Swine influenza: epizootiological and serological studies*

Bull. Org. mond. Sante 1972, 47, 481-487 Bull. Wld Hlth Org. Swine influenza: epizootiological and serological studies * ROBERT M. NAKAMURA,' BERNARD C. EASTERDAY,2 RONALDEAN PAWLISCH,3 & G. L. WALKER' Studies of naturally occurring respiratory diseases in the midwestern parts of the USA showed that swine influenza is still prevalent and that mild forms as well as the classical forms ofswine influenza occur. Outbreaks of respiratory disease of unknown etiology that are clinically similar to swine influenza were also found. On some farms, swine influenza occurred first in farrowing pens. It did not occur on some farms where the disease had occurred in previous years. This disappearance may have resulted from the elimination or hyperimmunization of breeder animals or from a change to the raising of swine obtained by caesarean section. Serological studies of swine with natural or experimental infections showed that antibody titres rose gradually for several months. This observation was corro- borated in serological studies of sera obtained at the abattoir, which showed that older breeder swine had consistently higher titres than the younger market swine. These results cannot be explained by the lungworm hypothesis proposed by R. E. Shope for the survival and transmission ofswine influenzavirus. It is suggested that breeder swine act as convales- cent carriers and as the reservoirs of swine influenzavirus between epizootics. Influenza in swine was first observed in the north- central part of the USA in the autumn of 1918 and its similarity to human influenza was noted and described by Dorset et al. (1922). Shope (1931) reported that the disease was caused by a virus act- ing in association with a bacterium, Hemophilus influenza, var. suis. Retrospective serological studies have suggested the close relationship of the virus causing swine influenza with the virus responsible for the human influenza pandemic of 1918-19 (Andrewes et al., 1935; Francis & Magill, 1936; Shope, 1936; Davenport et al., 1953). This disease has been reported to occur only in the autumn and early winter and it is difficult to * From the Department of Veterinary Science, University of Wisconsin, Madison, Wis., USA. This study is based on a dissertation submitted by the senior author in partial fulfil- ment of the requirements for the PhD degree at the University of Wisconsin. 1 Present address: Department of Animal Science, College of Tropical Agriculture, University of Hawaii, Honolulu, Hawaii. 2 Professor and Chairman. 'Veterinarian, Brodhead Veterinary Hospital, Brodhead, Wisconsin, Wis., USA. ' Research Assistant. account for the survival and transmission of the virus between the epizootics. A solution was offered in the widely accepted " lungworm hypothesis " of Shope (1941a, 1941b, 1943a, 1943b). Since the studies of Shope, relatively little new information on the epizootiological aspects of swine influenza has been reported. However, Blaskovic et al. (1970) reported the transmission of virus from pigs, infected 3 months previously, to susceptible pigs placed with them for a period of 1 month, thus indicating that virus shedding had taken place during the fourth month after infection. The results of epizootