后）几乎总是有效的（1）（亦可参见第 8 节）。

CCEEVs 包括在细胞基质中扩增的狂犬病病毒，这些细胞有如人二倍体细胞、 Vero 细胞、原代鸡胚细胞或鸭胚等。新近研发的基于鸡胚和 Vero 细胞的疫苗与人二倍体细胞疫苗有着同样的安全性和效力，并且价格更便宜。病毒在细胞（或鸡胚）中增殖后，将收获的病毒进行浓缩、纯化、灭活和冻干。在一些 CCEEVs 中，人血白蛋白或加工过的明胶用作稳定剂。肌肉注射用狂犬病疫苗不会采用一瓶多剂分装，并且经过了 WHO 预认证的狂犬病疫苗不含如硫柳汞等防腐剂。如果疫苗贮藏在 2℃至 8℃并避光，其保质期不低于 3 年。用配套的无菌稀释剂复溶后，这些疫苗应当马上使用，若疫苗保存在其要求的温度下，也需在 6 小时内使用（1），目的在于防止没有使用完的疫苗被污染。人用狂犬病疫苗应符合 WHO 生物制品标准专家委员会（ECBS）制定的指导原则中疫苗特性、生产及质量控制的要求（2）。目前， WHO 仅推荐使用细胞培养或鸡胚制备的灭活狂犬病疫苗。

神经组织疫苗可以引起更为严重的副反应并且其免疫原性低于 CCEEVs。自 1984 年以来， WHO 就已建议停止神经组织疫苗的生产和使用并由 CCEEVs 取代。许多发展中国家遵从了这项建议（见下面列出的停止使用神经组织疫苗的国家和日期）并通过进口疫苗、研发或获取 CCEEVs 生产技术来满足其需求。在少数国家中，主要是亚洲和拉丁美洲的国家，处于狂犬病高风险人群仍然依靠动物神经组织制备的疫苗用于暴露后预防。在拉丁美洲的厄瓜多尔和秘鲁以及亚洲的缅甸和巴基斯坦正在探索价格可以承受、并可持续提供的神经组织疫苗的替代品。此次磋商会再次强烈建议应终止包括乳鼠脑组织的动物神经组织培养的人用狂犬病疫苗的生产和使用，并由 CCEEVs 取代。一个用细胞或卵胚生产的现代狂犬病疫苗取代神经组 - 32 -

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织疫苗的四步策略已经形成（3），并且作为附件 3 附加在此报告中。

■ 孟加拉国（2011） ■ 不丹（1995） ■ 印度（2004） ■ 印度尼西亚（1992） ■ 尼泊尔（2006） ■ 斯里兰卡（1995） ■ 泰国（1987）

■ 柬埔寨（2005） ■ 中国（1990） ■ 老挝人民民主共和国（2005） ■ 菲律宾（1997） ■ 越南（2007）

■ 巴西（2002） ■ 智利（2003） ■ 多明尼加共和国（2009） ■ 萨尔瓦多（2009） ■ 墨西哥（1995） ■ 尼加拉瓜（2005） ■ 巴拉圭（2006）

通过联合国机构如联合国儿童基金会（UNICEF）等提供的疫苗应当先通过 WHO 预认证。这是由疫苗生产企业自愿发起的一项固定程序，目的是对获得国家许可的疫苗通过 WHO 初次和继续的评估。经过预认证后，每隔一段时间对产品进行再评价以确信其质量持续的保证。WHO 生物制品标准化专家委员会（ECBS）在 2010 年 10 月通过了一项修订的 WHO 疫苗预认证程序，该程序已于 2012 年 2 月 1 日起生效（4）。 - 33 -

国家药品监管机构应该承担疫苗监管的责任，且作为 WHO 预认证的先决条件，疫苗应当先获得国家的生产许可。 WHO 预认证确保疫苗的质量、安全性、有效性及其适宜应用于中低收入国家的疫苗免疫规划中。疫苗特性必须保证其在使用时符合如下规定，包括效价、热稳定性、外观、标签和冷链体积。生产者应当符合国际质量标准并遵从药品生产质量管理规范（GMP）的国际标准。预认证包括生产工艺及质量控制程序的审核，测定制品批间的一致性， WHO 以及国家药品监管机构的检查员对生产设施的审查，确保每隔一段时间仍然可以持续接受和再评估。监控持续的质量标准。在 2012 年，只有三种肌肉途径使用的狂犬病疫苗通过了预认证：纯化 Vero 细胞狂犬病疫苗、纯化鸡胚细胞疫苗和纯化鸭胚疫苗。相关内容列表，必要时会更新，并可从以下网址获取： http://www.who.int/immunization_standards/vaccine_quality/prequalification_vaccine_list_en/en/。 WHO 咨询委员会鼓励狂犬病疫苗制造商参与 WHO 预认证过程，并鼓励成员国购买通过 WHO 预认证的疫苗。

CCEEVs 的最低效价为每一剂肌注剂量达到 2.5 国际单位（IU），由小鼠保护性效价试验确定（5， 6）。基于血清中和试验的效力测定替代方法（7），包括 ELISAs （8）、使用更少的动物（9）、外周攻毒方法（10）及其他方法（11）尚在探索中。这些替代方法的效果需要在 WHO 合作中心、国家监管机构和质控实验室进行，并且要与疫苗制造商合作。标准的小鼠保护试验以及体外糖蛋白含量测定均需要使用狂犬病疫苗的国际标准品。 2008 年，一种候选疫苗采用第五代国际标准品进行了协作标定并成为狂犬病疫苗的第六代国际标准品。当进行小鼠保护试验时，该标准疫苗的效价是每安瓿 8 IU，即用 1 ml 蒸馏水复溶时为 8 IU/ml。其他体外测定方法，如酶免疫检测和单放射免疫扩散试验，用于确定狂犬病病毒糖蛋白抗原含量（12）。

已经有超过十几个种类或基因型的狂犬病病毒属的病毒被描述为狂犬病的病原体（参见第 2 节）。狂犬病病毒属基因组多变，到目前为止，狂犬病病毒是引起人狂犬病的最常见致病源并且也是至今应用于疫苗生产的唯一病毒种类。现有疫苗可能无法保护除系谱Ⅰ （参见 - 34 -

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第 2 节）中的病毒以外的其他种类的狂犬病病毒的感染。因此用于疫苗的病毒种类必须慎重选择，并且病毒株的抗原鉴别及用于培养病毒的细胞系的鉴别和纯度也应该定期评估。建议通过全基因组测序分析的方法全面掌握疫苗株的基因特性。灭活狂犬病疫苗的临床和非临床评估的一般原则已由 WHO 出版（4）。临床前试验是开展人体临床研究的先决条件，包括动物的免疫原性研究和安全性试验。狂犬病疫苗的临床发展应当包括疫苗用于暴露前和暴露后预防的评估，结合不同的免疫接种程序和接种途径，免疫保护的起始时间、程度及持续时间，加强免疫的需求及时间。临床试验应当遵守 WHO 良好临床试验规范（GLP）（13）所描述的原则以及 WHO 用于疫苗临床评估指南中的设计、执行和分析（4）。所有临床试验均应得到相关国家疫苗监管机构的批准。

目前狂犬病疫苗以单剂量肌肉注射剂型来生产。 CCEEVs 用 0.5 或 1ml 溶剂复溶至一个剂量肌肉注射的小瓶中，其效力大于等于每剂量 2.5IU，可以应用于暴露前和暴露后的预防。然而，用于肌肉注射给药的细胞培养疫苗的价格限制了其在狂犬病高发的许多地区的广泛使用。这些疫苗通过皮肤内给药也具有相同的安全性和免疫力。皮内注射途径用于暴露后预防的完整程序只需要 1 到 2 小瓶疫苗，因此与肌肉内注射相比较，这种方法使用的疫苗量及其直接费用减少了 60%到 80% （14-18）。尚未有证据表明皮内注射所使用的疫苗一定要比推荐用于肌注途径的疫苗有更高的效价（3,19,20）。皮内接种以较低的剂量引起同等的免疫反应，从而在暴露前和暴露后的预防中节约了疫苗使用量。应当通过适当的培训来确保疫苗完全皮内注射并且避免发生皮下注射。每点 0.1ml 的皮内注射剂量是肌肉内注射剂量的 1/10 到 1/5，具体取决于疫苗复溶后的体积。尽管肌肉内注射后的抗体滴度更高并且持续时间也更长，但两种给药途径在加强免疫时都可以诱导快速回忆应答。皮内接种不建议用于免疫功能缺陷的个体（21,22），因为潜在的疾病有可能损害抗原呈递树突细胞运输到引流淋巴结并因此减弱抗体反应水平。一旦开启，疫苗瓶储存不能超过 6 小时，否则会导致浪费，尤其是在那些每日注射疫苗人数较少的医疗中心。即便如此，皮内注射对于暴露前和暴露后的预防依然是既经济又有效（23）。 WHO 预认证的三种疫苗中只有两种 —纯化 Vero 细胞狂犬病疫苗和纯化鸡胚细胞疫苗—按照 WHO 推荐的暴露前或暴露后的预防程序以每剂量 0.1ml 进行皮内给药是安全和有效的。疫苗制造商应当提供临床证据来证明新产品在皮内给药时具有免疫性、有效性和安全 - 35 -

性。给药要遵守 WHO 给药途径的指导并预先获得国家药品监管当局的批准。特别是所使用的疫苗要与一种已知免疫原性、效力和安全性的疫苗进行比较，通过用快速荧光灶抑制试验进行血清学检测，并且这些结果已发表在国际性同行评审杂志。在那些已许可皮内注射用于暴露前或暴露后预防的国家，经证实通过皮内途径给药是安全并有效的产品，其制造商应当对产品可以用于皮内注射进行注册登记，并在产品说明书中注明该疫苗可以用于皮内注射。

一般说来， CCEEVs 是安全和耐受良好的。然而，仍然有可能发生不良反应，这在某种程度上取决于灭活狂犬病病毒的纯度，并可能存在批间差异（24）。 35%到 45%的疫苗接种人群中，会在注射部位出现轻微短暂的红疹、疼痛或肿胀，尤其是在皮内加强接种之后。在 5%到 15%的疫苗接种人群中还观察到一些轻微的全身不良反应，例如短暂的发热、头痛、眩晕及胃肠症状等。严重的不良反应非常少，包括 Guille-Barre 综合征和过敏反应（25）。

CCEEVs 建立的免疫记忆可能在接种者个体中持续一生，甚至在中和抗体滴度下降之后。临床数据证实，即使暴露前或暴露后预防的初始给药是在多年以前，无论初始免疫或加强免疫时的用药途径（肌肉内注射或皮内注射），无论在加强免疫时可检测到的狂犬病病毒特异性抗体存在与否，接种疫苗的人群都对加强免疫有应答（26-28）。此外，已公布的数据表明，在初次狂犬病疫苗接种后并不需要定期的疫苗加强剂量，但除了那些职业处于连续或频繁暴露风险的人群需要额外的防护（参见 8.4）。然而，所有接种过疫苗的个体随后又暴露于狂犬病，根据 WHO 对暴露的定义，应当接受一次简短的暴露后预防疗程，相关内容见第 8 节。

在估算的每年接受暴露后预防的 2 千万人中，病人及时接受了正确的方案但仍然死去，即暴露后预防失败是极其少见的。虽然这样的病例数肯定是低估了，只有少数被公布出来，但所有这些病例都发生在发展中国家并且绝大多数是因与 WHO 所推荐的预防接种程序有偏差(31,32)。大多数因与推荐程序有偏差而导致的死亡：耽搁了寻求狂犬病预防的时间；狂犬病免疫球蛋白的缺乏或不恰当使用（如没有成功注射至所有咬伤部位）；暴露后没有进行或进行了不规范的伤口处理，和 / 或狂犬病疫苗质量低劣（33）。 - 36 -

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为了保护人体免于狂犬病的感染，之前未注射疫苗或未完成疫苗注射的人、 Ⅲ度暴露或 Ⅱ度暴露但严重免疫功能缺陷（如艾滋病患者和移植受者）的人应当同时接受有效狂犬病疫苗和狂犬病免疫球蛋白(34)。狂犬病免疫球蛋白更适宜于注射在受伤的部位及其周围以中和残留的狂犬病病毒（参见第 8 节）。三种类别的生物制品可用于被动免疫：人狂犬病免疫球蛋白，马狂犬病免疫球蛋白和由马免疫球蛋白制成的高纯度 F(ab’ )2 片段（35）。后者的制备过程中 Fc 片段的缺失可能会减少抗体制备的免疫学功能，包括它的免疫原性及其产品的反应原性。人免疫球蛋白的第二批国际标准品由位于英国赫特福德郡波特斯巴的国家生物标准及控制研究所（NIBS）持有并按 WHO 国际生物标准实验室的需求分配（12）。目前 WHO 参考血清标定为每安瓿含有 30IU。狂犬病免疫球蛋白应当与第一剂疫苗同时应用并在伤口部位和伤口周围注射。人免疫球蛋白应当以每公斤体重 20IU 的剂量给药；而马免疫球蛋白在人体内的半衰期更短，要求按每公斤体重 40IU 给药。马免疫球蛋白比人免疫球蛋白产品要便宜许多，并且大多数新型马免疫球蛋白制备物是有效的、高度纯化的和安全的，几乎没有不良反应。血清病可在高纯度马狂犬病免疫球蛋白用药一周后发生，发生率少于 1%-3%的接种者。过敏反应风险低（1/150000），并且这样的过敏反应通常可以治疗。在马狂犬病免疫球蛋白给药前不建议做皮肤测试，因为这些测试很难预测严重的不良反应并且在需要给药的时候不应当作为不注射马免疫球蛋白的依据。马免疫球蛋白应当在具备应对过敏反应的条件下使用。狂犬病免疫球蛋白在全世界范围供应短缺。新技术可能引领单克隆抗体用于暴露后预防。WHO 推荐使用至少包含抗狂犬病病毒的两种抗体混合物的单克隆抗体 “鸡尾酒” ，作为狂犬病免疫球蛋白的替代品用于暴露后预防（36）。一种完全人源单克隆抗体混合物正在进行临床安全性及效力的评估（37）， WHO 正在设计一种人源化的鼠单克隆抗体鸡尾酒用于发展中国家的暴露后预防（38）。WHO 单克隆抗体已获得许可向一些商业开发合作伙伴开放，其中一家在 2012 年启动了鸡尾酒疗法的第一阶段临床评估。这些产品而有望在不远的将来投入使用。

1. WHO position paper on rabies vaccines. Weekly Epidemiological Record, 2010, 85:309-320. - 37 -

2. WHO Expert Committee on Biological Standardization. Fifty-sixth report, Annex 2. Geneva, World Health Organization, 2007 (WHO Technical Report Series, No. 941). 3. Human and dog rabies prevention and control: report of the WHO/Bill & Melinda Gates Foundation consultation, Annecy, France, 7-9 October 2009. Geneva, World Health Organization, 2010 (WHO/HTM/NTD/NZD/2010.1) (http://whqlibdoc.who.int/hq/2010/WHO_HTM_NTD_NZD_2010. 1_eng.pdf). 4. WHO Expert Committee on Biological Standardization. Fifty-third report,Annex 1. Geneva, World Health Organization, 2004 (WHO Technical Report Series, No. 924). (http://www.who.int/immunization_standards/vaccine_quality/pq_revision2010/en/index.html). 5. Seligmann EB Jr. Laboratory techniques in rabies: the NIH test for potency. Monograph Series. Geneva, World Health Organization, 1973,23:279-286 6. Wilber LA, Aubert MFA. The NIH test for potency. In: Meslin FX, Kaplan MM, Koprowski H, eds. Laboratory techniques in rabies, 4th ed. Geneva, World Health Organization, 1996:360-368. 7. Kamphuis E et al. Potency testing of inactivated rabies vaccines using a serological method. Developments in Biologics (Basel), 2012, 134:23-27. 8. Nimmagadda SV et al. Recombinant diabody-based immunocapture enzyme-linked immunosorbent assay for quantification of rabies virus glycoprotein. Clinical and Vaccine Immunology, 2010, 17:1261-1268. 9. de Moura WC et al. Potency evaluation of rabies vaccine for human use: the impact of the reduction in the number of animals per dilution. Journal of Virological Methods, 2009, 158:84-92. 10. Wunderli PS et al. The rabies peripheral challenge test: more accurate determination of vaccine potency. Vaccine, 2006, 24:7115-7123. 11. Stokes W et al. Report on the international workshop on alternative methods for human and veterinary rabies vaccine testing: state of the science and planning the way forward. Biologicals, 2012, 40:369-381. 12. National Institute for Biological Standards and Control. WHO international standard. Sixth international standard for rabies vaccine (NIBSC code: 07/162. Instructions for use, Version 1.0, dated 10/11/2008) (http://www.nibsc.ac.uk/products/biological_reference_materials/product_catalogue/detail_page.aspx?catid=07/162). - 38 -

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13. WHO Expert Committee on the Use of Essential Drugs. Sixth report,Annex 3. Geneva, World Health Organization, 1995 (WHO TechnicalReport Series, No. 850). 14. Warrell MJ et al. Economical multiple-site intradermal immunization with human diploid-cell-strain vaccine is effective for post-exposure rabies prophylaxis. Lancet, 1985, i: 1059-1062. 15. WHO Expert Committee on Rabies. Eighth report. Geneva, World Health Organization, 1992 (WHO Technical Report Series, No. 824). 16. Briggs DJ et al. Antibody response of patients after postexposure rabies vaccination with small intradermal doses of purified chick embryo cell vaccine or purified Vero cell rabies vaccine. Bulletin of the World Health Organization, 2000, 78:693-698. 17. Quiambao BP et al. Reducing the cost of post-exposure rabies prophylaxis: efficacy of 0.1 ml PCEC rabies vaccine administered intradermally using the Thai Red Cross post-exposure regimen in patients severely exposed to laboratory-confirmed rabid animals. Vaccine, 2005, 23:1709-1714. 18. Ambrozaitis A et al. Rabies post-exposure prophylaxis vaccination with purified chick embryo cell vaccine (PCECV) and purified Vero cell rabies vaccine (PVRV) in a four-site intradermal schedule (4-0-2-0-1-1): an immunogenic, cost-effective and practical regimen. Vaccine, 2006,24: 4116-4121. 19. Beran J et al. Potency requirements of vaccines administered intradermally using the Thai Red Cross regimen: investigation of the immunogenicity of serially diluted purified chick embryo cell rabies vaccine. Vaccine, 2005, 23:3902-3907. 20. Sudarshan MK et al. Assessing the relationship between antigenicity and immunogenicity of human rabies vaccines. Results of a meta-analysis. Human Vaccines, 2005, 1:187-190. 21. Kopel E et al. Inadequate antibody response to rabies vaccine in immunocompromised patient. Emerging Infectious Diseases, 2012, 18:1493-1495. 22. Tantawichien T et al. Failure of multiple-site intradermal postexposure rabies vaccination in patients with human immunodeficiency virus with low CD4+ T lymphocyte counts. Clinical and Infectious Diseases, 2001, 33:E122-E124. 23. Hampson K, Cleaveland S, Briggs D. Evaluation of cost-effective strategies for rabies post-exposure vaccination in low-income countries. PLoS Neglected Tropical Diseases, 2011, 5:e982. - 39 -

24. Finke S et al. Assessment of inactivated human rabies vaccines: biochemical characterization and genetic identification of virus strains. Vaccine, 2012, 30:3603-3609. 25. Grading of scientific evidence. Table III. Safety of cell-culture-based rabies vaccines. Geneva, World Health Organization, 2010 (http://www.who.int/entity/immunization/rabies_grad_safety.pdf). 26. Suwansrinon K et al. Survival of neutralizing antibody in previously rabies vaccinated subjects: a prospective study showing long lasting immunity. Vaccine, 2006, 24:3878-3880. 27. Brown D et al. Intradermal pre-exposure rabies vaccine elicits long lasting immunity. Vaccine, 2008, 26:3909-3912. 28. Naraporn N et al. Immune response to rabies booster vaccination in subjects who had postexposure treatment more than 5 years previously. Journal of Travel Medicine, 1999, 6:134-136. 29. Strady A et al. Antibody persistence following preexposure regimens of cell-culture rabies vaccines: 10-year follow-up and proposal for a new booster policy. Journal of Infectious Diseases, 1998, 177:1290-1295. 30. The immunological basis for immunization series, module 17: Rabies. Geneva, World Health Organization, 2011. 31. Wilde H et al. Failure of postexposure treatment of rabies in children. Clinical and Infectious Diseases, 1996, 22:228-232. 32. Wilde, H. Failures of post-exposure rabies prophylaxis. Vaccine, 2007,25:7605-7609. 33. Rupprecht CE et al. Evidence for a 4-dose vaccine schedule for human rabies post-exposure prophylaxis in previously non-vaccinated individuals. Vaccine, 2009, 27:7141-7148. 34. Guide for post-exposure prophylaxis. Geneva, World Health Organization,2012 (http://www.who. int/rabies/human/postexp/en/). 35. Lang J et al. Evaluation of the safety, immunogenicity, and pharmacokinetic profile of a new, highly purified, heat-treated equine rabies immunoglobulin, administered either alone or in association with a purified, Vero-cell rabies vaccine. Acta Tropica, 1998, 70(3):317-333. 36. Consultation on a rabies monoclonal antibody cocktail for rabies post-exposure treatment. Geneva, 23-24 May 2002. Geneva, World Health Organization (available at www.who.int/rabies/vaccine/en/mabs_final_report.pdf; accessed March 2013). - 40 -

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37. Bakker AB et al. First administration to humans of a monoclonal antibody cocktail against rabies virus: safety, tolerability, and neutralizing activity.Vaccine, 2008, 26(47):5922-5927. 38. Müller T et al. Development of a mouse monoclonal antibody cocktail for post-exposure rabies prophylaxis in humans. PLoS Neglected Tropical Diseases, 2009, 3(11):e542. Erratum in: PLoS Neglected Tropical Diseases,2009,3(11):10.1371/annotation/df98339d-6bdb-40ed-af83-cc38b249264a.

7.兽用疫苗兽用疫苗用于预防家养动物和野生动物中的狂犬病。兽用疫苗包括灭活疫苗、改良的活病毒疫苗以及基因工程重组疫苗。但无论何种方法生产的疫苗，原材料的质量和标准（如疫苗生产用毒株、无特定病原蛋、细胞种源）都应清楚记载，尤其是消毒和安全方面的信息。兽用狂犬病疫苗应当由国家有关主管部门批准并遵从国家对疫苗的要求。在没有适宜的国家兽用生物制品（上市前和上市后的要求）涉及效力、消毒、安全和有效性等相关法规时，应该参照相关的国际标准（1-8）。疫苗病毒株应该明确其遗传特征，最好进行全基因组测序分析。

疫苗应该由具备能力的人士，如兽医，来管理或监督，根据生产者的建议确定如最低年龄、给药途径（口服、肌注或皮下注射）、免疫持续时间以及给药间隔时间。然而，在有科学证据和无禁忌症的情况下，可允许根据生产者的建议适当修改免疫程序，如在大规模免疫计划中对小于 3 月龄动物的母体进行疫苗接种，目的是为了优化群体免疫力（见第 9 节）。

改良活疫苗是通过将改良的鸡胚适应病毒株（如 FLURY 株）在鸡胚中连续传代生产的。改良活疫苗还可以通过适应细胞培养的毒株（例如 SAD / ERA ）生产。这些疫苗因其有引起狂犬病的风险而不再被认为是安全的，因此应停止其在家畜中使用。

家养动物中最常用的疫苗是灭活的注射用疫苗，通常是安全又价廉。这种疫苗的安全性、效力和纯度在使用前应通过可靠的方法进行评估。灭活疫苗是通过狂犬病病毒适应株感染原代细胞或传代细胞系的细胞培养生产的。可使用多种方法灭活，最常用的是 β- 丙内酯或紫外线。建议使用佐剂，最常用的是氢氧化铝。灭活狂犬病疫苗有液体和冻干两种剂型。灭活狂犬病疫苗可与菌苗（如钩端螺旋体）以及其他病毒抗原（多价），如犬瘟热病毒，犬 - 41 -

腺病毒 II 型和犬细小病毒一起做成联苗使用。目前可用于猫的联合疫苗包括猫泛白细胞减少症病毒、猫杯状病毒和猫疱疹病毒等。一种狂犬病和口蹄疫联苗用于免疫牛、绵羊和山羊。一种狂犬病与抗波托马克发热（由里氏艾利希体造成）的联苗应用于马。

表达狂犬病病毒糖蛋白的金丝雀痘病毒已在美国获准作为猫注射用疫苗。商业化可以应用的疫苗是将金丝雀痘病毒载体 - 狂犬病疫苗与猫泛白细胞减少症、猫杯状病毒和猫疱疹病毒成分联合使用。

尽管口服疫苗在特定条件下可能是恰当的，犬的疫苗免疫仍首选注射途径。口服途径应该用来作为一种大规模注射接种疫苗的补充方法，主要针对无法注射疫苗的犬以提高犬群疫苗接种覆盖率（见第 9 节）。液体疫苗通常含在一小袋或胶囊中包装入一个诱饵，其味道、大小、质地应适合于犬。为最大限度提高其使用率并防止与疫苗相关的伤害人类不良事件的发生， WHO 已确立了针对候选口服疫苗的安全性和效力以及对犬用诱饵的设计、检测和分布的要求（9,10）。只有已知最低残余致病性的疫苗才能在犬中使用。至目前为止，一种减毒疫苗（11）和一种重组疫苗（12）符合 WHO 的建议（10）。目前只有一种犬口服疫苗获得许可。用于家犬口服免疫的疫苗应逐一进行评估，其根据是品种的结构（有主，无主）和介入犬群的可接受性（13-15）。由于口服狂犬病疫苗价格昂贵并且安全投放需要一些时间，将这些疫苗应用于犬的成本效益比率应仔细评估。

用于免疫野生动物的狂犬病疫苗通常是通过口服途径免疫的活疫苗。液体疫苗通常含在一小袋或胶囊中包装入一个诱饵，其味道、大小和质地应适于目标动物种类。获得许可用于家养动物的灭活疫苗也可以用于野生动物的捕获—预防接种—释放的项目中。

所有目前使用的减毒活疫苗均来源于 ERA/ SAD （ Street Alabama Dufferin）病毒株，经细胞培养传代后不同程度减毒。几种疫苗是通过在克隆的幼仓鼠肾细胞上连续体外筛选或小鼠体内传代减毒的。有一种疫苗是通过狂犬病毒糖蛋白单克隆抗体筛选出的在 333 位连续两次突变的减毒株发展而来（11,16 ）。不推荐使用可能会在野生动物中导致狂犬病的狂犬病病毒株。 - 42 -

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已经开发了几种重组疫苗：重组牛痘病毒以及新近的人腺病毒载体疫苗，这两种重组疫苗均表达狂犬病毒糖蛋白基因（17,18）。目前也有基于狂犬病病毒点突变（反向遗传学）的重组疫苗。这些重组构成中的一些疫苗已经获得国家主管部门批准。

在市场投放之前要确立批内释放滴度，这代表了在狂犬病强毒株的攻击下能使目标实验动物获得 100 ％保护的最低疫苗滴度。疫苗诱饵中的批投放滴度应至少 10 倍于 100%保护剂量（ 3,21 ）。参与疫苗批准或评估口服狂犬病疫苗免疫项目的国家实验室或政府机构可以在疫苗接种之前和期间对所有批次疫苗诱饵的病毒滴度进行核实（ 7,21,22 ）。这样的检测应该在有证明文件、经过验证的方法和适当标准的合格实验室中进行。

应该设立一个有效的药物警戒系统在授权上市后的过程中（野外使用）检测与疫苗相关的问题。安全测试应通过小鼠脑内接种进行或者，如果证明有效的话，通过细胞培养进行则更佳（ 2,5,7,19 ）。

疫苗的安全性是通过目标和非目标物种评估的，即在所在地区生活并可能消耗诱饵的野生啮齿动物及其他野生和家养动物，以及非人类的灵长类（10）。该疫苗无论在目标或非目标物种中均不应该产生任何不良反应。一些在野外用于野生动物的改良型狂犬病活病毒口服疫苗可能有残余致病性，这取决于病毒株的减毒程度。因此，任何从疫苗接种地区的动物中分离到的狂犬病病毒均应通过单克隆抗体或分子生物学技术进行鉴定以避免发生疫苗引起的狂犬病。在人类暴露于减毒狂犬病病毒疫苗的意外事件中，应寻求医疗处置并考虑进行暴露后预防。应评估、并在研究和开发的早期确定在痘病毒或腺病毒载体重组活疫苗中含有的成分对动物、人类和环境存在的潜在风险，缓解或治疗的方法，尤其是对于人类（10 ， 23）。当口服疫苗用于免疫犬时，疫苗病毒在目标和非目标物种（包括人类）之间的传播风险 - 43 -

应通过检测犬接种疫苗后 7 天内在其唾液和粪便样本中的狂犬病病病毒来进行评估。疫苗接种后不应检测到活病毒，如果有活病毒则提示病毒的复制和排毒。任何检测到的病毒都应该通过分子技术或单克隆抗体进行鉴定。

具体条件可以用于使用兽用疫苗在大规模疫苗接种计划中控制犬类狂犬病。犬类大规模疫苗接种运动的实施和监测在第 9 节阐述。为保证狂犬病疫苗的免疫原性，应该尊重制造商关于储存的建议。特别应避免在冷链的储存时间过长、暴露于阳光和温度波动。开封的疫苗应在 2-3 天内使用（灭活疫苗），使用无菌技术从多头份瓶中抽取疫苗。如果可能的话，犬也应接种预防其他疾病的疫苗，进行除虫，绝育手术，以改善它们的健康。这种 “有形” 的效果可能会促使人们在未来对所饲养的犬进行加强免疫。狂犬病病毒中和抗体通常在最初抗原刺激后的 4-6 周达到峰值。随后抗体的水平迅速下降并可能在疫苗接种后几个星期内很快低于检测阈值。在进行过几次疫苗接种的犬，包括相隔 12 个月接种两次的犬，不论何时进行血清学检测，抗体滴度普遍较高（24）。根据国家权威部门的意见，灭活疫苗的免疫有效期为 1-3 年。

1. WHO Expert Co mmittee on Rabies. Eighth report. Geneva, World Health Organization, 1992 (WHO Technical Report Series, No. 824). 2. Meslin FX, Kaplan MM, Koprowski H, eds. Laboratory techniques in rabies, 4th ed. Geneva, World Health Organization, 1996. 3. WHO Expert Consultation on Rabies. First report. Geneva, World Health Organization, 2005 (WHO Technical Report Series, No. 931). 4. Guidelines on nonclinical evaluation of vaccines, Annex 1. Geneva, World Health Organization, 2005 (WHO Technical Report Series, No. 927). 5. Rabies. In: Manual of diagnostic tests and vaccines for terrestrial animals (mammals, birds and bees), 7th ed. Paris, World Organisation for Animal Health, 2012:263-282. - 44 -

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6. Principles of veterinary vaccine production. In: Manual of diagnostic tests and vaccines for terrestrial animals (mammals, birds and bees), 7th ed. Paris, World Organisation for Animal Health, 2012: 52-63. 7. Code of Federal Regulations, Title 9, Parts 1-199 (1-1-12 edition). Washington DC, Government Printing Office, United States Department of Agriculture Animal and Plant Health Inspection Service, 2012. 8. Brown CM et al. Compendium of animal rabies prevention and control, 2011. Journal of the American Veterinary Medicine Association, 2011, 239(5):609-617. 9. Matter HC. Suggestion for the development of a research project for the field evaluation of several vaccine-bait delivery techniques to vaccinate dogs orally against rabies. Geneva, World Health Organization, 1993 (WHO/Rab.Res./93.40). 10. Guidance for research on oral rabies vaccines and field application of oral vaccination of dogs against rabies. Geneva, World Health Organization,2007. 11. Cliquet F et al. The safety and efficacy of the oral rabies vaccine SAG2 in Indian stray dogs. Vaccine, 2007, 25:3409-3418. 12. Blancou J et al. Innocuité et efficacité d’ un vaccine antirabique recombinant vaccine virus rabique administré par voie orale au renard, chien et chat [Safety and efficacy of an antirabies vaccine consisting of recombinant vaccinia-rabies virus administered orally to the fox, dog and cat.] Annales de Recherche Vétérinaire, 1989, 20(2):195-204. 13. Field application of oral rabies vaccines for dogs: report of a WHO consultation organized with the participation of the Office International des Epizooties, Geneva, Switzerland, 20-22 July 1998. Geneva, World Health Organization, 1998 (WHO/EMC/ZDI/98.15). 14. Matter HC, Fico R. Accessibility of dogs to oral and parenteral vaccination against rabies in Tunisia and Turkey. Geneva, World Health Organization,1992 (WHO/Rabies/93.206). 15. Matter H et al. Field evaluation of two bait delivery systems for the oral immunization of dogs against rabies in Tunisia. Vaccine, 1998, 16(7): 657-665. 16. Cliquet F et al. Eliminating rabies in Estonia. PLoS Neglected Tropical Diseases, 2012, 6(2):1-17. 17. Rosatte RC et al. Prevalence of tetracycline and rabies virus antibody in raccoons, skunks and red foxes following aerial distribution of V-RG baits to control raccoon rabies in Ontario Canada. Journal of Wildlife Diseases, 2008, 44:946-964. - 45 -

18. Yarosh OK et al. Human adenovirus type 5 vectors expressing rabies glycoprotein. Vaccine, 1996, 14:1257-1264. 19. Rabies vaccines (inactivated) for veterinary use. In: European Pharmacopoeia. Strasbourg, Council of Europe, European Directorate for the Quality of Medicines and Health Care, 2010:734-736. 20. Servat A et al. In vivo potency tests of rabies vaccines for veterinary use.A 2-year retrospective analysis of data according to the criteria of the European Pharmacopoeia. Pharmeuropa, 2008, 20(4): 655-664. 21. The oral vaccination of foxes against rabies. Report of the Scientific Committee on Animal Health and Animal Welfare. Luxembourg, European Commission, 2002. 22. Rabies vaccines (live, oral) for foxes. In: European Pharmacopoeia. Strasbourg, Council of Europe, European Directorate for the Quality of Medicines and Health Care, 2008:736-743. 23. Human vaccinia infection after contact with a raccoon rabies vaccine bait-Pennsylvania, 2009. Morbidity and Mortality Weekly Report, 2009, 58:1204. 24. Cliquet F et al. Neutralising antibody titration in 25,000 sera of dogs and cats vaccinated against rabies in France, in the framework of the new regulations that offer an alternative to quarantine. Revue Scientifique et Technique (International Office of Epizootics), 2003, 22(3):857-866.

8. 人类狂犬病的预防狂犬病几乎总是致命的。因此，狂犬病病毒暴露之前以及怀疑或证实的暴露后通过免疫来预防都非常重要。用于预防的狂犬病疫苗和免疫球蛋白应符合 WHO 对生产和质控以及对用于肌肉和皮内两种途径的免疫源性和安全性的建议（见第 6 节）。

怀疑或证实暴露于狂犬病病毒后，及时使用 CCEEVs 和对伤口进行正确的处理并同时注射狂犬病免疫球蛋白几乎总是可以有效地预防狂犬病，即便是在严重暴露的情况下。潜在暴露的评估是复杂和混乱的。怀疑发生暴露时，应启动暴露后预防，主治医师应向具有狂犬病专业知识的传染病专家咨询。针对由于职业、居住地或旅游等原因而存在狂犬病病毒高暴露风险的人群，强烈推荐暴露前的预防。 - 46 -

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疫苗可以在一定部位的肌肉或皮内注射给药。对于肌肉途径给药，成人和≥2 岁的儿童应将疫苗注入三角肌部位， <2 岁的儿童建议在大腿前外侧注射疫苗。狂犬病疫苗不应在臀部给药，因为对于诱导足够的免疫应答缺乏可靠性。对于皮内注射，建议的部位包括三角肌、大腿外侧或上肩胛区（见附件 4）。部位的选择是基于可提供的隐私程度以及社会文化的接受。有一些设施可用于促进皮内注射。

由于居住地或职业的原因而持续、频繁或增加的狂犬病病毒暴露风险的任何人，如接触狂犬病病毒及其它狂犬病病毒属病原的实验室工作人员、兽医和动物操作者，均建议进行暴露前预防。在高风险地区的旅游者在风险评估后应接种疫苗。在狂犬病流行地区居住或旅行的儿童有患病风险，当经济、行政许可及后勤保障等方面可行的时候，应对其进行个体或群体水平的暴露前预防（参见 8.8 节）。下面列出的疫苗接种方案应尽可能在规定的时间内完成，但如果某一针剂没有在给定的时间内注射，也没有必要重新启动接种方案（1）。肌肉注射：一个肌肉注射针剂分别在 0、 7 和 21 或 28 天注射。 0 天是疫苗首剂给药的日期。皮内注射：一次 0.1 毫升皮内注射剂量分别于 0、 7 和 21 或 28 天给予。为了最大限度节约，皮内暴露前预防的时段应该包括足够的人数以便 6 小时内用完所有已开启的疫苗。

暴露后尽快进行伤口局部治疗；注射一个疗程符合 WHO 推荐的有效的狂犬病疫苗；如果需要，注射狂犬病免疫球蛋白。决定是否进行暴露后预防应该考虑的因素包括涉及到的动物患狂犬病的流行病学可能性、暴露的严重程度（见 8.3.2 节）、动物的临床特点及其免疫接种状况（尤其是犬和猫）和对其进行观察和实验室检测的可行性。所有确定为存在狂犬病风险的暴露都需要暴露后预防。预防应立即开始。如果可能，应对可疑的动物进行识别、隔离观察（看起来是健康的犬和猫）或安乐死进行实验室检测。在等待实验室结果或在观察的期间内应继续进行预防。如果 - 47 -

实验室检测是阳性，应立即进行回顾性风险评估以确定所有可能暴露的人群，并应给予他们暴露后预防。如果可疑动物无法进行实验室检测或观察则应完成暴露后预防全过程，如果动物经适当的实验室检测证实未感染狂犬病则暴露后预防可以终止。当家养的犬、猫或黑足鼬在人暴露初始是健康的，正确接种过疫苗（至少用有效疫苗接种过两次的记录）并易于进行 10 日观察的情况下，应确保恰当的伤口处理而加强免疫接种可以推迟，特别是当患者曾经接受过暴露前预防或此前的暴露后预防是在过去的 3 个月内（2）。所有被咬的受害者以及其他与可疑动物接触的人在医疗保健机构一经发现，应立即报告给兽医专家，对动物进行调查并对怀疑患狂犬病的动物进行实验室检测确认，如果动物处在观察中要监测动物的健康状况。当在动物咬伤、抓伤以及其他接触（不包括接触蝙蝠）发生在没有食肉类动物狂犬病的地区并且在这些地区有良好的狂犬病监测，则可以不需要暴露后预防。这一决定应基于对当地狂犬病流行情况熟知的医学专家所做的风险评估。在犬和 / 或野生动物狂犬病存在地方性流行的地区，当疑似有暴露后应立即进行暴露后预防，除非实验室监测有效运行并且实验室和现场来源的数据均表明涉及的物种不是狂犬病的传播动物，例如，被啮齿类、兔子和野兔咬伤通常不需要进行暴露后预防。这里给出的建议是一个综合的指导，可以根据具体情况有所改变，如无法得到可靠接触史的时候（如婴儿或智障者）。尤其是在狂犬病为地方性动物病以及无法对咬人动物跟踪观察和 / 或实验室检测不能及时提供的地区更是如此。最好由合格的医学专业人员对每一个暴露于疑似狂犬病动物的病人进行细致的风险评估。

所有咬伤和抓伤的迅速的伤口局部治疗是暴露后预防中重要的一步。推荐的急救程序包括：立即彻底冲洗并用肥皂、水、洗涤剂、碘伏或其他有杀病毒活性的物质清洗伤口。如果没有肥皂或杀病毒制剂，则用水对伤口进行彻底广泛的冲洗。应该使住在狂犬病流行地区的人们知晓简单的局部伤口处理并告诫不要进行可能会导致伤口进一步污染或扩大的处理步骤。伤口流血无论在任何部位均提示潜在的严重暴露，必须注射人或马的狂犬病免疫球蛋白。大多数严重咬伤的伤口应每日换药，必要时再进行二期缝合。如果伤口清洗后必须缝合，伤口应首先浸润人或马狂犬病免疫球蛋白并延迟几个小时再缝合以便让免疫球蛋白在缝合 - 48 -

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前扩散进入组织。二期缝合感染的可能性小，如果在最佳条件下进行会有更好的美观效果。咬伤感染对于注射狂犬病免疫球蛋白是没有禁忌的（3）。咬伤在手指或脚趾尖、耳垂或鼻区等部位可以安全地注射狂犬病免疫球蛋白，前提是不会产生过大的压力，因为这可能会导致压迫综合征（4）。其他的治疗，如抗生素和破伤风的预防注射，适用于有污染可能的伤口。

在狂犬病地方性流行的国家或地区，怀疑或确认为狂犬病（家养或野生）动物的暴露分类如下： ■ I 级：接触或喂养动物，舔舐完好的皮肤，完好的皮肤与狂犬病动物或人的分泌物或排泄物接触。这些都不能视为暴露，不需要暴露后预防。 ■ II 级：轻咬裸露皮肤，无出血的轻微抓搔或擦伤，应该尽快注射疫苗。 ■ III 级：单个或多个贯穿皮肤的咬伤或抓伤，破损的皮肤被舔，通过舔而使唾液污染粘膜以及暴露于蝙蝠。应尽快就近注射疫苗和狂犬病免疫球蛋白。在第一剂疫苗注射后的 7 天内均可以使用免疫球蛋白。对于 II 级和 III 级暴露，彻底的伤口局部处理（见 8.3.1）是最重要的。暴露后预防，包括狂犬病免疫球蛋白，一旦确认是 III 级暴露均要实施，即便是在暴露后的数月或数年。当无法使用相同的细胞培养或鸡胚疫苗来完成暴露后预防时，应该使用符合 WHO 要求的细胞培养狂犬病疫苗完成接下来的预防程序。但是，这只是特殊情况。

第 0 天是指接种第一剂疫苗的日期。重要的是在 1 周内完成初始 3 剂疫苗的注射。

建议的方案包括 5 个剂量（1-1-1-1-1）或 4 个剂量（2-0-1-0-1 或 2-1-1）注射程序： ■ 5 剂量 ‘Essen’ 程序（1-1-1-1-1）包括在第 0 天， 3 天， 7 天， 14 天和 28 天各分别接种一剂。也有针对健康人群的简化为四个剂量（1-1-1-1-0）的免疫程序，该程序受到支持的证据包括同行评议的文献、未发表的数据、流行病学评议和专家的意见。这一缩短了的 Essen 方案，包括在 0， 3， 7 和 14 天分别给药，可以作为一种替代方法，用于健康、免疫健全的暴露者，前提是这些 III 级和 II 级暴露者可以获得伤口处理并注射狂犬病免疫球蛋白和 WHO 认可的狂犬病疫苗（5）。 ■ 4 剂量 ‘Zagreb’ 程序（2-0-1-0-1 或 2-1-1）包括在 0 天注射两剂量疫苗（两侧的三角肌或大腿部各注射一剂），随后分别在 7 和 21 天再各注射一个剂量。 - 49 -

更新的泰国红十字会两点注射方案（2-2-2-0-2）包括在第 0， 3， 7 和 28 天在两个不同的皮内位点分别注射 0.1ml 的疫苗（6）。此方案可在已经得到国家卫生当局许可的国家用于 II 级或 III 级暴露人群的皮内注射途径。

暴露或再暴露的患者如果具有先前用狂犬病 CCEEVs 完成全程暴露前或暴露后预防的证明文件应该接受： ■ 在 0 和 3 天分别在一个位点肌肉注射或皮内注射一个剂量的疫苗。这种情况下不需注射狂犬病免疫球蛋白。此方案也适用于接种了狂犬病疫苗并能够检测到狂犬病病毒中和抗体的人群。 ■ 作为替代方案，可以给予病人 ‘一次四点’ 皮内注射法，包括在一次就诊中同时将四针次 0.1 毫升疫苗均等注射在左和右侧的三角肌、大腿或上肩胛区（7,8）。对于那些曾经接受过全程暴露前或暴露后预防接种的人，在 3 个月的时间内发生咬伤或其他接触的暴露或再暴露，应该确保正确的伤口处理，如果咬人的犬或猫是健康的、已经免疫并且可以进行 10 天期限的观察，则疫苗加强注射可以推迟（2）。使用效力不确定的疫苗，包括神经组织疫苗，曾经进行过全程暴露前或暴露后预防接种的 III 级暴露者，或者没有完成暴露前和暴露后的全程预防接种，应给予全程暴露后疫苗接种，包括狂犬病免疫球蛋白。

对于生活在或旅行到狂犬病疫区而且用狂犬病 CCEEVs 进行过全程暴露前或暴露后预防接种的人群，没有必要定期进行狂犬病疫苗加强免疫注射。只有那些处于持续或频繁暴露风险的职业人群应接受定期的加强注射作为应对无法确认的暴露的特别防护。如果可行，对高危工作人员进行狂犬病毒中和抗体的监测要优先于常规加强接种。对于存在潜在的暴露于高浓度活狂犬病病毒风险的实验室工作人员，应每 6 个月测定其中和抗体滴度。如果血清抗体滴度低于 0.5IU/ml，应肌内注射或皮内注射接种一剂加强免疫的疫苗。如果不是处于持续暴露风险的专业人员，如某些兽医和动物卫生官员，应每 2 年进行血清学监测。由于疫苗诱导的免疫记忆大多数情况下持续多年，建议只有狂犬病病毒中和抗体滴度低于 0.5 IU/ml 时再给予一剂加强免疫的狂犬病疫苗。 - 50 -

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一些对艾滋病病毒携带者 / 艾滋病患者的研究表明， CD4 细胞计数非常低的患者狂犬病病毒的中和抗体反应明显低下或检测不到。当这些患者和其他免疫功能不确切的人群发生的 II 级和 III 级暴露，需要在正确彻底的伤口处理和消毒同时，局部浸润注射人或马的狂犬病免疫球蛋白以及全程 5 个剂量肌肉注射狂犬病 CCEEV。如果可行，在疫苗接种 2-4 周后检测狂犬病病毒中和抗体以评估是否需要额外剂量的疫苗。如有疑问，请咨询具有专业艾滋病病毒 / 艾滋病和狂犬病预防方面的传染病学专家。

狂犬病免疫球蛋白在被动免疫中的作用是在患者接种疫苗后生理上产生自身抗体之前在暴露的部位提供中和抗体。因此，所有 III 级暴露者均应使用狂犬病免疫球蛋白。狂犬病免疫球蛋白为一次性给药，最好是在起始暴露后疫苗接种后立刻或尽快开始。最好不要超出第一剂狂犬病疫苗接种后的第七天，无论第 3 天和第 7 天是否注射了疫苗，因为这时 CCEEV 的主动抗体反应已经开始，并因此可能在主动和被动免疫之间产生干扰。人狂犬病免疫球蛋白的剂量是 20 IU/ 公斤体重，而马免疫球蛋白和 F （AB'） 2 产品的剂量是 40 IU/ 公斤体重。所有的免疫球蛋白，如果解剖结构上允许（但避免可能的骨筋膜室综合征），应仔细地注射到伤口内及伤口周围。如果有剩余，将其注射到远离疫苗注射点的肌肉部位。必须避免使用同一注射器或混合狂犬病疫苗和狂犬病免疫球蛋白的情况。对于严重和多处创伤的情况，通常需要的免疫球蛋白比计算的剂量更多，这时可以用无菌生理盐水稀释免疫球蛋白至一定体积以确保有效、安全侵润注射到所有伤口（8）。暴露一经确认，即使在几个月或几年后，均应给予暴露后预防，包括 III 级暴露时应用狂犬病免疫球蛋白。

由于狂犬病是致命的，暴露后预防没有禁忌，应根据暴露程度处理，在工作人员进行了充分的预防处置和应对可能出现的不良反应等方面的培训的前提下实施。对于婴幼儿、孕妇或免疫功能低下的个体，包括儿童艾滋病毒携带者 / 艾滋病患者，也同样没有暴露后预防的禁忌。服用氯喹治疗或预防疟疾可能会使皮内接种狂犬病疫苗的免疫反应减弱，故这种情况下应该进行肌内注射。正如所有疫苗的接种一样，受种者在接种疫苗后至少需要 15-20 分钟的医学观察。如果对疫苗中的任何成分曾产生过严重副反应（除狂犬病免疫球蛋白以外），在进行暴露前或暴露后预防时应禁止使用同一种疫苗。 - 51 -

狂犬病流行的国家和地区的旅行者和居民应避免与自由流浪的动物接触，尤其是犬和猫，也要避免与野生、自由放养或捕获的动物接触。对于参加洞穴探险，偶然暴露于洞穴空气不必顾虑，但要告诫洞穴探险者不要触摸蝙蝠。与蝙蝠接触后进行暴露后预防。图 1 中地图显示了四个类别的国家或地区，这些国家或地区可以分为无风险、低、中、高风险的地区。这种分类是基于主要的宿主动物 / 传播者和所涉及的狂犬病病毒的种类，以及对这些宿主种类可靠的实验室基础上的监测数据的可获得性。获得规范的医疗处置和 CCEEVs 的可及性以及其他狂犬病生物制品的情况也在考虑之中。 ■ 1 类，无风险：无狂犬病病毒风险的国家或地区。 ■ 2 类，低风险：只有狂犬病相关病毒存在于蝙蝠中或者在蝙蝠（非吸血）和其他野生动物中存在狂犬病病毒的国家或地区。在上述两种国家或地区中，规范的医疗处置、 CCEEVs 和其他狂犬病生物制品均可方便获得，并且有可靠的基于实验室的监测数据。 ■ 第 3 类，中等风险：狂犬病病毒在蝙蝠（非吸血）和其他野生动物中流行的国家或地区。 ■ 第 4 类，高风险：持续的犬与犬间传播狂犬病病毒的国家或地区和 / 或有吸血蝙蝠狂犬病报告的地区（9）。

无风险：无需暴露前预防。低风险和中等风险：人们参与任何可能使他们直接接触非吸血蝙蝠和其他野生动物的活动，尤其是食肉动物（例如，野生动物专家、研究者、兽医和探险旅行者到访蝙蝠和其他野生动物经常出没的地方），应该接受暴露前预防。高风险：到农村地区旅行的人或参与跑步、骑自行车、露营或远足等活动的人，应该接受暴露前预防。对于有显著职业风险的人也推荐进行预防，如兽医以及有显著暴露于家养动物的高风险地区的居民，尤其是犬和猫以及包括吸血蝙蝠在内的野生动物。儿童由于存在更高的风险而应进行预防性免疫。

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世界卫生组织狂犬病专家磋商会

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图 1 从无风险到低、中、高四个风险水平的国家或地区分布地图当有可能在低、中或高风险国家或地区发生暴露，对于已经接受过暴露前预防的人应该接受加强疫苗注射（参见 8.3.4 节），而对于之前没有接种过疫苗的人应向医生咨询并且如果需要应在最短时间内接受暴露后预防（参见 8.3.3 节）。预防狂犬病的暴露前和暴露后疫苗接种的推荐建议见附件 6。

1. Recommendations for routine immunization. Summary tables. Geneva,World Health Organization, 2012 (www.who.int/immunization/policy/immunization_tables/en; accessed March 2013). 2. Sudarshan MK, Ravish HS, Ashwath Narayana DH. Time interval for booster vaccination following re-exposure to rabies in previously vaccinated persons. Asian Biomedicine, 2011, 5(5):589-593. 3. Wilde H et al. Is injection of contaminated animal bite wounds with rabies immune globulin a safe practice Transactions of the Royal Society of Tropical Medicine and Hygiene, 1992, 86:86-88. 4. Suwansrinon K et al. Is injecting a finger with rabies immunoglobulin dangerous? American Journal of Tropical Medicine and Hygiene, 2006,75:363-364. 5. Rupprecht CE et al. Use of a reduced (4-dose) vaccine schedule for postexposure prophylaxis to prevent human rabies, recommendations of the Advisory Committee on Immunization Practices. Morbidity and Mortality Weekly Report, 2010, 59(RR02):1-9. - 53 -

6. Madhusudana SN et al. Comparison of safety and immunogenicity of purified chick embryo cell rabies vaccine (PCECV) and purified Vero cell rabies vaccine (PVRV) using the Thai Red Cross intradermal regimen at a dose of 0.1 ml. Human Vaccines, 2006, 2(5):200-204. 7. Rabies vaccines: WHO position paper. Weekly Epidemiological Record, 2010, 85:309-320. 8. Human and dog rabies prevention and control, report of the WHO/Bill & Melinda Gates Foundation Consultation, 2009, Annecy, France. Geneva, World Health Organization, 2010 (WHO/HTM/NTD/NZD 2010.1). 9. Schneider MC. et al Rabies transmitted by Vampire bats to humans: an emerging zoonosis in Latin America Pan American Journal of Public Health, 2009, 25(3):260-269.

9．控制犬狂犬病的国家项目犬狂犬病可以被消灭，北美洲、西欧，日本和南美洲的许多地区和亚洲部分地区都已经证明了这一点。但犬狂犬病仍然在 80 多个国家和地区广泛分布，主要为发展中国家。 99％以上的人类狂犬病病例的病毒都来自于犬，全球半数的人口都居住在犬狂犬病流行地区，面临感染狂犬病的风险。控制并最终消除该疾病在犬类中的传播，对人类健康有重大意义，可从源头上预防因犬狂犬病造成的绝大多数的人类病例。现在已经开发出可提供较长时间免疫保护的兽用疫苗，大规模的注射接种项目是犬狂犬病控制的主要手段。近年来，通过大规模疫苗接种的犬狂犬病的控制和消除方案已导致人类狂犬病病例显着减少或消除（1-4）。在一些情况下，犬接种狂犬病疫苗结合消毒灭菌措施已消除当地病例（5）或已经预测将消除当地人狂犬病病例（6）。除去单独接种疫苗，消毒灭菌在犬狂犬病控制中的贡献尚未得到充分评估。疫苗接种项目应考虑到当地犬群的生态特点，包括与人类的关系（家养并有限制，家养并漫游，集体拥有或无主）。这方面的信息必不可少，用以确保项目最大化为犬进行疫苗接种，并开展适合当地文化特点的教育活动。拉丁美洲疫苗接种活动取得成功的关键，就在于公共卫生部门和社区在狂犬病控制中的中心协调作用。此外，由于狂犬病控制项目必须涉及多个机构和部门，包括动物和公众健康，他们需要一个 “同一个健康” 的方式，以及有效的部际合作。尽管大规模犬类接种疫苗已经被多次证明可有效控制犬狂犬病，但没有证据表明，捕杀犬只对于犬群密度或狂犬病的传播有显著的影响。大规模灭犬行动不应被纳入狂犬病控制策略：它是无效的且可能在疫苗接种计划中适得其反。 - 54 -

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对怀疑患狂犬病的犬进行安乐死可降低人类健康风险以及防止动物的进一步痛苦。在第 11 部分中给出了犬类狂犬病典型的临床症状。当诊断不明确时，可以对犬类进行隔离和观察；但是如果症状加剧，应进行安乐死（7）

为了实现控制并最终消灭狂犬病，项目必须确保经常性的活动（通常是每年），并实现疫苗接种覆盖率至少 70％（8,9）。不管犬的群体数量变化（出生，死亡，迁出，迁入），这样的覆盖范围足以保持两次接种活动期间接种群体的群体免疫力所需的水平（8,10）。拉丁美洲是成功控制狂犬病的几个国家的一个例子。自其在 1983 年正式承诺消除由犬传染的狂犬病人死亡，该区域各国已经减少了 90％以上的犬狂犬病病例，人类死亡病例随之有相应的下降（ 9,12）。通过每年对超过 45 万只犬进行大规模疫苗接种和对存在狂犬病潜在风险的人进行适当的处置（暴露前和暴露后预防）以及流行病学监测，绝大多数目标已经实现。近期其他成功案例包括夸祖鲁 - 纳塔尔省（南非），米沙鄢群岛（菲律宾）和巴厘岛（印度尼西亚）。南非夸祖鲁 - 纳塔尔省被犬类狂犬病一直困扰了几十年。在 1983 年至 2007 年期间，南非 79％的经实验室确认的人类病例发生于该省，人口估计为 10.6 万。消灭犬类狂犬病项目说明了省级政府，捐助者（例如，比尔和梅林达 · 盖茨基金会）和世界卫生组织之间的合作的有效性。该项目在 2009 年年初以来已接种超过 150 万只犬。在 2012 年，超过 630 万只犬接种了疫苗，这是在一年内由省级兽医服务免疫接种的最高数字。在 3 年内动物狂犬病的发生已经减半，人类感染病例开始减少（11）： 20 年来夸祖鲁 - 纳塔尔省首次报告在 2010 年 - 2011 年连续 12 个月期间没有一个人类病例（12）。尽管面临诸多挑战，该项目目前伴随新的支持和动力正在整个非洲南部扩展。在米沙鄢群岛消灭狂犬病的区域方案是在全国狂犬病行动 9482 的基础上，由农业、卫生和教育部门共同实施的国家狂犬病项目的一部分，由农业部畜牧局主持。与合作伙伴如世界卫生组织，比尔和梅林达 · 盖茨基金会，全球狂犬病控制联盟（GARC）和 Optimus 基金会开展合作， “无狂犬病米沙鄢群岛项目” 正在进行中。该项目涉及超过 5 年为超过 3 万只犬接种疫苗，并且已经在西米沙鄢岛，米沙鄢中部包括薄荷岛（13）和东米沙鄢岛进行了活动。已开展了密集的信息和教育活动，加强社会的支持和志愿者参与，以增加犬类疫苗接种和负责任的宠物主人，改善人类狂犬病的临床管理以及监测和诊断能力。在米沙鄢群岛，人类因狂犬病死亡的人数已大幅下降，从 2008 年的 48 例下降为 2012 年的 13 例，减少了 70％（12）。 - 55 -

在 2008 年狂犬病传入巴厘岛并迅速蔓延到整个岛屿，至 2012 年年底造成 141 人死亡。遏制该疾病蔓延的首次尝试包括不分青红皂白的大规模扑杀犬。从 2010 年底引进以大规模犬类接种为主要策略以来，人类和动物的狂犬病病例的数量已急剧下降： 2010 年和 2011 年之间的人类病例数下降了 72％，在 2010 年和 2012 年之间下降了 90％。已经完成两轮大规模疫苗接种运动，第三次接种已接近尾声。由当地的非政府组织巴厘岛动物福利协会，在世界保护动物协会的资助下，证明了全岛接种活动的可行性。在粮农组织的技术援助下印度尼西亚政府承担了第二轮接种和后续活动的责任。项目成功的原因是：具有一个明确的行动目标，即每次接种运动中在岛上每个地方为 70 ％的犬接种疫苗；通过短信和报纸在接种运动期间每日报告疫苗接种和接种后的调查结果；在接种活动期间的每天，每周和每月进行政府协调会议;具有活动特定的标准操作程序以及现场工作人员的在职培训（14,15）。

疫苗接种活动必须进行战略规划，具有良好的管理以及充分的资源和资助。狂犬病预防伙伴制作的 “狂犬病蓝图” 为犬接种活动的规划和实施提供了指导（16）。

要规划疫苗接种活动，必须估计犬的数量并明确犬的保定方式，以计算所需要的资源并确定对犬进行免疫所需的适当方法（17 ）。可以从人与犬的比例对犬类数量进行估计，但这些比率在社区间有很大的不同。在市区中报告养犬的水平低以及养犬状态多变，使得难以准确估计城市犬群。估算犬群的其他方法包括问卷调查、提供家养犬的信息，以及捕获—标记—再捕获方法涵盖无主的流浪犬（18）。这些方法的详情可从国际伴侣动物管理联盟（19）和狂犬病预防合作联盟（20）获得。这种调查结合疫苗接种后的调查对于评估疫苗接种覆盖率往往是有益的，还可以为以后的活动修正犬群估计数。从犬类登记获得的信息是有用的，但是，这些不包括未登记犬或无主犬，仅使用此种资源会导致总犬群数量的低估。

目标犬群的疫苗接种覆盖率低或不均的情况与狂犬病的持久存在直接相关，这将妨碍整个地区消除狂犬病，即便其他地区的疫苗覆盖率很高。如果在连续的小区域全面进行疫苗接种可能会比在许多分开的领域更有效。在这样的情况下狂犬病传播模型对于确定最佳策略是有帮助的（21）。不建议反应性接种疫苗，除非越来越多的监测显示在几个残余重点地区的发生率已经降低到很低的水平。反应性接种战略需要更长的时间来控制狂犬病，且与在整个区域系统化接种相比成功控制狂犬病的可能性要小。 - 56 -

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所需的免疫覆盖率可以通过精心设计的教育活动，跨部门和跨学科的合作，社会参与，地方规划和执行的承诺，提供高品质的疫苗，媒体的大力支持，以及有关当局有效的协调和监督活动实现。

在大型接种活动中，无论年龄，体重或健康状况，所有的犬都应该接种疫苗。虽然我们的目标应该是对尽可能多的犬接种疫苗，通过至少 70％的犬群进行疫苗接种即可获得群体免疫力。由于猫是人类狂犬病的重要载体，在疫苗接种运动时也应该给猫接种疫苗。覆盖率低的一个常见的原因是误认为幼犬不宜接种（22-24）。在许多犬狂犬病流行国家，幼犬占了相当大的比例，犬主和接种疫苗的队伍必须知道，幼犬包括新生犬也应接种疫苗，以确保有足够的群体覆盖率。已经单独或联合使用三种基本的方法对犬进行疫苗接种活动：走家访户，社区内容易识别的定点接种站，流动医疗队设立的临时接种站。经验显示，只有在 500 米或步行约 10 分钟路程内的人通常会光顾这些接种点（25）。方法的选择应取决于社区的具体情况。可能需要各种方法相结合。

狂犬病疫苗接种活动通常每年进行一次，但在犬群流动率很高的地方，可以进行更频繁的接种活动。 1 天到 1 个月的持续的疫苗强化接种运动已有效控制狂犬病，特别是在拉丁美洲。然而，活动必须覆盖至少 70％的犬群，对速度的追求不应该影响覆盖率。可以在周末或学校放假期间组织活动以提高参与率，因为孩子往往会带来他们的犬接种疫苗。

建议对接种疫苗的犬进行登记和永久性标识，然而，有效的鉴别方法还没有得到广泛的使用，需要进一步研究。即使缺乏对犬进行永久性标识的资源或能力，也不应该影响疫苗接种运动的实施。使用彩色标签或塑料项圈作为临时标志已被证明有助于识别接种疫苗的犬（25）以及激励业主带宠物去接种疫苗。对已接种疫苗的犬的鉴别是必要的，以便评估接种率 - 57 -

和区分未接种疫苗的犬以便进行后续疫苗接种。例如，在巴厘岛，红项圈或喷漆（为仍在发育的幼犬准备）被用来标记每一个接种的犬。然后在接种运动 3 天内进行了一项调查，评估标记和未标记的犬的数量; 计算出的覆盖率低于 70 ％的地区要组织复种活动，对未接种疫苗的犬进行接种。在大规模犬类疫苗接种活动的背景下，如果： ■ 已使用了一种有信誉保证的疫苗（即一种疫苗已被证实单次注射后 2 年或更长时间后，进行一次造成至少 80 ％对照组死亡的致命的攻毒，仍然能产生保护作用）； ■ 接种疫苗的队伍已经经过训练，能够使用正确的注射技术，犬类保定和疫苗瓶管理; ■ 自始至终保持冷链状态。则不建议进行常规血清学监测。如果每年重复地达到了目标覆盖率的接种活动没有导致动物狂犬病病例的数量减少，上述一个或多个元素可能没有被遵守。则应该保证进行精心设计的血清学检测和其他研究（如疫苗效力，冷链监控等）。

一些理论研究表明，使用犬用疫苗结合暴露后预防与单独进行暴露后预防相比，在防止狂犬病导致的人类死亡方面更具成本效益（26,27）。然而这一结论仍然不确定，因为不同活动的成本有很大的不同，而且运作成本实际上可能大大高于那些建模研究中所需的成本，如在坦桑尼亚联合共和国的农村，要花费 1.73-5.50 美元不等（ 20,24 ）。此外，暴露后预防的需求并不总是随犬狂犬病的发病率减少而减少，这种关系还需要进一步调查。已经做了关于要求犬主为登记或活动经费（尤其是疫苗成本）捐款的效果，或根据犬主的支付能力进行选择差额贡献方面的研究，进一步评价这种方法将是有益的。当犬主人不愿或无力支付时，会危及免疫覆盖率的临界水平，必须免费提供干预（即登记，标记，疫苗接种证书交付），并且与公共卫生福利的成本相平衡。

因为疫苗对于极端的温度，包括冻结非常敏感，应小心谨慎以确保冷链保持在可接受的温度范围之内。应采用免疫力最短持续时间为 2 年的长效疫苗，在每年进行的免疫中重新免疫所有的犬。重复接种无不良影响，而且每年活动提供简单有用的信息。排除这些主人和犬可能会混淆消息，而且与免疫活动的成本相比，重复接种的直接成本微乎其微。 - 58 -

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处理犬的疫苗接种团队中的所有成员应在活动前接受暴露前预防。应向接种活动期间暴露的人提供足够的暴露后预防。

当常规的对犬进行预防接种的途径被认为效果不佳时，可以使用其他措施。增加社区参与度和动员可以提高疫苗接种活动的参与率，成本效益和可持续性，以及狂犬病病例的监测和管理。当一部分的犬主人不能自行处理犬或没有犬主负责该犬的疫苗接种时，专业犬类管理人员可以人道捕捉和控制犬进行疫苗接种。有很多捕犬方法。专业捕犬员需要进行适当的培训，以确保他们能高效、可靠和人道的捕获犬;不熟练的处理可能伤害捕犬员和犬，并可能使未来的疫苗接种更加困难。犬口服疫苗可能在犬不能被控制或捕获的情况下提高覆盖率。需要进一步实地考察评估口服疫苗在不同场合与不同投放策略下实现目标覆盖率的成本效益（28）。

犬群的人道主义管理主要通过可靠的犬主登记、提供绝育以及基本的犬的健康护理来实现的（29）。在控制犬狂犬病的背景下，犬群管理的目的是改善和维持疫苗接种覆盖率和降低犬的危险行为。由于没有证据证明狂犬病的传播取决于犬群密度，通过人道的方式减少犬群规模未必是最重要的因素，尽管它可能有其他的好处（例如，关于犬的幸福或减少滋扰行为）。因此犬群管理可能对于犬类狂犬病控制是有益的。关于人道主义犬群管理项目对犬狂犬病（和其他相关益处）的影响的研究还较为有限（ 5,6,29 ），进一步评估这种做法将是有益的。人道主义犬群管理对于减少犬群的增长及创造一个健康的可持续的种群是一种有效的策略。由于犬群的地位和组成因国家而异，没有一种能在所有的情况下行之有效的方法。政府应与了解当地犬群的人一起合作以了解犬的所属关系、犬群数量统计学和当地社区对犬的态度。此信息可以为形成长期的，可持续的管理提供量身定制的人道主义犬群管理奠定基础（ 16,19,29 ）。印度通常有非常高的无主犬比例。犬群管理已在动物出生控制计划中用于控制犬类狂犬病，自由流浪犬被捕获，绝育，接种疫苗后被释放。几个地点已报告在这样的项目中因狂犬病死亡的人数减少（ 5,30,31 ）。但是没有充分评估除接种之外单独采取绝育措施对于犬狂犬病控制的贡献。 - 59 -

专家建议在犬的狂犬病控制计划中包括以下部分：建立国家中心和国家消灭狂犬病委员会来制定、实施和监督存在风险人员暴露前和暴露后的有针对性的预防治疗方案的管理，大规模犬群疫苗接种以及犬群的人性化管理的长期计划。加强包括快速诊断技术在内的监测和诊断设施的建设。确保社区、地区、国家和区域的狂犬病控制项目可持续发展。发展有效的控制和消灭狂犬病的跨境合作。通过宣传和儿童教育计划提高关于负责任的犬所有制的好处，疑似患有狂犬病的咬伤的基础护理和避免动物暴露的公众意识。促进所有相关部门包括兽医服务，公共健康，野生动物管理和生态学家的合作，发展有科学依据的消灭人类和动物狂犬病的方法。支持各级医疗卫生服务部门的狂犬病控制活动的整合，使其与其他公共卫生项目，如细菌（如结核病），寄生虫（如脑囊虫病，囊性包虫病）和媒介传播的疾病（如人非洲锥虫病，利什曼病）进行结合。项目之间的协同作用提高了人力，物力和财力的利用。向双边和多边机构和其他技术合作或人道主义援助体系中的捐助者寻求资金。加强与国际组织的协调与合作，如世界卫生组织，联合国粮食和农业组织（ FAO ），世界动物卫生组织及其合作中心和参考实验室以及非政府的全球和区域组织的专门网络（如 GARC ，世界兽医协会，联邦兽医协会，世界动物保护协会，动物福利以及其他国际组织和联盟）。鼓励制药行业和社会公共机构的合作，提供人用和兽用疫苗和确保疫苗的储存，交付和管理的技术合作。

犬狂犬病控制的运作研究在干预过程（如疫苗接种，犬群控制）中进行，可以利用动物被保定并能进行检查和标记的优势。在运作研究中，要努力遵守适当的协议，并确保严格的统计标准和公正的采样。在可能的情况下，应设置对照组。 - 60 -

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需要进一步进行运作研究的主要领域如下：应进行有主犬问卷调查和征求关于无主犬的意见，以确定干预前后的犬群数量（每人，每个家庭，每一面积），人口统计学，动力学以及分配（32,33）。疫苗接种和其他兽医干预活动为进行暂时或永久性可视标记提供机会，如项圈，耳缺口，耳标或纹身以便再次研究。对犬进行快速、低成本的标记和随后的标志 - 重捕分析需要更好的方法，包括例如犬的短期走势（ 1 天到 1 周的活动范围）的信息。需要整合问卷调查和野生动物普查方法，以更好地确定可能不是很方便接种的无主犬真正的数量。问卷调查也可作为一个教育计划的一部分收集关于狂犬病的认识，社会对犬的态度和犬群数量控制方法的信息。直接观察和问卷调查应该在明确界定的监管范围内用来收集数据。这些信息可以被用来估计对犬进行兽医干预的难易，其决定于监督范围的广度，文化，栖息地和生态环境（气候，气象）。应对犬消毒或接种后进行临时（如项圈，染色，微芯片）或永久（如纹身，耳标）标记的方法加以探讨。在通过手持设备，定位设备或计算机软件记录犬群的绝对数量和不同类别的比例（如年龄，性别，治疗）和管理快速评估疾病的发病率和疫苗接种覆盖率关系的结果方面需要更好的方法。应探索将犬的条件、疾病和寄生虫进行分类和记录的方法（如在兽医干预措施期间，实地调查，家访），以确定犬群的健康情况。可以评估疾病对于犬类种群动态的影响以及对犬进行健康管理。在犬类疫苗接种经济学，方案的可持续性，支付意愿，不同文化背景下成本效益和成本效益分析，生态和经济环境等方面需要进行应用研究（ 13,26,27,34,35 ），包括建立大型和区域模型。应包括项目实施的社会经济障碍研究，政策和实践的转化研究，狂犬病控制项目与其他由犬传播的人畜共患病如包虫病和黑热病的潜在整合研究。应对相关信息及其传播方法进行评估。可通过活动之前和之后的问卷调查判断教育活动的影响。其他可以用来评估教育效果的途径包括被犬咬伤的案例数、医院就诊数量和自由流浪犬的数量变化。 - 61 -

随着犬群管理逐渐转向手术或非手术绝育或避孕，通过控制生育对于控制犬群规模和控制狂犬病的影响仍然存在问题，包括对犬群动态，社会行为和疾病传播的影响（ 5,36 ）。应进行研究活动以评估生育控制是否降低了接触率，家庭范围和侵略性（尤其是雄犬）和疾病传播率。最近开发的非手术避孕药和避孕措施，如免疫避孕药和睾丸内避孕药，应在可以严密监测犬类的情况下进行测试，以确定其是否人道，对群体水平上寿命的影响以及使用和提供这些药物的可行性（37 ）。应对与手术或非手术绝育相结合的狂犬病疫苗接种的成本、可行性和可持续性进行评估。与此同时，应进行成本效益分析，以比较不同的犬群管理选择，并确定在某些情况下是否以及如何控制生育来作为一种优化狂犬病消灭计划的辅助手段。

1. Lembo T et al. Renewed global partnerships and redesigned roadmaps for rabies control. Veterinary Medicine International, 2011(doi:10.4061/2011/923149). 2. Lembo T et al. Zoonoses prevention, control, and elimination in dogs. In: Macpherson CNL, Meslin F-X, Wandeler AI, eds. Dogs, zoonoses and public health, 2nd ed. Wallingford, Oxon., CAB International, 2013:205-258. 3. Nel L, Le Roux K, Atlas R. Meeting the rabies control challenge in South Africa. Microbe, 2009, 4 (2):61-65. 4. Wandeler AI et al. Dogs and rabies. In: Macpherson CNL, Meslin F-X,Wandeler AI, eds. Dogs, zoonoses and public health, 2nd ed. Wallingford,Oxon., CAB International, 2013:43-66. 5. Reece JF, Chawla SK. Control of rabies in Jaipur, India, by the sterilization and vaccination of neighbourhood dogs. Veterinary Record, 2006, 159:379-383. 6. Totton SC et al. Stray dog population demographics in Jodhpur, India following a population control/rabies vaccination program. Preventive Veterinary Medicine, 2010, 97:51-77. 7. Terrestrial animal health code. Chapter 7.7. Stray dog control. Paris, World Organisation for Animal Health, 2011 (http://www.oie.int/index. php?id=169&L=0&htmfile=chapitre_1.7.7.htm; accessed 29 November 2012). 8. Coleman PG, Dye C. Immunization coverage required to prevent outbreaks of dog rabies. Vaccine, 1996, 14:185-186. - 62 -

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9. Cleaveland S et al. Dog rabies vaccination campaign in rural Africa: impact on the incidence of dog rabies and human dog-bite injuries. Vaccine, 2003, 21:1965-1973. 10. Tamayo H et al. Case report (4) Americas. Elimination of human rabies transmitted by dogs in Latin America and the Caribbean: achievements. In: OIE Global Conference on Rabies Control, Republic of Korea, 7-9 September 2011 (http://www.oie.int/eng/A_RABIES/presentations.htm; accessed 29 November 2012). 11. Report of the 4th meeting of the international coordination group of the Gates Foundation/WHO project for human and dog rabies elimination in low-income countries, 2-4 October 2012, Cebu, Philippines. Geneva, World Health Organization, 2013 (http://www.who.int/rabies/bmgf_who_ project/en). 12. Report of the 3rd meeting of the international coordination group of the Gates Foundation/WHO project for human and dog rabies elimination in low-income countries, 19-21 October 2011, PieterMaritzburg, KwaZulu-Natal, South Africa. Geneva, World Health Organization, 2011 (http://www. who.int/rabies/bmgf_who_project/en/). 13. Lapiz SMD et al. Implementation of an intersectoral programme to eliminate human and canine rabies. The Bohol Rabies Prevention and Elimination Project. PLoS Neglected Tropical Diseases, 2012, 6 (12):e1891.14. Suseno PS et al. Dog vaccination and campaign management for effective rabies control: the Bali experience. In: International Conference on Emerging Infectious Diseases, 11 14. March 2012, Atlanta, Georgia. Atlanta, Georgia, United States Centers for Disease Control and Prevention, 2012. 15. Suseno PP et al. Integrated bite case management for rabies in Bali: putting one health into action. In: International Conference on Emerging Infectious Diseases, 11 14 March 2012, Atlanta, Georgia. Atlanta, Georgia, Centers for Disease Control and Prevention, 2012. 16. Lembo T et al. The blueprint for rabies prevention and control: a novel operational toolkit for rabies elimination. PLoS Neglected Tropical Diseases, 2012, 6(2):e1388. 17. Report of a WHO consultation on dog ecology studies related to rabies control. Geneva, World Health Organization, 1988 (WHO/Rab.Res./88.25). 18. Hiby LR et al. A mark resight survey method to estimate the roaming dog population in three cities in Rajasthan, India. BMC Veterinary Research, 2011, 7:46. 19. Humane dog population management guidance. International Companion Animal Management Coalition, 2008 (http://www.wsava.org/PDF/2008/Misc/AWC_ICAM_Coalition.pdf). 20. Blueprint for rabies prevention and control [canine rabies blueprint]. Partners for Rabies Prevention (www.rabiesblueprint.com; accessed March 2013). - 63 -

10. 野生动物狂犬病的预防和控制在过去，虽然有偶尔的报告表明野生动物狂犬病的发生，狂犬病主要见于家犬。在 20 世纪 40 年代欧洲和北美由于严格执行犬类大规模疫苗接种和其他措施，使得实际上由犬传播的狂犬病消失，但该疾病在野生动物中意外的重新出现。随着鉴定病毒变种和系统发育的分子生物学方法的发展，对于狂犬病流行病学的了解显著改善。狂犬病是一种病毒性人畜共患病，与许多种食肉目和翼手目物种有关，它们是狂犬病病毒的主要宿主；只有翼手目物种是几乎所有其他狂犬病病毒属的主要宿主（见第 2 节）。

普遍认为目前在世界范围内流行的犬狂犬病病毒在欧洲殖民统治时期已蔓延至整个非洲大陆。在非洲，家养犬仍然是狂犬病病毒主要的宿主（1）。虽然整个非洲大陆已有野生动物狂犬病散发病例记录，只有在非洲南部发现了狂犬病在野生食肉动物中流行的有说服力的证据，那里的野生犬科动物，如豺狗（侧纹胡狼和黑背胡狼）和蝙蝠耳狐狸（大耳狐）被认为獴科家族的成员是狂犬病病毒的主要宿主（2,3）。此外，（如猫鼬）似乎是非洲南部另一种狂犬病病毒变种的传播媒介（4）。犬狂犬病毒感染已证明是纳米比亚捻角羚（林羚属弯角羚）的重大死亡原因，怀疑是通过捻角羚之间传染性的唾液进行直接通过口腔传播的（5,6）。从犬向外扩散的狂犬病病毒正威胁着非洲濒危野生犬科动物如埃塞俄比亚狼（草原胡狼）和非洲野狗（非洲野生猎犬）（7-10）。

虽然在亚洲中部和热带地区犬狂犬病占主导地位，但在亚洲大陆的森林草原和草原区狂犬病由野生犬科动物携带，主要是赤狐（赤狐属）和俄罗斯远东地区的貉（11,12）。在中国南部，雪貂（鼬獾）很多年来与人类狂犬病相关，被认为是这一地区的主要宿主（13）。尽管在中东地区和亚洲中部，南部和东南部偶尔有野生食肉动物的狂犬病病例报告，目前尚不清楚野生动物狂犬病在这些地区是否独立于犬的狂犬病传播循环。狐狸狂犬病目前存在于以色列，约旦河西岸和加沙地带，并已在土耳其出现，当地大多数牛狂犬病病例都是通过患狂犬病的狐狸接触传播的（14 ）。此外，中东地区的某些国家的野生动物狂犬病的病例报告越来越多，包括伊朗伊斯兰共和国，阿曼，沙特阿拉伯和也门。在这些地区红狐狸和金豺通常会受到牵连（15-17）。 - 64 -

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在欧洲犬狂犬病被消灭后出现了野生动物狂犬病，新的宿主为赤狐（赤狐属）。从东部开始，狐狸狂犬病在几十年内在整个欧洲大陆残酷地蔓延。 20 世纪 80 年代中期，欧洲中部和西部大部分地区都受到了影响。疾病的向西扩张在法国和意大利北部等地区终止，在这些地区用口服狂犬病疫苗对狐狸进行免疫（17）。感染狂犬病病毒的狐狸是狐狸种群内狂犬病存在和传染其他野生动物物种和家养动物的原因。在受感染的区域中，狂犬病在各种各样的物种中以不同的频率被检测到。最有可能接触到带毒狐狸的动物如狍子，牛和其他家养反刍动物占受侵害动物的绝大多数。有迹象表明，貉可能是另一个主要的野生动物宿主，因为它是在欧洲中部和波罗的海地区第二大经常报道的感染的物种（18）。目前，经狐狸传播的狂犬病仍然盛行于欧洲东部和东南部，而欧洲西部和中部大部分地区已通过实施国家和区域口服狂犬病疫苗接种计划摆脱了狐狸狂犬病（19）。虽然 2012 年 10 月在希腊北部报告了一起病例，一些地中海南部和岛屿国家从来没有发生过狐狸狂犬病疫（20）。其他国家从未有过狐狸狂犬病，例如瑞典和英国（17）。

随着 20 世纪中期在加拿大和美国成功地消灭犬狂犬病以及在墨西哥犬类狂犬病的预防和控制取得实质性进展，像欧洲一样，野生动物狂犬病开始出现在北美。与世界其他地区相比，北美洲的温带的野生动物狂犬病涉及许多的主要宿主循环，它们往往具有重叠的地域范围，对于控制动物狂犬病来说是一项重大挑战。在阿拉斯加部分地区和加拿大，常见主要宿主是红狐狸（赤狐），而在东部则是浣熊。虽然北美洲的狐狸狂犬病疫情在加拿大范围扩大，在佛罗里达州（美国）的浣熊中出现了狂犬病毒的另一个变种并蔓延到相邻的洲。在 20 世纪 70 年代患有狂犬病的浣熊迁徙到大西洋中部地区加速了其蔓延，疾病在广大的南部和北部地区爆发，最远到达了魁北克。而在加拿大东南部狐狸狂犬病最终在二十世纪末被消灭，这主要是由于进行了口服狂犬病疫苗接种，但浣熊狂犬病仍然在该地区造成了严重的问题（21-23 ）。在大陆的极地地区北极狐（蓝狐）是一个主要宿主，而条纹臭鼬是整个中部平原和加州的主要宿主（22,23）。此外，灰狐狸（ Urocyon cinereoargenteus ）尤其是在美国西南部也与狂犬病相关，在墨西哥若干种臭鼬被确认为主要宿主。每一个野生物种携带着一种占主导地位的宿主特异性狂犬病毒变种，但也可以携带明显不同的其他主要宿主物种向外扩散的狂犬病毒变种。在各个地区病毒向其他野生和家养动物的扩散非常频繁。至目前为止，口服狂犬病疫苗在红狐狸和浣熊狂犬病的预防和控制方面发挥了重大的作用，并消灭了得克萨斯州的郊狼和灰狐狂犬病。 - 65 -

在几个区域已经有野生食肉动物狂犬病的记录，从很多物种中分离到的狂犬病毒分离株的基因组进化研究表明存在很多不同的野生动物主要宿主，包括狨猴（狨属）和食蟹狐狸（食蟹狐属）。但是野生动物狂犬病的监测一般不足以得出大型流行病学推论。可以从泛美卫得到狂犬病存在的信息。生组织（ http://new.paho.org/rabies ）

十九世纪下半段为控制鼠害从南亚引入许多加勒比岛屿的印度小猫鼬（ Herpestes auropunctatus ）是部分加勒比地区的狂犬病主要宿主。例如，目前在古巴，多米尼加共和国，格林纳达和波多黎各已报道了猫鼬狂犬病。其他加勒比岛屿被认为在家养和野生食肉动物中没有狂犬病。

尽管还不是很清楚这些不被完全监控的人口稀少地区的流行病学，北极狐（蓝狐）、家犬和红狐参与了北极狂犬病或 “极地疯病” 的传播。有趣的是，北极狂犬病毒谱系还在亚州中部和东南亚被发现。

虽然在不同地区蝙蝠有不同的品种，狂犬病病毒已在世界各地的蝙蝠中被发现（24; 见第 2 节表 1 ）。蝙蝠已经被确定为除 Mokola 病毒和 Ikoma 狂犬病病毒之外所有狂犬病毒属病毒的载体（见第 2 节），这两种病毒的真正宿主还没有被发现。这一观察结果强烈表明，蝙蝠是狂犬病病毒的主要宿主。不同于那些食肉类动物狂犬病宿主，蝙蝠有几个特点（如体积小，寿命长，固有的较低群体增长率和各种界限分明的生态小环境）。因此，蝙蝠狂犬病病毒的特点肯定不同于那些导致食肉动物狂犬病的病毒。在蝙蝠中携带狂犬病所涉及的因素还没有得到充分探讨。

已知在非洲食虫及食果蝙蝠种群中至少有四种狂犬病病毒流行（见表 2 ，第 2 节）。主要与各种大型非洲食果蝙蝠（大蝙蝠亚目）相关的一种狂犬病病毒—— — 拉各斯蝙蝠病毒，最初于 1956 年在尼日利亚的黄毛果蝠中分离得到，后来从中非共和国、塞内加尔和南非的其他种类的蝙蝠中也分离到了该病毒。导致重大死亡的肩毛果蝠狂犬病的流行在纳塔尔、南非被观察到，在这些地区目前仍能偶尔分离到这种病毒。拉各斯蝙蝠病毒也偶尔从食虫冈比亚裂颜蝙蝠（裂颜蝠属）中分离到。目前尚没有证实存在人类病例，可能是由于监控或病毒特性鉴定不足。很少有拉各斯蝙蝠病毒向外传播到其他哺乳动物的报道（1,25 ）。 - 66 -

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Duvenhage 病毒首次于 1970 年在南非德兰士瓦省一个被长翼蝠属食虫蝙蝠咬伤后死于狂犬病脑炎的人中分离得到。已报道了另外两宗 Duvenhage 病毒感染引起的人类狂犬病病例，分别发生在南非和荷兰，后者是在肯尼亚被感染的（1）。在 2009 年，被称为希莫尼蝙蝠病毒的蝙蝠相关狂犬病病毒在肯尼亚从食虫叶鼻蝠（小蹄蝠）中分离。与莫科拉病毒和拉各斯蝙蝠病毒一样，它属于遗传谱系 II（26）（参见 2.3 节）。在 1996 年，从澳大利亚东海岸的食果蝙蝠（飞狐，中央狐蝠）中分离到了澳大利亚蝙蝠狂犬病病毒，而澳大利亚自 1867 年以来被认为是 “无狂犬病国家” 。在 1996 年和 1998 年被证实由澳大利亚蝙蝠狂犬病病毒引起的狂犬病导致了两个人的死亡。澳大利亚蝙蝠狂犬病病毒已从全部四种食果狐蝠（狐蝠属，大蝙蝠科）及一种食虫蝙蝠 - 黄腹鞘尾蝙蝠（囊喉墓蝠属）中分离出来（27,28）。在欧洲过去的 60 年中，在蝙蝠中有零星狂犬病散发病例已被确诊。大多数病例发生在大棕蝠（棕蝠属），该病毒被确定为欧洲蝙蝠狂犬病病毒 1 型，而那些从鼠耳蝠（ M. dasycneme M. daubentonii ）分离到的病毒则被定义为欧洲蝙蝠狂犬病病毒 2 型（29,30）。在欧洲蝙蝠狂犬病病例的出现没有新大陆那么频繁，然而，尽管有国际建议，在欧洲的监控水平仍然是非常庞杂的。总体上，在欧洲共发现了四例由本土蝙蝠传播的人狂犬病病例：俄罗斯联邦有两个病例（ 1977 年和 1985 年），一例在芬兰（1985），另一例在苏格兰（2002）（ 24 ）。在 2002 年，一只普通的弯曲翼蝙蝠（普通长翼蝠）在俄罗斯联邦格鲁吉亚边境附近被抓获，并在随后的狂犬病病毒感染测试中呈阳性。名为西高加索蝙蝠病毒的该病毒与其他来自蝙蝠狂犬病病毒属的病毒的基因不同，代表了第三遗传谱系的病毒，与其他狂犬病病毒没有血清学交叉反应性（31）。于 2010 年德国和 2012 年法国从纳氏鼠耳蝠 (Myotis nattereri) 中分离到的 Bokeloh 蝙蝠狂犬病病毒，已经显示出不同于在欧洲发生所有已知的狂犬病病毒，但在抗原性和遗传学上接近欧洲蝙蝠狂犬病病毒 2 型和 Khujand 病毒（32,33）。在 2012 年，从伊比利亚半岛上的长翼蝠中分离到了一株 Ikoma 型狂犬病毒（34）。在中亚，已分离出三中与蝙蝠相关的狂犬病病毒。在 1991 年，在吉尔吉斯斯坦阿拉万地区抓获的明显不是很健康的鼠耳蝠（狭耳鼠耳蝠），狂犬病小鼠接种试验检测呈阳性。十年后，塔吉克斯坦 Khujand 镇附近，一只须鼠耳蝠（Myotis mystacinus）同样检测呈阳性。随后对病毒分离株的鉴定显示两个新的狂犬病病毒种类： Aravan 病毒和 Khujand 病毒（35）。在 2002 年，一株来自俗称为管鼻蝙蝠的管鼻蝠属蝙蝠的病毒被鉴定为一种狂犬病毒属病毒，并以伊尔库茨克省的一个村庄命名为伊尔库特病毒。2007 年在俄罗斯远东地区报告了一例由于感染类似原始的的伊尔库特病毒造成的人狂犬病病例 31）。由于只分离到一次，我们对蝙蝠狂犬病病毒的流行病学知之甚少。 - 67 -

迄今为止，在美洲所有蝙蝠狂犬病病毒均被归类为狂犬病毒。许多基因和抗原类型不同的变种在蝙蝠中流行，有很多发生在同一物种内，而且变种的地理分布相互重叠。然而跨种间传播和食虫蝙蝠种群进化距离之间存在负相关关系（36,37 ）。经常观察病毒向其他动物扩散。虽然在北美洲温带地区人类狂犬病的发病率很低，近 50％的病例是由蝙蝠狂犬病相关病毒引起的（38）。银发蝙蝠（ Lasionycteris noctivagans ）和东部三色蝙蝠（ Parasrellus subflavus ）在蝙蝠狂犬病传播人类的活动中扮演关键角色。

吸血蝙蝠狂犬病是在从墨西哥到阿根廷的美洲亚热带和热带地区的一个重大的公共卫生问题。与其他美洲蝙蝠病毒相关的一种狂犬病毒变种在吸血蝙蝠，主要是圆头叶蝠中存在（37），并经常传播给家畜和人类。吸血蝙蝠传播的牛麻痹型狂犬病对畜牧行业有显著的经济影响。目前，大多数在亚马逊河流域的人类狂犬病是由吸血蝙蝠引起的（39 ）。

在世界各地狂犬病疫区对几十万野生和伴侣啮齿动物的检测显示，只有特殊情况下存在狂犬病毒感染的终端溢出，说明这些动物既不是主要宿主，也不能在该疾病的流行病学和传播中发挥作用。

当狂犬病在高度濒危的贝尔山国家公园埃塞俄比亚狼（ C. simensis ）、非洲东部和南部的非洲野犬（Lycaon pictus）和以色列的布兰福德狐狸中爆发后，其已成为保护这些种群的一个威胁。埃塞俄比亚狼和非洲野犬属于世界最濒危的食肉动物物种，而且从数量更丰富的主要宿主（如家犬）的狂犬病毒的传播被认为是一种致使几个种群灭绝的威胁。在北半球出现野生动物狂犬病的地区，到处都有狼(C. lupus)发生狂犬病的记录，因此常常认为它们在传播中发挥了重要作用。虽然狼易感且容易死于该疾病，但它们不能独立于其他野生动物维持狂犬病毒的循环，因为狼的群体密度和动力不能支持流行病的发生，而且狼的高度领土天性防止疾病从一个狼群蔓延到另一个。一只狼群成员一旦被感染，这种疾病可以消灭整个狼群，因为其具有高度的社会性，动物之间会进行定期接触。从狼分离的狂犬病毒的基因构成与其附近的数量更丰富的食肉动物主要宿主中发现的病毒是相同的（无论是家犬还是野生物种）。虽然狼是疾病的受害者而不是一个真正的主要宿主，它们可以向其他幼年易感宿主传播狂犬病毒。狼群发生狂犬病往往经历了一个戏剧性的过程，尤其是涉及到 - 68 -

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人。因为它们进行长距离迁移，携带狂犬病毒的狼被认为能够向无狂犬病区域重新引入野生动物狂犬病。

狂犬病病毒在能够维持一个感染周期的野生食肉动物中的传播被认为具有密度依赖性。通过急剧的大量毁灭野生食肉动物种群控制狂犬病的常规方法来消除狂犬病均以失败告终（ 17,40 ）。食肉类动物在被消灭时的恢复力，高繁殖力和环境提供食物，水和栖息地的能力往往使群体控制的努力付之东流。关于人性化、经济和生态方面的考虑将防止低效的大规模扑杀活动。

主要野生动物宿主的大规模疫苗接种是一种比剔除更有效的控制方法。该方法分别出现在欧洲和北美（ 22,40 ）。自 20 世纪 70 年代末，最初为狐狸狂犬病开发的口服狂犬病疫苗接种策略在欧洲西部和中部大部分地区，加拿大和美国被应用来消灭狐狸狂犬病。它的成功是由于工具开发的研究，包括有效、安全的疫苗，可以吸引很多物种的机器生产的诱饵，计算机支持的自动化空中布饵，有足够的接种策略和强有力的政策承诺（ 22,41 ）。对于一种食肉动物主要宿主物种的口服狂犬病疫苗接种策略，不一定要适用于其余物种。改良的口服疫苗策略在红狐狸和其他主要野生动物宿主，包括土狼，灰狐狸和貉中已相当成功，但是还需要优化浣熊策略（23）。其他主要野生动物宿主需要不同的战略。由于口服狂犬病疫苗接种计划的目的是通过建立一个免疫屏障（围堵政策，防疫封锁线）消除划定区域内的狂犬病或防止疾病的传播，应该诱导足够的群体免疫力以减少在目标主要野生宿主中的疾病传播（即疾病有效复制率低于 1）。要求群体免疫水平随着目标物种和种群中疾病的特定传播动力学和当地的条件而变化。野外使用的疫苗必须符合国家或国际监管机构对于生物制品的要求，如疗效，安全性和稳定性，并获得许可或注册（见第 7 节）。诱饵的设计必须针对每个目标野生动物物种，以确保可在该疫苗的易感靶组织（口咽粘膜或扁桃体）中释放，引起免疫反应。诱饵外壳必须满足三个功能：能够引诱目标物种，含有一种可以对摄取诱饵的目标种群进行标记的生物标志物（通常是四环素），可以保护疫苗泡、胶囊或小包免于紫外线照射，以确保稳定的病毒滴度。诱饵外壳的要求在相关的标准中进行了规定（42-46 ）。饵料必须是耐高温的，以保证其适口 - 69 -

性，而且它应该在获得上市许可前在不同温度下进行测试（44）。大多数狂犬病疫苗诱饵在投放到指定区域后 7 天之内消耗，诱饵外壳应在当地这个时候的天气条件下保护疫苗小包或小泡。泡罩或诱饵基质上应印有警告。诱饵的吸收和目标种群群体免疫力的产生依赖疫苗的有效性和稳定性、诱饵外壳和吸引力，引诱方法，诱饵的空间分布，口服狂犬病疫苗接种活动的时间和饵料竞争对手的丰富程度。诱饵分配的模式应保证能作用于大部分目标物种。口服狂犬病疫苗活动在欧洲通常每年进行两次，分别在春季和秋季，在北美每年一次，主要通过固定翼飞机或直升机投放诱饵（ 23,44 ）。应通过手动分配对空中投放进行补充或在种群稠密地区作为分发诱饵的唯一方式。

口服狂犬病疫苗已成为当主要宿主是野生动物时防止地域性扩散，控制和消灭狂犬病的的必备工具。由于口服狂犬病毒的疫苗和诱饵开发主要针对一个主要宿主物种，可能不能作用于其他物种，应评估针对每一个新的目标物种的疫苗的疗效、诱饵设计和吸引力。口服接种项目的评估应包括对公众健康的成本效益分析。已经公布了大规模疫苗接种活动或现场试验的规划、实施和评估的基本要求（43,44）并且最近进行了修订（45）。口服狂犬病疫苗接种程序或田间试验启动之前，必须获得基于针对目标和非目标物种（野生和家养）狂犬病病例的可靠监测和实验室研究的流行病学数据。

强有力的政治承诺是口服狂犬病疫苗接种计划的先决条件，因为法律体制、规划、组织和评估是其成功的关键。全国狂犬病委员会应由所有的利益相关者构成。一个有效的方案基于一个全面的计划，阐明理由（利益），目标，角色（哪些机构应参与），责任（谁应该负责什么）和命令以及基础设施（实验室需求和设备以及冷链），估计成本（预算要求）和资金。该计划还必须包括连续多年被覆盖的领域信息，要考虑到野生动物种群的运动模式，该地区的地理特征，邻国狂犬病的情况，疫苗接种策略的详细信息（时间，布饵模式，饵料密度，航线距离），安全考虑，活动的监测和监控。在疫苗接种前应当估计目标种群的大小以及生物标志物（如果适用）的基础水平。作为惯例，口服狂犬病疫苗接种计划应包括两个阶段：攻击阶段（消灭）和维护阶段。一项长期，大规模的方法是最有效的，必须担保项目可以长期维持。计划应分发给主管部门提前做好审议和评估。世卫组织可以根据需要提供必要的专业知识。

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他人员）和物流（冷链的维护），并确保足够数量的诱饵均匀覆盖在广阔的土地上。应该由全国狂犬病委员会为所有利益相关者，包括猎人，捕猎者，野生动物服务人员，森林官员，医生，兽医和地方当局组织启动会议，详细讨论活动计划，并对各利益相关者应付的责任达成一致意见。相关负责部门和人员应接受关于狂犬病监测、数据库管理、数据分析和解释的训练以便监控干预措施的进展，向主管机关报告和传递信息 ;监控疫苗的诱饵，目标物种和人类组成部分，并在适当的条件下进行标本采样。训练有素的人员和实验室设施，应可以开展推荐的常规诊断狂犬病的标准测试（见第 4 节）和在质量保证体系中监测接种活动（生物标志物检测，血清学，病毒滴度，狂犬病病毒分离株特性）。应该提高猎人、捕手、公众、医生和兽医的活动意识，使他们能够在意外暴露于疫苗的情况下采取适当的措施。应建立医疗或兽医咨询小组。强烈鼓励委派专家调查人类和动物的流行病学情况的现状和变化，对活动进行评估，并定期向主管部门报告。应定期举行国家会议，与所有利益攸关方讨论活动的进展以及未来的活动所需的任何变动。

口服狂犬病疫苗接种活动的监测和监督对于评估其成功是至关重要的。需要一个持续不断、精工细作的方法。足够的监测非常重要，因为狂犬病的发病率是一项活动产生的作用的一个指数。应使用一种以风险为基础的抽样方案，关注生病的、怀疑有狂犬病的、表现出异常行为的、被发现死亡或与人类暴露有关的所谓 “哨兵动物 “。虽然无法预先确定所需动物的确切数目，它应该处于可以得出可接受的确定性的统计结果的范围（18）。监控一般应在疫苗使用之前、期间和之后进行，不应只在疫苗接种的地区，而且应在邻近地区尤其是那些没有狂犬病的地区进行，以尽早检测动物疫情的蔓延或再感染，以便迅速做出响应和对策（45）。在免疫区从动物中分离到的狂犬病病毒应进行鉴定。协商会议强调了在接种地区和更远的地方增强监督的重要性，并要求各国政府考虑采纳上述指南。 - 71 -

监控口服狂犬病疫苗接种程序的效果（诱饵摄取，血清转换率），需要有足够猎取的或陷阱捕获的目标物种动物样本。建议的采样数为每年每 100 平方公里 4 只靶动物（ 18 ）；但是经验表明，这个样本大小可能很难实现，取决于疫苗接种区的地形特点，基础设施和物流。如果不能采集到此数目的样本，可以选择样本大小可以达成的参考区域。应该收集发现的所有动物的基本或共同数据，例如物种，发现和提交日期，地点（高斯克鲁格坐标或最低国家领土单位），年龄，性别，实验室的调查结果（荧光抗体或组织培养感染试验，病毒特征，生物标志物检测，血清学）并进行分类和适当的流行病学（时间和空间）分析。为了消除野生动物狂犬病，应建立 “逐步控制途径” 和国际无狂犬病状态认证程序。

在各级口服狂犬病疫苗接种计划的规划，实施和评估中，国际合作与协调对于获得成功和成本效益是必要的。当政策决定时应与邻国进行初步接触，并保持这些接触直到消灭本病。与邻近地区和国家的公共卫生和兽医主管部门的代表保持定期的多边会议可以确保协调共同边界的活动和透明度。建议邀请世卫组织合作中心和其他国际组织参与。应在国际会议上展示接种项目的结果，因为在国际舞台上展示可以向各国政府施加压力，迫使政府斥巨资致力于消灭狂犬病。

除了口服疫苗，战略诱捕野生食肉动物并在注射接种后释放它们（陷阱 - 预防接种 - 释放）已在北美的一些地区主要是臭鼬和浣熊上应用，并获得了明显成功（46,47 ）。

在蝙蝠中消灭狂犬病的目标被过多的狂犬病病毒种类和翼手目在全球生态方面实质性的作用如在种子传播、授粉和节肢动物捕食中的作用所阻碍。因此在目前消灭蝙蝠狂犬病不可能的。虽然感染的后果也很严重，与蝙蝠狂犬病（除吸血蝙蝠传播）相关的公共健康风险低于食肉动物狂犬病。因此，应该避免不分青红皂白地消灭蝙蝠的方法，特别是蝙蝠在大多数国家是受保护的。公众教育是防止蝙蝠传播的人狂犬病的关键。它应包括避免与蝙蝠潜在的传染性接触，在暴露后寻求适当的医疗照顾以及防止蝙蝠在敏感建筑（如医院和学校）建立栖息地的基本信息。吸血蝙蝠传播的牛麻痹型狂犬病可以通过对牛免疫接种控制。通过在捕获的蝙蝠的背 - 72 -

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上或对牛直接肌肉注射疫苗或以抗凝剂来拣选主要宿主物种的方法来控制吸血蝙蝠传播狂犬病的方法是有问题的、过时的。建议在人接触到吸血蝙蝠的情况下严格应用暴露后预防方法。应考虑对在狂犬病高度流行以及抗狂犬病生物制品有限的地区生活的人进行预防接种。

市民应更好地了解一般情况下避免与野生动物直接接触，特别是行为异常的病畜。任何人被野生或家养动物咬伤，尤其是在野生动物狂犬病呈地方性流行的地区，应主动就医（见第 7 节）。应禁止或极力阻止除保护野生动物以外的任何目的的迁移行为。

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36. Streicker DG et al. Host phylogeny constrains cross-species emergence and establishment of rabies virus in bats. Science, 2010, 329:676-679. 37. Streicker DG et al. Ecological and anthropogenic drivers of rabies exposure in vampire bats: implications for transmission and control. Proceedings of the Royal Society B. Biological Sciences, 2012, 279:3384-3392. 38. De Serres G et al. Bat rabies in the United States and Canada from 1950 through 2007: human cases with and without bat contact. Clinical Infectious Diseases, 2008, 46(9):1329-1337. 39. Schneider MC et al. Rabies transmitted by vampire bats to humans: an emerging zoonotic disease in Latin America Revista Panamericana de Salud Pública, 2009, 25(3):260-269. 40. WHO Expert Consultation on Rabies. First report. Geneva, World Health Organization, 2005 (WHO Technical Report Series, No. 931). 41. Müller T et al. Rabies elimination in Europe-a success story. In:Compendium of the OIE Global Conference on Rabies Control, Seoul,Korea, 7-9 September 2012. 42. Manual of diagnostic tests and vaccines for terrestrial animals. Chapter2.1.13. Rabies. Paris, World Organisation for Animal Health, 2012 (http://www.oie.int/international-standard-setting/terrestrial-manual/access-online/; accessed 4 December 2012). 43. Report of a WHO seminar on wildlife rabies control, Geneva, Switzerland, 2-5 July 1990. Geneva, World Health Organization, 1990 (WHO/CDS/VPH/90.93). 44. Report of the WHO/APHIS consultation on baits and baiting delivery systems for oral immunization of wildlife against rabies. Geneva, World Health Organization, 1990 (WHO/Rab. Res./90.36). 45. Blueprint for rabies prevention and control [fox rabies blueprint]. Partners for Rabies Prevention (www.rabiesblueprint.com; accessed March 2013). 46. Rosatte RC et al. Trap vaccinate release and oral vaccination for rabies control in urban skunks, raccoons and foxes. Journal of Wildlife Diseases, 1992, 28(4):562-571. 47. Slavinski S et al. Trap vaccinate release program to control raccoon rabies, New York, USA. Emerging Infectious Diseases, 2012, 18(7):1170-1172.

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11．狂犬病监测所谓监测，即系统、连续的收集，分析和解释数据，并将其传递给合适的人以采取行动（1）。其目的是为了论证疾病不存在或确定疾病存在或其分布，在不同部门之间及时传递信息以便进行整体行动（2）。监测与监控的概念截然不同，世界动物卫生组织对监控的定义是间歇性的执行和对日常测量和观察的分析，以发现环境或群体健康状况的变化。狂犬病控制的监控可能包括通过家庭调查、观察大规模注射接种时应用于犬身上的标记（见第 7 节）以及野生动物口服免疫活动中的诱饵摄取来评估狂犬病疫苗接种覆盖率。在第 8 部分给出了关于在野生动物监测狂犬病和监控口服接种项目的进一步细节。因此狂犬病的监测包括衡量疾病在人类和动物中的发病率。发病率的测量对于狂犬病预防和控制中确保对于病例和暴发进行适当管理，监测发展趋势以评估干预措施的有效性，以及估计疾病负担是必不可少的。狂犬病的监测还包括通过适当的渠道如世界动物卫生信息系统和的数据库（ WAHIS 和 WAHID），全球预警系统（ GLEWS ）， Empress 嵌入式数据系统，欧洲狂犬病通报以及官方区域数据库进行数据共享。狂犬病应纳入国家卫生和兽医服务法定传染病。及时对监测活动和结果作出反应将激励现场和医院工作人员继续报告病例（见 4.1 节病例定义标准和 4.2 节人类临床诊断）。最低限度的反应应包括报告的迅速确认、对诊断测试结果的反馈以及对病例和疫情管理的建议。与在现场的医疗和兽医工作人员保持联系可以确保适当的管理和病例的跟进，提高病例检出率。为了确保有效，狂犬病的监测必须立足于人类和动物疑似及可能病例的确认诊断。建议诊断设施缺乏或不足的国家通过 OIE 实验室组队项目及与世卫组织合作中心联络来加强其实验室的能力。私人和公共兽医、动物卫生工作者，狩猎督导员和其他专业人员的参与是必不可少的，因为他们是最有可能看到了临床狂犬病患犬的专业人士。他们应该知道疑似病例的临床症状，样品的采集方法和报告过程。收集和提交样品的基础设施和资源的缺乏比诊断设备的缺乏对于狂犬病监测来说往往是一个更大的障碍（3）。应根据风险进行动物狂犬病的监测，因此应重点调查和诊断疑似病例。当动物出现狂犬病临床症状如无故咬人、动物呈病态或死亡时，可能怀疑为狂犬病。在动物中狂犬病的临床症状有很大的不同。经典的迹象包括异常行为，发声改变，异食癖，性欲亢进，流口水唾液，漫无目的的游走， ' 飞身猛咬 ' ， ' 如骨在喉 ' 综合征，侵略性，动作不协调，瘫痪和抽搐。在狂犬病流行地区，因压抑和异常行为死亡的野生动物（如白天活动的夜行性动物）的损失应怀疑为狂犬病。在动物尸体上，沾满土壤的嘴可以表明撕咬行为异常。神经过敏不是狂犬病的动物的一个特征。 - 77 -

即使在已经成功地消灭了犬狂犬病的国家也应保持监测。最近在印度尼西亚几个无狂犬病岛屿出现的犬狂犬病（4,5）和欧洲由于从狂犬病流行地区非法进口宠物和伴侣动物传播造成的代价惨重的爆发（6）表明了这种监测的重要性。特别是考虑到国际旅行和动物运动的增加，鼓励对狂犬病病例和暴发的病毒分离株进行常规鉴定，以确定动物宿主的起源，感染来源及地域分布（ 7,8 ）。不推荐将狂犬病的特异性抗体检测用于常规狂犬病的监测。除了实验室确诊病例，还应记录和报告疑似和可能患病的动物、动物咬伤人数以及寻求和接受暴露后预防的人的情况。此信息应当与医学和兽医部门共享，以方便管理动物咬伤、疫情调查和控制措施的实施。

1. Making surveillance work [modules 1-4]. Geneva, World Health Organization Department of Vaccines and Biologicals, 2001 (V&B/00.08to 00.11). 2. Terrestrial animal health code [Chapter 1. Animal disease diagnosis,surveillance and notification, section 1.4. Surveillance]. Paris, World Organisation for Animal Health, 2012 http://www.oie.int/internationalstandard-setting/terrestrial-code/access-online/; accessed 26 November2012). 3. Halliday J et al. Bringing together emerging and endemic zoonoses surveillance: shared challenges and a common solution. Philosophical Transactions of the Royal Society of London B, 2012, 367: 2872-2880. 4. Windiyaningsih C et al. The rabies epidemic on Flores Island, Indonesia (1998-2003). Journal of the Medical Association of Thailand, 2004,87(11):1389-1393. 5. Susilawathi NM et al. Epidemiological and clinical features of human rabies cases in Bali 2008-2010. BMC Infectious Diseases, 2012, 12:81. 6. Lardon Z et al. Imported episodic rabies increases patient demand for and physician delivery of antirabies prophylaxis. PLoS Neglected Tropical Diseases, 2010, 4(6):e723. 7. Bourhy H et al. The origin and phylogeography of dog rabies virus. Journal of General Virology, 2008, 89:2673-2681. 8. Talbi C et al. Phylodynamics and human-mediated dispersal of a zoonoticvirus. PLoS Pathogens, 2010, 6(10):e1001166. - 78 -

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12. 无狂犬病国家和地区为协助公共卫生部门进行与动物接触后感染狂犬病的风险评估，本次研讨会定义了三种类型的无风险国家或地区：无犬类狂犬病地区，无野生动物（除蝙蝠外）狂犬病地区和无狂犬病病毒地区。这些无狂犬病国家的定义与世界动物卫生组织目前的以动物运动为目的的定义不同（1）。

应公布所有动物物种和人类的狂犬病，并保持持续有效的监测系统的运行。系统已有或随时可以与能够应用世界卫生组织（2）或世界动物卫生组织（3）推荐的用于狂犬病诊断技术的狂犬病实验室联系。应对主要易感家畜和野生动物品种中的疑似病例进行足够数量的样本检测。应由适当的国家机关设置用于定义样本大小的统计学意义的水平。国家主管部门应确保在全国各地收集样本。有效的进口政策到位，如采取防止引进狂犬病的措施，尤其是在第 13 部分提到的。一个无狂犬病风险的国家或地区的定义如下：在过去 2 年的任何时间，未发生人类、犬或猫或其他动物物种的本地获得的狂犬病病毒的确诊感染。任何本地阳性病例必须通过分子鉴定表明是由野生动物向外传播的。如果确诊了进口食肉动物病例，分子鉴定证实该病毒为非本土来源以及反复的流行病学追踪没有显示出犬类发生二次感染的证据，则该国家或地区的地位不会受到影响。

在过去 2 年的任何时间，未发生人类或任何家养或野生动物物种的本地获得的野生动物狂犬病病毒的确诊感染。任何本地阳性病例必须被证明是由蝙蝠或犬向外扩散的。如果确诊了进口动物的病例，分子鉴定证实该病毒为非本土来源以及反复的流行病学追踪没有显示出野生或家养食肉动物发生继发感染的证据，则该国家或地区的地位不会受到影响。 - 79 -

一些野生动物（如猫鼬）感染的血清学证据应作为考虑是否存在狂犬病的一个指标。

在过去 2 年的任何时间，未发生人类或任何家养或野生动物物种包括蝙蝠的本地获得的狂犬病病毒的确诊感染。如果确诊了进口动物的病例，分子鉴定证实该病毒为非本土来源以及反复的流行病学追踪没有显示出任何物种发生继发感染的证据，则该国家或地区的地位不会受到影响。蝙蝠感染的血清学证据，应作为考虑是否存在狂犬病的一个指标。在上述任何类别的国家或地区，应采取额外措施如对犬和其他宠物接种疫苗。随着时间的推移，在先前界定的无狂犬病病毒风险的国家或地区的边界发生的几起病例应足以令国家当局怀疑狂犬病可能来自本土而非进口。应参考第 8 部分来决定是否使用人类暴露前或暴露后预防。

1. Terrestrial animal health code [vol. 2, chapter 8.10: Rabies]. Paris, World Organisation for Animal Health, 2011 (http://www.oie.int/index.php?id =169&L=0&htmfile=chapitre_1.8.10.htm; accessed 21 September 2012). 2. Laboratory techniques in rabies, 4th ed. Geneva, World Health Organization, 1996. 3. Manual of diagnostic tests and vaccines for terrestrial animals [vol. 1,chapter 2.1.12]. Paris, World Organisation for Animal Health, 2011 (http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.01.13_RABIES.pdf; accessed 21 September 2012).

13. 动物的国际间转运从无狂犬病国家或认为存在狂犬病感染的国家进口家养、野生和野生哺乳动物的条例应符合世界动物卫生组织的标准（1），包括展示一份有效的国际兽医证书（2）。国际标准取决于起源于该国家的狂犬病的状态和所涉及的动物物种。

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国家进口当局应该需要一个国际兽医证书证明动物在装运时没有狂犬病迹象，进行了永久鉴定，接种或重新接种疫苗以及在装运前进行阳性血清学试验。应遵循 OIE 国际标准。附件 7 给出了国际狂犬病疫苗接种证书的模版。

这些动物应符合世界动物卫生组织标准（3），其中包括家养动物，实验用啮齿类动物 / 兔类和野生动物的兽医证明、永久身份证明、家养反刍动物、马属动物、骆驼和猪的非强制疫苗接种以及一份动物未在运输当天出现狂犬病迹象的声明，特别是对实验室和野生动物，它们需要在运输前至少 12 个月内在隔离场所进行封锁或其他相关隔离措施，运输前 6 个月内无狂犬病病例的出现。无狂犬病国家应该禁止特定种类哺乳动物（特别是食肉类和翼手目动物）的进口，或根据在隔离区检疫的情况以及在政府兽医部门批准的条件下，仅允许有执照的动物进入。允许入境停留的时间可以有一定的期限或为永久性。鉴于作为宠物的野生动物出现狂犬病病例数的增加，国家有关部门应限制这种动物的贸易。应该对饲养此类动物作为宠物的行为进行劝阻。

对于无狂犬病国家已有的、残疾人使用的经过认证的导盲犬和其他服务犬（如军犬和搜寻犬），如果犬已接种了满足 WHO 和 OIE 标准的细胞培养疫苗、且经 OIE （3）和 WHO （4）推荐的两种方法之一的检测结果显示有足够的病毒中和抗体滴度，则该犬应被允许陪伴主人进入有狂犬病感染的国家。这些犬必须有微芯片的身份证明。如果主人确定犬在另一个有狂犬病的国家时，一直有犬链束缚，或从未脱离主人的视线监督，则可允许该犬在该国家停留最长 6 个月的时间，而且除了需要再确认抗体滴度之外，无需任何其他的再入境条件。

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1. Terrestrial animal health code [vol. 2, chapter 8.10: Rabies]. Paris, World Organisation for Animal Health, 2011. 2. Terrestrial animal health code [vol. 1, chapter 5.11: Model international veterinary certificate for dogs and cats originating from rabies infected countries]. Paris, World Organisation for Animal Health, 2012 (http://www.oie.int/index.php?id=169&L=0&htmfile=chapitre_1.5.11.htm;accessed 21 September 2012). 3. Manual of diagnostic tests and vaccines for terrestrial animals [vol. 1,chapter 2.1.12]. Paris, World Organisation for Animal Health, 2011 (http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.01.13_RABIES.pdf; accessed 21 September 2012). 4. WHO Expert Consultation on Rabies. First report. Geneva, World Health Organization, 2005 (WHO Technical Report Series, No. 931).

14．有关狂犬病的全球性和地区性活动自从第一届 WHO 狂犬病专家磋商会报告公布以后（1），针对于狂犬病在国家、地区乃至国际层面都进行了很多活动。越来越多的成员（包括政府间和非政府组织，公立和私营性质的机构和基金会等）参加到预防、控制和消除人间和动物狂犬病的队伍中来，例如：联合国粮食与农业组织（FAO）、国际兽医局（OIE）、东南亚国家联盟（ASEAN）、南亚区域合作联盟（GARC）、英联邦兽医协会、国际人道协会、亚洲狂犬病基金会、无国界兽医组织、世界动物保护协会、比尔和梅林达 · 盖茨基金会等。这些成员已经筹备了全球性标准和政策，辅助资源动员，提供地区协调或者直接支持国家性项目等。

1990 年创立的 WHO 全球狂犬病调查，通过后续建立的计算机数据管理系统来采集处理国家层面的数据，即 Rabnet，而得到了进一步加强。这个系统在 2000 年后得到进一步改良，增加了一些新特点，如产生全球和国家级的相互影响的地图以及自定义的图、表和地图。设计这个数据库是为了分析狂犬病的全球趋势以及地区和国家的变化。然而，每年由指定的国家级狂犬病中心提供的独立报告能输入该系统的过于少，导致对这些数据的分析没有意义，因此该系统在 2010 年关闭。WHO、 WHO 地区办公室和狂犬病合作中心以及 GARC 正在寻求其它方法来收集资料并产生人和动物狂犬病的年度报告。人、家养动物和野生动物的狂犬病信息应该在部门间共享。 - 82 -

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“被忽视的人兽共患病” 概念是在 2005 年 9 月份 WHO 总部的一次会议（2）上提出的，而后在 2007 年（3）和 2010 年（4）的国际会议上重申。这组疾病 “被忽视的” 术语是指政府和国际社会未充分给予应有的重视，这类疾病主要局限于居住在发展中国家偏远农村或城市贫民窟的人群。这个术语现在已经得到了国际广泛认可。不幸的是狂犬病具备被 “被忽视的人畜共患病” 的所有特点。然而，由于具有可应用的预防措施，狂犬病又是最应该得到控制的。狂犬病是被忽视疾病中排在第一位的要求地区并全球性最终消灭的疾病。一个跨部门的会议提议在 “优先被忽视人兽共患病控制” 方面进行投资，其中包括在拉丁美洲和亚洲区域性消除犬—人传播的狂犬病（5）。前期预算表明在接下来的 5 年大约每年要投入 1000 万美金才能达到预期的 2016 年的结果。狂犬病在第一届和第二届 WHO 被忽视热带病的报告中已充分讨论过（6,7），在 2012 年出版的 “实施规划” 的执行总结中（8）关于地区性消除的目标疾病的清单中也包括了狂犬病。狂犬病也是 2012 年 WHO 发表的热带病研究和培训特别项目（TDR）中人兽共患病参考组的技术报告中的主要病毒性人兽共患病之一（9,10）。 WHO 发表的《国际旅行与健康》提供关于降低狂犬病风险的建议和地图（参考 6.8 章节）并且会定期更新，以提醒国际旅行者根据自己的目的地进行必要的暴露前预防（11）。自从 2004 年上一届专家磋商会以来，为实现其为各成员国在狂犬病预防方面提供指导的目的， WHO 在预防国际公共卫生重要性疾病的疫苗和复合疫苗的系列定期更新意见书中已经发表了针对狂犬病疫苗的意见书（12）。该意见书于 2010 年发表，是基于 2009 年在法国阿纳西举办的预防和控制人间和动物狂犬病的 WHO 磋商会的结果（13）。这份意见书，取代了 2002 年出版的意见书，并经 WHO 在疫苗和免疫方面的战略咨询专家组审阅和签署（12）。该意见书主要供国家公共卫生部门和免疫规划管理者使用，以及对此感兴趣的国际基金机构、疫苗制造企业、医学团体、科学媒体和大众等使用。自 2002 年起， WHO 就建立并定期维护一个提供人间和动物狂犬病，人和动物疫苗以及暴露前和暴露后预防信息的网站。这个网站也囊括了 WHO 报告和经同行评议的文章。自 2009 开始，这个网站提供了在所选择的发展中国家（南非的夸祖鲁 - 纳塔尔省，坦桑尼亚联合共和国西南部和菲律宾的米沙鄢群岛）中实施的消除人和犬狂犬病五年（2009-2013）试点项目实施过程所取得的进展的信息，该项目由比尔和梅琳达.盖茨基金会提供资金支持并由 WHO 组织实施（14,15）。

WHO 驻东南亚地区办公室已积极地在为区域内成员国人和动物狂犬病的预防和控制 - 83 -

制定标准和指南、提出建议并提供技术支持等。提倡使用成本效益高的皮内疫苗免疫以改进现代狂犬疫苗的可及性和可负担性，并逐步停止神经组织疫苗的生产和使用。自 2005 年以来，神经组织疫苗的生产和使用已经在孟加拉、柬埔寨、印度、老挝人民民主共和国、尼泊尔和越南被取缔。在孟加拉、印度的 WHO 狂犬病诊断合作中心与美国疾病预防控制中心的 WHO 狂犬病参考研究合作中心进行合作，将直接快速免疫组化（DRIT）的方法引进到这一地区，并通过 2010 年在班加罗尔组织的区域性狂犬病诊断的手把手培训将该检测方法的应用进行了实验室人员的培训。为了巩固在成员国中控制人兽共患病，尤其是狂犬病，方面取得的成就，地区办公室还组织了会议（16,17）并制定了消除由犬传播的人狂犬病的区域性策略。目的是在 2020 年，在狂犬病地方性流行的国家通过逐步控制犬的狂犬病和人的预防实现消除人间狂犬病并且维持该地区的无狂犬病状态(17)。

泛美健康组织 /WHO 驻巴西里约热内卢美洲地区办公室的兽医公共健康部领导实施了犬传播的人狂犬病的消除计划。这项计划在拉丁美洲主要城市消除狂犬病的项目，起始于 1983 年，一直延续到 1992 年在小的聚居区和乡村地区消除犬传播的狂犬病。自 1983 以来，犬传播狂犬病的发生在稳步下降，与此同时人和犬的病例减少了约 90%。兽医公共卫生部举办了一系列关于卫生和农业的美洲国家间部长级会议，讨论部门间政策，其中包括地区狂犬病消除计划。每隔两年，这个部门还召开国家狂犬病消除计划的负责人会议，在这个会议上讨论并更新狂犬病的流行形式和预防策略。这些结果和建议会在部长会议上提交给卫生和农业部长们以引起他们的关注和认可。 2006 年在巴西利亚召开的第十一届国家计划负责人会议建议在 2012 年之前在半个地球内消除犬传播的人狂犬病， 2008 年在里约热内卢召开的第十五届部长级会议上得到了与会的卫生和农业部长们对这一目标的承诺（18）。2009 年，泛美卫生组织第四十九届指导委员会在 CD49.R9 决议中将 2015 年定为区域性消除所有被忽视疾病和其他与贫穷相关的感染的目标日期，这其中包括了狂犬病（19）。会根据各成员美洲狂犬病流行病学监测的区域性信息系统（http://siepi.panaftosa.org.br/）国的卫生和农业部门输入该系统的官方数据产生人和动物狂犬病的报告。1970 年以后的数据可以通过在线咨询获得。

狂犬病合作中心网络几乎从 WHO 成立伊始就建立起来用以支持 WHO 在国家、国家之间、地区、地区之间和全球层面的活动。这些合作中心也参与加强成员国机构能力，包括信 - 84 -

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息、服务、研究以及狂犬病相关活动的培训，如诊断、监测、研究以及消除人和动物狂犬病的项目和计划的监控和评估。这些中心是由 WHO 根据共同商定的工作计划基础上正式任命，通常为 4 年，在经 WHO 的年度评估后可延续。这些工作计划取决于这些中心的专长或特点，通常包括以下几方面： ■ 狂犬病信息的收集、整理和宣传； ■ 狂犬病诊断试剂、预防和治疗制剂以及它们的应用方法和步骤的标准化； ■ 适当技术的设计和应用； ■ 提供参考品及其他服务； ■ 参与所组织领导的合作研究； ■ 培训，包括研究培训； ■ 由多机构进行的协调活动。现有 12 个 WHO 指定的合作中心，大多数为狂犬病参考和研究中心。其中 5 个在亚洲， 4 个在欧洲， 3 个在美国（见附录 8）。由德国 Friedrich-Loeffler 研究所主持的 WHO 狂犬病监测和研究合作中心出版了 WHO 狂犬病欧洲公报（参照 14.2.2 章节）。

在过去的十年里，一些旨在控制并最终消除狂犬病的全球性和区域性活动迅速展开并持续蓬勃发展。以下举例讲述。

FAO 在一些非洲和亚洲国家通过提高动物狂犬病控制的意识并提供政策性建议和技术支持为狂犬病控制做出了贡献。 FAO 在塞拉利昂支持学校动物健康俱乐部，致力于预防和控制狂犬病的合作和联盟，如狂犬病预防合作团体和 GARC。FAO 已经联同世界动物保护协会、 OIE 和 WHO 一起，组织全球利益相关者进行通过犬群管理控制狂犬病的磋商，以期寻求致力于消除犬传播的人狂犬病的 ‘逐步控制途径’ 并最终消除狂犬病（20）。

OIE 是一个政府间组织，旨在提供基于科学的标准、指南和建议用于控制动物间传染病，包括可传播至人的传染病，如狂犬病。《OIE 陆生动物诊断试验和疫苗手册》（21）公布了国际上认可的实验室诊断方法和对动物狂犬病疫苗及其它生物制品的生产和控制要求。《 OIE 陆 - 85 -

生动物卫生法典》（22）列出了国际上已采用的狂犬病控制措施。通过参比实验室互联网（http://www.oie.int/en/our-scientific-expertise/reference-laboratories/list-of-laboratories）和合作中心（http://www.oie.int/en/our-scientific-expertise/collaborating-centres/list-of-centres）， OIE 提供政策性建议、策略计划和技术支持用于动物狂犬病的诊断、控制和消除。 2011 年， OIE 联同 WHO 和 FAO 在韩国组织召开了题为 “从源头上可持续性预防 ” 的全球狂犬病会议，由此提高了负责部门和决策者对从动物源头控制狂犬病的重要性的意识，并再次强调了国家兽医服务在预防和控制狂犬病中的作用（23）。

GARC 是唯一一个致力于减轻全球狂犬病负担的注册慈善机构。它有两个分支机构：在美国的全球狂犬病控制联盟和建于苏格兰的狂犬病控制联盟。GARC 的使命是消除狂犬病。所致的人类死亡并减轻动物狂犬病尤其是犬狂犬病的负担（http://www.rabiescontrol.net/） GARC 与非洲和亚洲的政府和团体进行合作，计划和引导部门间（或 “ 一个健康 ” ）可持续的狂犬病控制项目，这些项目的基金来源于政府间的合作、国际性基金、个人捐赠和动物福利组织。GARC 已建了一个教育物资储备库用于世界狂犬病日网站提供给个人和组织对于提高其所在区域公众意识的材料需求。 GARC 是狂犬病预防合作伙伴的一员并有助于其建立过程（24）。这个非正式团体包括涉及狂犬病的主要国际机构： WHO、 FAO、 OIE、 WHO 狂犬病合作中心、科学家、比尔和梅琳达.盖茨基金会的代表、 UBS （瑞士联合银行集团） Optimus 基金和工业代表。狂犬病预防合作伙伴已经出版了狂犬病预防和控制的蓝皮书（25）。世界狂犬病日由 GARC 于 2006 年发起。现在它包括了人类和动物健康方面的所有合作伙伴，包括国际、国家、州和地方、兽医、医学和其他职业性或学生组织、团体的和非营利组织成员。它的目标是提高公众意识并动员人狂犬病预防和动物狂犬病控制的资源。2007 年 9 月的开幕典礼活动有分布于 74 个国家的将近 400000 人参加。这对狂犬病预防和控制是重要的一步并且表明了广为认同的对于这一易于预防的疾病采取行动进行控制的必要。已有 150 个国家举行过世界狂犬病日活动，对 182000000 人进行了健康教育并对 7700000 只犬进行了疫苗注射。

东南非洲狂犬病组始建于 1992 年，旨在控制犬狂犬病。每两年举行一次正式会议，以标准的国家报告形式介绍狂犬病数据，这些报告随后发布在一个开放登录的网站上（http: //www.searg.info）。这些国家报告描述了人类、家养和野生动物的狂犬病情况并概述对疫苗采 - 86 -

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购或生产以及疫苗免疫策略的需求。这些会议有助于改进诊断、监测和意识并强调了在非洲缺乏对狂犬病真实负担的认识。第十届会议于 2011 年在莫桑比克的马普托召开，下一届会议将于 2013 年在坦桑尼亚联合共和国的达累斯萨拉姆召开。是非洲法语国家狂犬病专家的一个非正式网非洲狂犬病专家局（http://www.afroreb.info/）络，建立于 2008 年。成员们会定期会晤以回顾这些国家的狂犬病形势、分享经验并讨论任何遇到的问题和可能的解决方法。这些会议的报告发表在国际期刊上。该专家局提供了一个法语狂犬病专家交流信息和与其他狂犬病专家网络联系的平台。

FAO 为一些亚洲国家提供了控制动物狂犬病的技术支持，特别是印度尼西亚。 OIE 建议全国兽医服务于亚太地区犬狂犬病控制和犬群管理并在由欧盟支持的一个项目内向特定国家提供犬的狂犬病疫苗。这个由 FAO、 OIE 和 WHO 实施的 4 年期项目（2009-2013）的目的是加强各国以及两个主要区域组织， ASEAN 和南亚区域合作联盟，的能力，以提高包括狂犬病在内的动物源疾病的区域间合作。逐步控制亚太地区跨界动物疾病的全球框架已经将狂犬病作为人与动物间优先考虑的疾病，并呼吁加强国家和地区的政治保证。 ASEAN 和南亚区域合作联盟的成员国也将狂犬病确定为优先解决的公共卫生问题，并且政府也表达了对消除人狂犬病的关注与承诺。 ASEAN 成员国采纳了一份预防和控制狂犬病的行动倡议，目标是在 2020 年消除狂犬病（26）。亚洲狂犬病基金会在 2009 年会议上决定采取七个步骤实现 2020 年消除人和犬狂犬病，要求东南亚和西太平洋地区的 WHO 区域委员会满足各成员国对技术支持和技术转让的需求，并发起在亚洲协同区域性组织控制和消除犬狂犬病的活动。 ASEAN、 FAO、 OIE 和 WHO 于 2012 年 1 月在泰国清迈组织了一个狂犬病研讨会，共有 12 个亚洲国家的动物和人类卫生的负责官员参加了该会议。各国介绍了自己的进展，并决定通过控制和消除计划一致努力消除区域性狂犬病（27）。亚洲狂犬病专家局是一个创建于 2004 年的狂犬病专家的非正式网络。其成员定期会晤以回顾所在国家的狂犬病形势，分享经验并讨论遇到的任何问题和解决的方法。有关的会议。报告可以在其网站上查阅（http://www.areb.info）

美洲国家狂犬病国际会议 (http://www.rabiesintheamericas.org/) 每年举办一次以回顾和讨论该地区的狂犬病研究和控制。该会议有一个由巴西、加拿大、墨西哥和美国的代表组成的 - 87 -

国际委员会。第二十三届会议于 2012 年十月在巴西召开。

中东和东欧狂犬病专家局（http://www.meereb.info）是一个创建于 2010 年的狂犬病专家非正式网络。成员们定期会晤以回顾所在国家的狂犬病形势、分享经验并讨论遇到的任何问题和可能的解决方法。有关的会议报告会发表在国际期刊上（28）。

WHO 和德国的 Friedrich-Loeffler 学会于 1977 年创立了 WHO 欧洲狂犬病通报这一狂犬病报告系统，该系统由学会的 WHO 狂犬病监测和研究合作中心主办。这个系统不断更新，并且所有报告的数据都会自动转入数据库，由行政单位总结和合计每个国家的数据。有超过 40 个欧洲国家每个季度报告官方确认的狂犬病例数，包括发生在野生和家养动物以及人的病例。欧洲狂犬病通报每个季度都会刊印，也可以从 www.who-rabies-bulletin.org/ 获取免费的电子版本。该网站也可进行动态数据查询。自 1990 年起，狂犬病病例地图已经在线显示，并且从 2009 年开始监测数据也可以绘制成图。该通报为普通大众和科学团体提供有价值的信息。欧盟成员国在食物链和动物健康常务委员会会议上定期交换狂犬病信息。 2013 年，欧盟在一个计划加强监测动物疾病根除用以评估共同筹资在成员国和邻近非成员国中口服狂犬病疫苗免疫战役的任务中建立了一个狂犬病分组。这个分组包括了私有的和政府的狂犬病专家，这些专家受欧盟委员会之邀访问各成员国。其关于改进口服狂犬病疫苗免疫项目的结论和意见递交给欧盟委员会和各成员国以供参考。这些报告可以提供给公众（http://ec. europa.eu/food/animal/diseases/eradication/taskforce_en.htm）。欧盟委员会食物和兽医办公室对各成员国共同筹资的狂犬病消除项目在各个水平的执行情况进行现场检查。其报告可在网站上获得（http://ec.europa.eu/food/fvo/inspectprog/policy_papers/index_en.htm）。在法国南锡的欧盟狂犬病参比实验室自 2008 年起组织欧洲国家狂犬病实验室年会以统一和规范诊断技术。欧盟也通过欧盟扩大委员会的一个仪器管理总局，进行技术支持和信息交流以支持成员国的工作。有关狂犬病的近期任务和研讨会相关信息可在线查询（http: //ec.europa.eu/enlargement/taiex/dyn/taiex-events/index_en.jsp）。独立的多边会议会和执行狂犬病口服疫苗免疫项目的邻近国家公共卫生和兽医主管部门代表联合组织。欧洲疾病预防与控制中心的建立是为了增强欧盟预防和控制传染病的能 - 88 -

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力，该组织于 2009 年 1 月与 WHO 一起组织了一个磋商会回顾欧洲狂犬病流行形势，找出管理暴露后预防的途径并寻找解决狂犬病生物制品短缺的方法，包括建立一个虚拟贮存的可能性（29）。欧洲监测杂志介绍和讨论人狂犬病病例及狂犬病流行病学，这是一个由同行评审刊载与欧洲有关的传染病流行病学、监测、预防和控制方面文章的杂志。该杂志由欧洲疾病预防与控制中心出版（http://www.eurosurveillance.org/）。

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22. Terrestrial animal health code. Paris, World Organisation for Animal Health, 2011 (http://www. oie.int/index.php?id=169&L=0&.htm; accessed 29 November 2012). 23. Globalconferenceonrabiescontrol.Towardssustainablepreventionatthesource,Incheon-Seoul,Republic of Korea, 7 9 September 2011 [recommendations]. Paris, World Organisation for Animal Health, 2011 （ http://www.oie.int/fileadmin/Home/eng/Conferences_Events/docs/pdf/recommendations/A_Recommendation_Global%20Rabies%20Conference%20Seoul_final.pdf ） . 24. Lembo T et al. Renewed global partnerships and redesigned roadmaps for rabies prevention and control. Veterinary Medicine International,2011 (ID 923149, doi:10.4061/2011/923149). 25. Lembo T et al. The blueprint for rabies prevention and control: a novel operational toolkit for rabies elimination. PLoS Neglected Tropical Diseases, 2012, 6(2):e1388. 26. Call for action: towards the elimination of rabies in the ASEAN Member States and the Plus Three Countries. Jakarta, Association of Southeast Asian Nations, 2010 (http://www.aseanplus3-eid. info/ Rabies_Call_for_Action; accessed 10 December 2012). 27. Report of the ASEAN/FAO/OIE/WHO rabies workshop, January 2012,Chiang Mai, Thailand. Jakarta, Association of Southeast Asian Nations,2012. 28. Report of the second meeting of the Middle East and Eastern Europe Rabies Expert Bureau (MEEREB), Paris, France, June 5-8, 2012. Lyon, 2012 (http://www.meereb.info/meetings-concrete-actions; accessed March 2013). 29. Meeting report: expert consultation on rabies post-exposure prophylaxis, Stockholm, 15 January 2009. Stockholm, European Centre for Disease Prevention and Control, 2009 (http://www.ecdc.europa. eu/en/publications/Publications/0906_MER_Expert_Consultation_on_Rabies_Post-exposure_Prophylaxis.pdf; accessed March 2013).

15、研究

虽然在过去的 10 年里，推荐了一些新的技术和方法用于狂犬病诊断，尤其是人的狂犬病，但报告的实验室确诊人狂犬病病例数还是有限的，显示出对于这一被忽视的人兽共患病真实影响的低估，尤其是在非洲和亚洲。因此更好的检测方法，即快速、经济而又不失敏感性和特异性的诊断方法，会受到欢迎（1-3）。在分子检测方法，尤其是在对狂犬病病毒的多样性认识不足的发展中国家，需要更多的针对 N 和 G 基因之外的其它病毒基因的通用引物、实时定量 RT-PCR 和巢式 PCR 方法以及改进序列测定等方法。 - 91 -

由于缺少评价这些技术敏感性的国际标准，使得比较这些方法会有困难。在狂犬病流行国家的本土实验室内应该制定相关标准，以确保对新的分子技术的恰当评估（3）。在国际层面应该组织精准检测评估，以确保狂犬病诊断和发病率资料的可靠。侧流和其它检测技术应该适用于现场狂犬病病毒抗原的快速检测，并且这些技术应按国际标准获得认可。

疾病负担数据是设定地区和国家研究和控制疾病优先等级的根据，而疾病负担准确数据的缺乏导致了对这类疾病的不重视和忽视的恶性循环（4）。因此需要更好的疏散的监测方法和更敏感特异的实验室检测技术，包括以下几个方面： ■ 基于验证过的方案及标本处理方法并经过现场条件评估的诊断方法； ■ 用以更好的估计狂犬病发病率的流行病学模型。近期的研究显示狂犬病的发病率在一些国家要高出官方所报告的 15 倍之多（5、 6）。因此鼓励就这些模型的设计和在当地的应用作进一步的工作，并且将更准确的现场数据整合到这些模型中（7）。应用这些方法和技术可产生如下信息： ■ 疾病发病率的数据，判断性输入狂犬病流行病学模型和当前对疾病负担准确评估的主要限制； ■ 在自然哺乳动物宿主种群中狂犬病的流行病学和种群动力学信息（8、 9、 10）； ■ 生态状况、感染动物活动的频率和范围等信息，可以用来预测狂犬病的传播； ■ 广范围基因组和进化分析以确立狂犬病病毒种类和变异的多样性，以便于鉴析狂犬病传播的决定因素。种系地理学资料与病毒遗传学数据的整合是了解狂犬病空间传播特征、预测并最终实现预防和控制的强有力方法。最近的研究提示蝙蝠是狂犬病病毒属重要的贮存宿主，与翼手目相关的病毒变异株偶尔会溢出至其它哺乳动物，并具有适应和生存下去的潜在危险（11）。在人狂犬病感染中直接暴露于蝙蝠的证据有时会缺乏，需要对蝙蝠狂犬病病毒的流行病学（11）和这类溢出感染的潜在致病机制进行研究。近期没有关于此类宿主与病毒或者替代途径以及不寻常传播的综合研究。

全世界有关新病毒分离的报道越来越频繁（参看章节 2）。鼓励鉴定到新狂犬病病毒的 - 92 -

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科学家及时揭示新分离株的特点并将其与之前认识的种类进行比较。尤为重要的是判断新病毒种类的流行病学（宿主范围、地理分布和对家养动物及人类的重要性）以及明确商品化的狂犬病生物制剂，如疫苗和抗体，是否能保护抵抗这些新病毒。

目前，对于严重的狂犬病病毒暴露人群推荐狂犬疫苗和免疫球蛋白的联合应用。两种产品对于很大一部分目标人群来说仍然昂贵。因此，需要寻求降低其费用并探究新治疗途径的方法和手段。另外，几乎所有的兽用产品都是暴露前应用，但有些情况下这些产品也可能用于幼崽的暴露后预防。应该制定相关产品和种类的验证方法。已经提出了一些新的方法。包括使用负链 RNA 病毒作为克隆和表达载体的反向遗传学，以及新的、更安全、更有效的重组病毒，如基于腺病毒、 DNA 和植物来源的疫苗，一直受到关注（12、 13）。所有基因工程设计的狂犬病疫苗都必须符合国家和国际的生物安全指导方针。如果持续发现新的狂犬病病毒，尤其是源自蝙蝠的病毒，将会需要有更广谱保护范围的疫苗。应该探索用传统的细胞培养方法或者分子技术（表达嵌合 G 蛋白的重组病毒，在狂犬病病毒 G 蛋白上插入不同表位）生产多价疫苗。应该进一步研究应用新型疫苗载体和佐剂激活先天免疫反应并将其应用于暴露后预防中的保护作用。狂犬病免疫球蛋白是人狂犬病暴露后预防的关键因素，尤其是被患狂犬病的食肉类动物严重或多处面部咬伤后。在狂犬病的预防中需要有更多关于适宜免疫球蛋白或者替代品，比如人源单克隆抗体，的研究、开发和评估（14）（参看 6.8 章节），以保证更广泛获得降低成本的被动免疫。除了标准的实验室效能检测狂犬病免疫球蛋白和其他产品，用于确定每单位体积中病毒中和抗体的浓度，还需要一些预期效果的检测方法。应该寻找可再生的动物模型用于评价各种免疫球蛋白和其他产品（混合单克隆抗体）在感染后原位中和病毒的效果。确定抗体制剂在相关靶组织的体内半衰期可用于新的制剂。需要确定被动免疫及其持续时间所需要的抗体水平，尤其是基于人源单克隆抗体的制剂。当前用于评估疫苗效价的鼠保护实验存在很多困难，需要有更合适的方法用于评价抗原含量及其与保护作用的关系（参看 6.3.1 和 7.2 章节）。应该寻找适合的动物模型用于研究人狂犬病病例的致病机理及加强监护（参看 5 章节）。 - 93 -

目前尚无商品化抗病毒治疗可以应用。针对基于阻断病毒蛋白之间的相互作用、以及以病毒复制复合体为靶标的相关治疗方法的研究，可能会开发出新的小分子化合物。目前针对短效干扰 RNA 的研究（15）应该扩大。全盘着眼应该包括快速活体诊断、加强病人监护、疫苗免疫、免疫球蛋白注射、细胞因子和抗病毒的治疗，如果可行的话，这些研究应基于可以应用的动物模型和参照成功治愈的人狂犬病病例。当前有关兽用灭活疫苗的转换和应用研究以及新的生产模式应该提供更好、更简易的方法用于控制热带地区和资源匮乏地区贮存宿主动物中的狂犬病。需要集中研究和发展用于口服或其它途径的具有复制能力的活疫苗，这种疫苗对于主要的野生宿主动物，如浣熊、猫鼬和臭鼬更有效，而对于包括人类在内的非目标物种则是安全的。

评估较短期的暴露后预防方案，比如对于免疫功能低下病人的简化的 Essen 肌肉注射程序以及每个注射点 0.1 ml 的四点皮内注射程序联合使用狂犬病免疫球蛋白（16-18）。如果该方法适合，则将减少前往诊所接受全程多剂狂犬疫苗注射的交通费用，并可能提高人们的依从性（19）。该程序的产业支持定会受到欢迎。 WHO 鼓励在犬狂犬病成为主要公共卫生问题的地区将狂犬病疫苗纳入婴儿和儿童的免疫规划，这些地区无经济能力、后勤或计划会有障碍。进一步研究的重点应该放在疫苗接种的可替代路径，恰当又价廉的设备和预充式注射器等以促进狂犬病的暴露前和暴露后预防（20、 21）。

自然情况下狂犬病病毒感染神经元，导致功能障碍和死亡（22、 23）。需要进一步的研究来阐明狂犬病病毒在神经元和其他组织中的病理学分子基础。病理学方面的深入研究可用于设计治疗狂犬病的其他方法（24、 25）。对于狂犬病病毒病理学的全面认识尚缺乏。许多研究关注于狂犬病病毒及其宿主之间关系的本质，但不同病毒蛋白的作用以及这些蛋白怎样影响宿主细胞机制仍然是一个难解 - 94 -

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之谜（26、 27）。关于自然界中发现的狂犬病病毒致病过程与人类在易感性上表现出差异的回顾性和前瞻性比较有可能帮助揭示其中的奥秘。进一步需要研究决定狂犬病病毒跨物种屏障能力的相关因素，即从野生宿主动物到家养动物和人类、以及传播至新宿主动物种类的能力。先天性免疫反应在控制宿主转换方面的作用也需要进一步研究。需要研发相应的细胞系以便更适宜翼手目及其病原体的免疫生物学。

不同的哺乳动物物种适宜不同的病毒变异株，通常识别宿主很难（比如在蝙蝠物种间）。鉴别不同宿主物种的方法仍需要研究。研究的重点包括宿主的鉴定、分布和行为（比如和疾病传播的关系）以及种群动力学与疾病持续的相关性。应该进行新型的具有吸引力的诱饵以及改进疫苗诱饵布放至猫鼬、臭鼬、浣熊和犬的动物物种的研究。在狂犬病流行的广大地区应持续进行具有成本效益的大规模口服狂犬病疫苗的策略，同时要考虑主要宿主的生态学、诱饵密度和免疫活动的时间及频率。新型免疫避孕产品可能改进大规模免疫接种策略中的动物种群的管理（28）。

1. Durr S et al. Rabies diagnosis for developing countries. PLoS Neglected Tropical Diseases, 2008, 2:e206. 2. Lembo T et al. Evaluation of a direct, rapid immunohistochemical test for rabies diagnosis. Emerging Infectious Diseases, 2006, 12:310-313. 3. Dacheux L et al. More accurate insight into the incidence of human rabies in developing countries through validated laboratory techniques. PLoS Neglected Tropical Diseases, 2010, 4(11):e765. 4. Knobel DL et al. Re-evaluating the burden of rabies in Africa and Asia.Bulletin of the World Health Organization, 2005, 83:360-368. - 95 -

5. Cleaveland S et al. Estimating human rabies mortality in the United Republic of Tanzania from dog bite injuries. Bulletin of the World Health Organization, 2002, 80:304-310. 6. Ly S et al. Rabies situation in Cambodia. PLoS Neglected Tropical Diseases, 2009, 3:e511. 7. Hampson K et al. Transmission dynamics and prospects for the elimination of canine rabies. PLoS Biology, 2009, 7:e53. 8. Zinsstag J et al. Transmission dynamics and economics of rabies control in dogs and humans in an African city. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106:14996. 9. Bourhy H et al. The origin and phylogeography of dog rabies virus. Journal of General Virology, 2008, 89:2673-2681. 10. Kuzmin IV et al. Molecular inferences suggest multiple host shifts of rabies from bats to mesocarnivores in Arizona during 2001-2009.PLoS Pathogens, 2012, 8(6):e1002786. 11. Streicker D G et al. Rates of viral evolution are linked to host geography in bat rabies. PLoS Pathogens, 2012, 8(5):e1002720. 12. Bahloul C et al. Field trials of a very potent rabies DNA vaccine which induced long lasting virus neutralizing antibodies and protection in dogs in experimental conditions. Vaccine, 2006, 24:1063-1072. 13. Wu X et al. Development of combined vaccines for rabies and immunocontraception. Vaccine, 2009, 27:7202-7209. 14. Bakker AB et al. First administration to humans of a monoclonal antibody cocktail against rabies virus: safety, tolerability, and neutralizing activity. Vaccine, 2008, 26:5922-5927. 15. Israsena N, Mahavihakanont A, Hemachudha T. Rabies virus infection and microRNAs. Advances in Virus Research, 2011, 79:329-344. 16. Sudarshan MK et al. Evaluation of a one week intradermal regimen for rabies post-exposure prophylaxis: results of a randomized open label, active controlled trial in healthy adult volunteers in India. Human Vaccines and Immunotherapeutics, 2012, 8(8):1-5. 17. Prapimporn S et al. Postexposure rabies prophylaxis completed in 1week: preliminary study. Clinical Infectious Diseases, 2010, 50(1):56-60. 18. Warrell M et al. A simplified 4-site economical intradermal postexposure rabies vaccine regimen: a randomised controlled comparison with standard methods. PLoS Neglected Tropical Diseases, 2008, 2:e224. - 96 -

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19. Hampson K, Cleaveland S, Briggs D. Evaluation of cost effective strategies for rabies post-exposure vaccination in low-income countries. PLoS Neglected Tropical Diseases, 2011, 5:e982. 20. Laurent PE et al. Safety and efficacy of novel dermal and epidermal microneedle delivery systems for rabies vaccination in healthy adults. Vaccine, 2010, 28(36):5850-5856. 21. Program for Appropriate Technology in Health (PATH). Intradermal delivery of vaccines. Seattle, Washington, 2010-2013 (http://sites.path.org/deliverytech/id/). 22. Schnell MJ et al. The cell biology of rabies virus: using stealth to reach the brain. Nature Reviews Microbiology, 2010, 8:51-61. 23. Rieder M, Conzelmann KK. Interferon in rabies virus infection. Advances in Virus Research, 2011, 79:91-114. 24. Jackson AC. Update on rabies diagnosis and treatment. Current Infectious Disease Reports, 2009, 11:296-301. 25. Jackson AC. Therapy of rabies encephalitis. Biomedica, 2009, 29:169 176.

26. Thanoms ridetchai N et al. Comprehensive proteome analysis of hippocampus, brainstem, and spinal cord from paralytic and furious dogs naturally infected with rabies. Journal of Proteome Research, 2011,10(11):4911-4924. 27. Reinke SN et al. Metagenomic and metabolomic characterization of rabies encephalitis: new insights into the treatment of an ancient disease. Journal of Infectious Diseases, 2012 (doi: 10.1093/infdis/jis479). 28. Carroll MJ et al. The use of immunocontraception to improve rabies eradication in urban dog populations. Wildlife Research, 2010, 37:1-12.

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结束语本次会议在世界卫生组织艾滋病、结核、疟疾和被忽视热带病的助理总干事 Hiroki Nakatani 博士及 F.X. Meslin 博士的主持下落下帷幕。 Nakatani 博士代表世界卫生组织总干事，陈冯富珍博士，对参会者、评论员及其他国际官方和非官方组织的代表表示了感谢，感谢他们为会产生实质性公共卫生和经济影响的受忽视人兽共患病所做的工作和给予的支持。还特别感谢了 WHO 合作中心的负责人以及 WHO 狂犬病专家顾问小组的成员。Nakatani 博士强调在狂犬病控制上需要人与动物管理部门之间的紧密有效的合作，并欢迎联合国粮食与农业组织（FAO）和世界动物卫生组织（OIE）对于本次磋商会的参与。他还特别对人用和兽用狂犬病疫苗的私有生产商同行表示感谢。 Nakatani 博士指出了因狂犬病而导致的重大疾病和经济负担，使得约 2 千万人使用 7 千万剂人狂犬病疫苗，其中大部分是在发展中国家；全世界范围狂犬病的社会负担估计超过 60 亿美元，其中 16 亿美元用于暴露后预防。随着对安全可靠的细胞培养人用狂犬病疫苗需求的增加，这些数字将持续增长。Nakatani 博士强调了本次磋商会的结论，即经犬传播的人狂犬病已具备控制的条件，在中期达到区域性消除进而在远期达到全球消除。Nakatani 博士在结束语中讲到一项包括狂犬病在内的主要关于受忽视热带病的决议正准备提交到 2013 年 5 月举办的世界卫生大会（WHA），期望成员国承诺控制、消除或根除这些疾病。该项决议的采纳将开启在狂犬病预防和控制中令人欣喜的新局面。

专家磋商会以及世界卫生组织秘书处感谢为文件起草做出特殊贡献的 K. Hampson 博士、 I. Kuzmin 博士、 T. Hemachuda 博士、 C. Rupprecht 博士、 S. Madhusudana 教授、 D. Briggs 博士、 H. Ertl 博士、 H. Wilde 博士、 F. Cliquet 博士、 M.K. Sudarshan 博士、 B.Quiambao 博士、 A. Rahman 博士、 E. Russell 博士、 G. Massei 博士、 A. Wandeler 博士、 T.Mü ller 博士、 S. Cleaveland 教授、 M. Vigilato 博士以及 H. Bourhy 博士。特别感谢比尔及梅林达 · 盖茨基金会给予的资金支持。

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Dr Hervé Bourhy, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, Paris, France Dr Florence Cliquet, WHO Collaborating Centre on Research and Management on Zoonoses Control, AFSSA-LERPAS, Laboratoire d’ études sur la rage et la pathologie, des animaux sauvages, Malzéville, France Dr Bernhard Dietzschold, WHO Collaborating Centre for Neurovirology, Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, USA Dr Hildegund Ertl, WHO Collaborating Centre for Reference and Research on Rabies, Immunology Program Leader, The Wistar Institute, Philadelphia,USA Dr Anthony Fooks, WHO Collaborating Centre for the Characterization of Rabies and Rabies-related Viruses, Animal Health and Veterinary Laboratries Agency, Weybridge, England Dr Thiravat Hemachudha, WHO Collaborating Centre for Research and Training on Viral Zoonoses, Member of the WHO Expert Advisory Panel on Rabies, Professor of Neurology, Neurology Division, Department of Medicine, Chulalongkorn University Hospital, Bangkok, Thailand Dr Rattan Lal Ichhpujani, WHO Collaborating Centre for Rabies Epidemiology, Member of the WHO Expert Advisory Panel on Rabies, Additional Director, Microbiology Department, Centre for AIDS and Related Diseases, National Centre for Disease Control, Delhi, India Professor S.N. Madhusudana, WHO Collaborating Centre for Reference and Research in Rabies, Member of the WHO Expert Advisory Panel on Rabies, Department of Neurovirology, National Institute of Mental Health and Neurosciences, Bangalore,India Dr Thomas Müller, Head, WHO Collaborating Centre for Rabies Surveillance and Research, Friedrich-Loeffler Institut, Federal Research Institute for Animal Health, Greifswald-Insel Reims, Germany

Dr Ahmad Fayaz, Former Head, Rabies Laboratory, Pasteur Institute, Tehran, Islamic Republic of Iran - 99 -

Professor Louis Hendrik Nel, Professor of Virology, Department of Microbiology and Plant Pathology, Faculty of Natural and Agricultural Sciences, University of Pretoria, Hillcrest, South Africa, President of the South Eastern Africa Rabies Group (SEARG) (Chair) Dr Beatriz P. Quiambao, Chief, Clinical Research Division, Research Institute for Tropical Medicine, Philippines. President of the Rabies Asia Foundation (RIA) Dr Charles E. Rupprecht, Former Chief, Rabies Section, Centers for Disease Control and Prevention, Atlanta, USA Dr Naseem Salahuddin, Indus Hospital, Korangi, Karachi, Pakistan (Rapporteur) Professor Dr Mysore K. Sudarshan, Dean, Principal and Professor of Community Medicine, Kempegowda Institute of Medical Sciences, Bangalore, India. President of the Rabies Asia Foundation (RIA) Dr Alexander Wandeler, Former Head, rabies laboratory, Canadian Food Inspection Service, Scientist Emeritus, Carp, Canada

Professor Sarah Cleaveland, Consultant, University of Glasgow, Glasgow, Scotland Dr Raffy Deray, National Program Manager, National Rabies Prevention and Control Program, National Center for Disease Prevention and Control, Department of Health, Manila, Philippines Dr Katie Hampson, Research Scientist, Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland Mr Kevin Le Roux, Rabies Project Management, Veterinary Services, Pietermaritzburg, KwaZulu-Natal, South Africa Dr Giovanna Massei, Ecologist, Food and Environment Research Agency, York, England Dr Mathew Maziku, National Project Coordinator, Rabies, WHO Country Office, Dar es Salaam, United Republic of Tanzania Dr Maria P. Rebollo, Scientific Manager, Monitoring and Evaluation, Liverpool School of Tropical Medicine, Centre for Neglected Tropical Diseases, Liverpool, England Dr Graham Smith, Senior Researcher, Food and Environment Research Agency, York, England Dr Mathurin Cyrille Tejiokem, Epidemiologist, Epidemiology and Public Health Laboratory, Centre Pasteur du Cameroun, Yaoundé, Cameroon -100-

世界卫生组织狂犬病专家磋商会

第二版报告

Dr Henry Wilde, Professor of Medicine, Senior Consultant, WHO Collaborating Centre for Zoonoses and Rabies, Faculty of Medicine, Chulalongkorn University, Bangkok,Thailand

Dr Marta Martinez, Veterinary Epidemiologist Dr Dietrich Rassow, Chargé de mission, WHO

Dr A. Rahman, President, Commonwealth Veterinary Association for Prevention and Control of Rabies in India, Department of Community Medicine, Bangalore, India

Dr Betty Dodet, Caluire et Cuire, France

Dr Kim Doyle, Trustee of the Alliance for Rabies Control, Global Alliance for Rabies Control c/o Balfour and Manson, Edinburgh, Scotland Ms Maylin Meincke, Development Studies, University of Helsinki, Helsinki, Finland Dr Elizabeth Miranda, Asian Coordinator, Global Alliance for Rabies Control, Laguna, Philippines Marwar Trust Mr Federico Spinola, Founder, Partnership for Animals, Geneva, Switzerland

Dr Darin Zehrung, Technical Officer, Portfolio Leader Vaccine Delivery Technologies, Seattle, USA

Dr Elly Hiby, Scientific Advisor, World Society for the Protection of Animals, Cambridge, England Dr Esmée Russell, Campaign Manager, World Society for the Protection of Animals,London, England

Dr Michael Attlan, Director, Traveler Endemic and Emerging Vaccines Franchise, Sanofi-Pasteur, Lyon, France Dr Jac Bergman, Global Marketing Director, Small Animal Vaccines, Global Companion Animal -101-

Business Unit, Boxmeer, the Netherlands Dr Rachel Chikwamba, Technical Lead, Rabies Initiatives, Pretoria 0001, South Africa Dr Pradip Desai, Director, Span Diagnostics Ltd, Surat, India Dr Alexandra Giesen, Novartis Vaccines and Diagnostics, Global Medical Affairs, Munich, Germany Dr Reinhard Glueck, CSO, Zydus Cadila Healthcare, Zydus Research Centre, Gujarat, India Dr Francoise Guinet-Morlot, Project Director New Vaccines, Sanofi Pasteur, Marcy l’ Etoile, France Dr Gaurav Gupta, Head, Viral Vaccines, Zydus Cadila, Zydus Research Centre, Gujarat, India Dr K. Jager, Intervet, Boxmeer, the Netherlands Dr Philipe Mahl, Rabies Programme Manager, Virbac, Carros, France Dr Joanne Maki, Veterinary Public Health, Global Public Health Director, Athens, Georgia, USA Dr Claudius Malerczyk, Head, Medical Affairs, Middle East and Africa, Novartis Vaccines and Diagnostics, Marburg, Germany Dr Stephanus Francois Marais, Commercialisation Manager, CSIR Biosciences, Pretoria, South Africa Dr Wilfred Marissen, Programme Director, Crucell Holland B.V., Leiden, the Netherlands Dr Anvar Rasuli, Medical Product Leader, Global Medical Affairs, Sanofi Pasteur, 2 Avenue Pont Pasteur, Lyon, France Dr Micha Roumiantzeff, Fondation M. Merieux, 1 rue Dangon, 69004 Lyon, France Dr Carolin Schumacher, Director, Corporate Public Affairs, Merial, Lyon, France Dr Daniela Todorova-Balvay, R&D Manager, Span Diagnostics SARL, Compiegne, France Dr Adriaan Vos, Head, Vaccine Development Technologies, IDT Biologika GmbH, Dessau- Rosslau, Germany

Dr Hiroki Nakatani, Assistant Director-General, HIV/AIDS, Tuberculosis, Malaria and Neglected Tropical Diseases -102-

世界卫生组织狂犬病专家磋商会

第二版报告

Dr Lorenzo Savioli, Director, Department of Control of Neglected Tropical Diseases Dr Francois Meslin, Team Leader, Neglected Zoonotic Diseases, Department of Control of Neglected Tropical Diseases (Organizer and convener) Dr Bernadette Abela-Ridder, Foodborne Disease and Epidemiology, Food Safety, Zoonoses and Foodborne Diseases Dr Simone Magnino, Foodborne Disease and Epidemiology, Food Safety, Zoonoses and Foodborne Diseases Dr Arve Willingham, Special Programme for Research and Training in Tropical Diseases (TDR) Dr Martin Friede, Innovation, Innovation, Evidence and Research Ms Erin Sparrow, Technology Transfer Initiative, Innovation, Information, Evidence and Research Dr Philippe Duclos, Immunization, Vaccines and Biologicals Dr Ivana Knezevic, Quality, Safety and Standards, Immunization, Vaccines and Biologicals Dr Jinho Shin, Quality, Safety and Standards, Immunization, Vaccines and Biologicals Dr David Wood, Coordinator, Quality, Safety and Standards, Immunization, Vaccines and Biologicals Dr Ana Padilla, Quality Assurance and Safety: Medicines Mrs Beatrice Wamutitu, Secretary, Neglected Zoonotic Diseases, Department of Control of Neglected Tropical Diseases

Dr Landry Bidé, Medical Officer, Neglected Tropical Diseases, Brazzaville, Congo

Dr Alfonso Clavijo, Veterinary Public Health, Panamerican Centre for Foot-and-Mouth Disease, Sao Bento, Duque de Caxias, Rio de Janeiro, Brazil Dr Marco Vigilato, Veterinary Public Health, Panamerican Centre for Foot-and-Mouth Disease, Sao Bento, 25045-002 Duque de Caxias, Rio de Janeiro, Brazil

Dr Gyanendra Gongal, Scientist, Veterinary Public Health, Disease Surveillance and Epidemiology, New Delhi, India -103-

Dr Katinka de Balogh, Senior Officer, Veterinary Public Health, Food and Agriculture Organization of the United Nations, Rome, Italy Dr Philip Binu, Business Development, Zydus Research Centre, Gujarat, India Dr Deborah Briggs, Executive Director, Global Alliance for Rabies Control, c/o Balfour and Manson, Edinburgh, Scotland Dr Yu Hongjie, Director, Division of Infectious Disease Control, Centre for Disease Control, Beijing, China Dr Ivan V. Kuzmin, Rabies Program, Centers for Disease Control and Prevention, Atlanta, Georgia, USA Dr Tiziana Lembo, Institute of Comparative Epidemiology, Faculty of Veterinary Medicine, University of Glasgow, Glasgow, Scotland Dr Anastasia Pantelias, Bill & Melinda Gates Foundation, Seattle, Washingthon, USA Dr A. Rowan, Humane Society International, Washington, District of Columbia, USA Dr Louis Taylor, PRP Coordinator, Global Alliance for Rabies Control, Manhattan, Kansas, USA

病例编号：

日期：

时间：

姓名：联系地址：

年龄：

性别：

家庭座机和移动电话：工作电话：医生及其联系电话：

乡村和城市：暴露日期：旅行日期：暴露性质：咬伤 / 舔 / 唾液 / 抓伤 / 其他（请具体描述） -104-

世界卫生组织狂犬病专家磋商会

第二版报告

暴露部位：皮肤是否破损？伤口是否流血？伤口数量：伤口深度：暴露类型：浅表 / 深是/否是/否

动物 / 物种类型：野生 / 家养：激惹的 / 未激惹的（给出细节）：动物的主人 / 家庭是否知晓？是否尝试追踪该动物？是/否是/否

最后一次见到该动物存活状态是什么时候？该动物的疫苗接种情况（如果知晓，请填写）：

是否有过 3 剂肌内注射 / 皮内注射的暴露前狂犬病疫苗接种？是 / 否细节：是否先前接受过抗狂犬病暴露后预防？是 / 否用的是哪一种疫苗？细节（年份、日期等）：是否接受过狂犬病免疫球蛋白？是 / 否局部 / 全身：其他信息：就以上信息联系病毒学家 / 医生寻求建议：若无法获得，请联系：

■ 1. 使用水、肥皂或抗病毒制剂清洗伤口。 ■ 2. 狂犬病疫苗接种： ■ 为先前接受过暴露前预防者的调整程序：第 0 天与第 3 天 -105-

■ 为未接种过疫苗者的标准程序： - 肌内注射—— — 第 0、 3、 7、 14、 28 天或第 0、 3、 7、 14 天 - 肌内注射—— —第 0 （两个剂量）、 7、 21 天 - 皮内注射—— —第 0 （两个部位）、 3 （两个部位）、 7 （两个部位）、 28 天（两个部位） ■ 3. 狂犬病免疫球蛋白：人狂犬病免疫球蛋白，每公斤体重 20IU；马狂犬病免疫球蛋白，每公斤体重 40IU/kg 注射部位：患者体重（kg）：是否安排了暴露后程序？是/否是/否建议剂量（IU 与 ml）：

是否通过信件、电子邮件、电话或短信通知了医生？完成以上工作的医师姓名、电话及签名

依然生产或使用神经组织疫苗的国家应当遵从如下所推荐的四步策略，用现代疫苗取代神经组织疫苗。：由国家卫生主管机构领导的相关国家权威机构必须做出将神经组织疫苗替换为现代疫苗的最终决定。在评价现代疫苗的安全性、免疫原性及有效性之后，相关权威机构应当评估当地状况，分析取代神经组织疫苗的可行性及其费用。应当考虑在狂犬病暴露前和暴露后的预防中使用可节省成本的皮内注射法。：国家相关指南应该对使用现代疫苗进行暴露前和暴露后预防给予清晰说明，包括使用适应症及给药途径；对狂犬病免疫球蛋白和其他产品也应当给出相应的指导。这些指南应当由具备技术水平的专家依据 WHO 狂犬病专家顾问小组、其他 WHO 顾问小组、最新的科学文献、国际及国内专家和观察的经验来拟定。指南应发放到所有提供暴露前和暴露后预防的机构去。这些指南应当基于明确的相关政策，如疫苗补贴（如果有的话）以及剩余疫苗的处理等，并应定期更新。：各狂犬病机构应当从总部得到安全、有效、 WHO 推荐使用的狂犬病疫苗和免疫球蛋白的持续供给。一旦做出停止神经组织疫苗的生产和使用的决定，就应当开始采购现代疫苗，以避免神经组织疫苗停止供应以后狂犬病疫苗在供给与治疗上出现任何缺口。监管部门对新型狂犬病生物制剂的注册及新型狂犬病疫苗和狂犬病免疫球蛋白上市后监督的协调也很重要。 -106-

世界卫生组织狂犬病专家磋商会

第二版报告

：应当建立一个咬伤专科的网络，其员工应接受过提供暴露前和暴露后预防及处置不良反应的培训；应确保这些中心有足够量的狂犬病生物制剂。应建立转诊系统来扩大皮内注射法的效益并减少剩余的疫苗量。应设立一个质量保证系统，所有机构都遵从其标准。省级和市级政府应参与新机构的建立，确保狂犬病疫苗、免疫球蛋白和其他物资的可持续供给，确保人狂犬病病例的上报和调查以及监控狂犬病项目。

皮内注射可用于所有按规定批准使用该途径进行狂犬病暴露前或暴露后预防的国家。所使用的疫苗必须通过皮内途径的用药许可，并得到 WHO 推荐（参见 5.1）。皮内注射不应当用于免疫功能低下、接受氯喹等抗疟疾治疗及长期接受皮质类固醇或其他免疫抑制治疗的人。由于单次皮内注射所用疫苗剂量小于单次肌内注射的剂量，因此皮内途径尤其适宜治疗短时间内多名患者就诊于同一机构的情况，即在推荐的疫苗稀释后 6 到 8 小时内。由于目前应用的疫苗不含防腐剂，一旦稀释后必须冷藏保存并在 6 到 8 小时后丢弃。皮内途径比标准肌肉途径更加节省成本，因此适用于疫苗和经费短缺的情况以及治疗暴露后患者的机构。

皮内注射狂犬病疫苗前： ■ 所有员工必须接受了充分的皮内注射技术培训。 ■ 若疫苗作为暴露后预防的一部分，还应采取其他处置步骤，即清洗伤口，根据情况注射适当剂量的狂犬病免疫球蛋白。 ■ 应当使用一个适当的 1.0ml 注射器（胰岛素或结核菌素注射器）和一个短小完好的注射针头。使用固定针头的注射器可节省大部分花费，因为减少了空隙容量。 ■ 应该对皮内注射程序有所选择。 WHO 推荐更新的 2-2-2-0-2 泰国红十字会程序（参见 8.3.3）。

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给药前，在无菌条件下取用制造商提供的适当剂量的稀释剂稀释疫苗。不可使用别的稀释剂。不能使用不同剂量的稀释剂。采用恰当的无菌措施，吸取足够的疫苗到注射器中用来给一位患者注射。小心排出所有气泡。用抗菌剂消毒注射部位，然后绷紧皮肤表面并插入针头尖端（尖端斜面朝上）到皮肤上层（真皮层），确保针头和注射器与皮肤表面几乎平行。

开始注射疫苗。若针头处于正确部位，会感受到一定阻力。一个橘皮样皮丘会马上出现，直径在 6 到 8 毫米。若疫苗很容易注射进去，或是没有出现皮丘，说明误为皮下给药，即打得过深。遇到这种情况，应当重复上述正确的注射方法。

当给同一患者的所有 0.1ml 剂量的疫苗注射完毕时，丢弃针头和注射器。稀释过后的疫苗可用于不止一位患者；然而，针对每一位患者都必须使用一套无菌的注射器和针头来吸取疫苗。稀释后的疫苗必须保存在 2℃ 到 8℃ 的冰箱中，并于 6 到 8 小时内使用。

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世界卫生组织狂犬病专家磋商会

第二版报告

暴露类型

与可疑或确认为狂犬病的家养或野生动物 a、或无法检测的动物的暴露类型

推荐采用的暴露后预防

Ⅰ

触碰或喂食动物舔舐完好皮肤无需治疗。如果具有可靠病史，完好皮肤接触狂犬病动物或人狂犬病病例的分泌物或排泄物裸露的皮肤被轻咬无出血的轻微抓伤或擦伤立即接种疫苗 b 如果动物在 10 天 c 的观察期内保持健康或经可靠的实验室使用恰当诊断技术证则停止治疗。明该动物未患狂犬病，

Ⅱ

Ⅲ

单处或多处贯穿皮肤的咬伤 d 或擦伤，破损皮肤被舔被唾液污染粘膜（即粘膜被舔） e 暴露于蝙蝠

起始暴露后预防以后尽快立即接种狂犬病疫苗并注射狂犬病免疫球蛋白。狂犬病免疫球蛋白可在首剂量疫苗接种后的 7 天内注射。如果动物在 10 天的观察期内保持健康或由可靠的实验室经恰当诊断技术证则停止治疗。明该动物未患狂犬病，

a 暴露于啮齿类动物、家兔或野兔时不要求按照常规接受狂犬病暴露后预防。 b 如果观察过程中明显健康的犬或猫，并且是在低风险地区或来自低风险地区，则治疗可以延迟。 c 此观察期仅适用于犬和猫。除濒临灭绝的物种之外，其他怀疑患有狂犬病的家养和野生动物均应当实施安乐死并使用恰当的实验室技术对其组织进行狂犬病抗原检查。 d 发生在头部、颈部、面部、手部和生殖器的咬伤归于暴露类型Ⅲ，原因是这些部位有丰富的神经分布。 e 当人与蝙蝠之间发生接触时应考虑进行暴露后预防，除非暴露者排除咬伤、抓伤或粘膜的暴露。

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可作为模板的疫苗接种证书提供如下。该证书应当与疫苗接种者个人健康档案一同由本人妥善保存。空白证书应该由疫苗制造商提供。

姓地

名 _______________________________________________ 性别 _______ 职业 ________ 址 _______________________________________________ _______________________________________________

出生日期 / 年龄（周岁） ______

电话号码 _____________________ 签名 _________________________

接种日期接种中心 / 地点疫苗类型 / 名称制造商（批号） / 有效期限剂量（ml）给药途径（肌内注射或皮内注射）接种部位（如有）不良反应（如已测定）狂犬病病毒中和抗体滴度 / 方法医师签名

第0天

第7天

第 21 或 28 天

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世界卫生组织狂犬病专家磋商会

第二版报告

加强免疫日期接种中心 / 地点疫苗类型 / 名称制造商（批号） / 有效期限（ml）剂量给药途径（肌肉内注射或皮内注射）接种部位不良反应（如果有的话）狂犬病病毒中和抗体滴度（如已测定） / 方法医师签名

姓地

名 ________________________________________________ 职业 ______________ 址 ________________________________________________ ________________________________________________

出生日期 / 年龄（周岁） ______ 性别 ____

电话号码 _____________________ 暴露日期 _____________ 咬人动物 _____________ 动物疫苗接种状态 ___________ 狂犬病病毒中和抗体滴度 / 方法 ____________________________________ 10 天观察期后情况（如果有关的话） __________________________________ __________________________________ 1. 用清水、肥皂或抗病毒制剂清洗伤口 ____________________ 2. 狂犬病免疫球蛋白：治疗日期 _______________ 门诊 / 医院名称 ____________________ -111WHO 暴露分级 ____________ 健康 / 患病情况 ____________

地

点 ___________________________ ______________________________

狂犬病免疫球蛋白名称 / 类型（人 / 马） ______________________________ 制造商（批号 / 有效期） ___________________________________________ 患者体重 _______kg. 剂量（IU） ___________ 总量（ml） ____________ 狂犬病免疫球蛋白浸润注射入伤口内及周围 / 肌内注射（ml） ___________ 剩余免疫球蛋白在远离疫苗接种的部位肌内注射（ml） _________________ 3. 狂犬病疫苗：疫苗程序：五剂 Essen 法（1-1-1-1-1）或四剂 Essen 法（1-1-1-1-0） Zagreb 法（2-1-1）更新的泰国红十字会双部位皮内注射法（2-2-2-0-2）

接种日期接种中心 / 地点疫苗类型 / 名称制造商（批号） / 有效期限（ml）剂量给药途径（肌肉内注射或皮内注射）接种部位不良反应（如果有的话）狂犬病病毒中和抗体滴度（如已测定） / 方法医师签名

第0天

第3天

第7天

第 14 天

第 21 天

第 28 天

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世界卫生组织狂犬病专家磋商会

第二版报告

可作为模板的疫苗接种证书提供如下。这是基于 OIE 证书制定[Terrestrial animal health code [vol. 2, chapter 8.10: Rabies]. Paris, World Organisation for Animal Health, 2011 (http://www.oie.int/index.php?id=169&L=0&htmfile=chapitre_1.8.10.htm; accessed 21 September 2012).]。一些国家可能会要求附加信息。

姓名和住址 ____________________________________________________ ____________________________________________________ ____________________________________________________ ____________________________________________________ 物种 ___________ 年龄或出生日期（如果知道的话） ____________ 性别 ___________ 品种 ______________ 毛色 ____________ 皮毛类型和斑纹 / 特征标志 ________________________________ 微芯片编号 _________________ 微芯片扫描器类型 __________ 微芯片位置 ______________________________________________ 纹身编号和位置（如果适用） ________________________________ 签名者特此声明，他或她已对第一页中所描述的动物进行了抗狂犬病免疫接种，详情如下。该动物在疫苗接种之日表现健康。 (1) 接种日期 (2) 疫苗名称 (3) 制造商名称 (4) 批号 (5) 有效期限 (6) 官方兽医签字及盖章 (7) 有效期至

兽医声明 1.Terrestrial animal health code [vol. 2, chapter 8.10: Rabies]. Paris, World Organisation for Animal Health, 2011 (http://www.oie.int/index.php?id=169&L=0&htmfile=chapitre_1.8.10.htm; accessed 21 September 2012). -113-

本人证明看过对采集于（日 / 月 / 年） _________ 的样本进行动物血清学检验获得结果的官方记录，该检验是在经批准的实验室进行的，记录表明狂犬病中和抗体滴度等于或大于 0.5 IU/ml。授权兽医的姓名、日期和签字：进一步的检验：日期结果经批准的实验室兽医签字和盖章

日期

疫苗种类

批号

兽医签字和盖章

来源国家 ______________________________________________________ 由主人申报的该动物去过的国家（说明日期） ________________________ ______________________________________________________________ ：本证书不一定能满足目的地国家的所有要求。请阅读第 VII 节。授权印制的单位（说明相应国家负责部门）：本证书每页上必须有打孔的数字方生效。

携带动物出国之前，动物的主人必须要了解目的地国家规定的兽医卫生要求，因为本证书不一定能满足目的地国家的所有要求。本证书的有效期为首次疫苗免疫后的第 30 天至第 12 个月月底；若于有效期内进行了再次免疫，则有效期自再次免疫日顺延 12 个月。本证书必须以法语和英语印制和填写，必要时使用来源地国家的语言。 -114-

世界卫生组织狂犬病专家磋商会

第二版报告

WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris, France Head, Dr Hervé Bourhy; e-mail: herve.bourhy@pasteur.fr WHO Collaborating Centre on Research and Management on Zoonoses Control, AFSSALERPAS, Laboratoire d’ études sur la rage et la pathologie, des animaux sauvages, Domaine de Pixérécourt, BP 9, 54220 Malzéville, France Head, Dr Florence Cliquet; e-mail: florence.cliquet@anses.fr WHO Collaborating Centre for the Characterization of Rabies and Rabies-related Viruses, Animal Health and Veterinary Laboratories Agency, Weybridge, Surrey KT15 3NB, United Kingdom Head, Dr Anthony Fooks; e-mail: t.fooks@ahvla.gsi.gov.uk WHO Collaborating Centre for Rabies Surveillance and Research, Friedrich-Loeffler Institut, Federal Research Institute for Animal Health, Sudufer 10, 17493 GreifswaldInsel Reims, Germany Head, Dr Thomas Müller; e-mail: thomas.mueller@fli.bund.de WHO Collaborating Centre for Control, Pathogenesis and Epidemiology of Rabies in Carnivores, 106 Pineridge Road, Carp, ON, Canada Head, Dr Christine Fehlner-Gardiner; e-mail: Christine.Fehlner-Gardiner@inspection.gc.ca WHO Collaborating Centre for Neurovirology, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19105, USA Head, Dr Bernhard Dietzschold; e-mail: bernhard.dietzschold@jefferson.edu WHO Collaborating Centre for Reference and Research on Rabies, Wistar Institute, 3601 -115-

Spruce Street, Philadelphia, PA 19104, USA Head, Dr Hildegund Ertl; e-mail: ertl@wistar.upenn.edu WHO Collaborating Centre for Reference and Research on Rabies, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA Head ad interim, Inger Damon, Chief, Poxvirus and Rabies Branch, CDC WHO Collaborating Centre for Rabies Epidemiology, Centre for AIDS and Related Diseases, National Centre for Disease Control, 22-Sham Nath, Delhi 110054, India Head, Dr Rattan Lal Ichhpujani; e-mail: ichhpujani@hotmail.com WHO Collaborating Centre for Reference and Research in Rabies, Department of Neurovirology, National Institute of Mental Health and Neurosciences, PO Box 2900, 560029 Bangalore, India Head, Professor S.N. Madhusudana; e-mail: mshampur@hotmail.com WHO Collaborating Centre for Research and Training on Viral Zoonoses, Chulalongkorn University Hospital, Rama 4 Road, Bangkok 10330, Thailand Head, Dr Thiravat Hemachudha; e-mail: fmedthm@gmail.com WHO Collaborating Centre for Research on Rabies Pathogenesis and Prevention, Queen Saovabha Memorial Institute, Thai Red Cross Society, 1871 Rama IV Road, 10330 Bangkok, Thailand Head, Professor Visith Sitprija; e-mail: sitprija@yahoo.com; and Dr Pakmanee Narumol; e-mail: npakmanee@yahoo.com

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世界卫生组织技术报告丛书 982

世界卫生组织狂犬病专家磋商会第二版报告主译: 余宏杰

副主译: 殷文武主审: 唐青

编译人员:（按章节顺序排列）王传林李玉华扈荣良王力华张怡滨佟丽北京大学人民医院中国食品药品检定研究院军事医学科学院军事兽医研究所中国疾病预防控制中心病毒病所辽宁成大生物股份有限公司辽宁成大生物股份有限公司

辽宁成大生物股份有限公司印制内部资料仅供专业人员参考 MKT(Z)1311-01

对狂犬病健康负担的评估虽然存在争议，但对于狂犬病引起的直接死亡和伤残调整寿命年（DALY）的估测仍然是被忽视热带病中最高的。不完善的监测、许多发展中国家病例的漏报、经常发生的狂犬病误诊以及缺乏相关部门间的协调均有可能导致对狂犬病负担的低估。无论怎样，狂犬病在不同程度上影响着贫困地区，尤其是儿童，是显而易见的。绝大部分暴露后预防的费用是由这些支付能力最差的人负担。随着犬的数量和人口数量的继续增长，如果没有对狂犬病控制的一致努力和投入，因狂犬病造成的人类死亡的负担和经济损失将会继续增加。自从 2004 年第一次 WHO 狂犬病专家磋商会以来， WHO 及其狂犬病合作中心网络、国家专业机构、 WHO 狂犬病专家咨询小组和包括盖茨基金会、全球狂犬病控制联盟和狂犬病预防的合作者们，一直倡导区域性和全球性消除狂犬病的可行性，并促进具有良好投入产出比的可持续策略的研究。这些共同的努力已经开始打破狂犬病被忽视的现状，狂犬病正逐渐成为优先投入的领域之一。本次磋商会认为控制经犬传播的人狂犬病并不困难，可在短期内达到区域性消除，进而最终实现全球性消除。一项主题为被忽视的热带疾病（包括狂犬病）的提议已经准备好提交到 2013 年 5 月的世界卫生大会，这项提议致力于确保成员国对这些疾病的控制、消除或消灭的承诺。对该决议的支持和响应将开启狂犬病预防和控制发展方面的令人欣喜的新时期。

辽宁成大生物股份有限公司印制

Key facts

Organisation World Health Organization (WHO)

Document type Publications

Adoption date July 3, 2013

Source World Health Organization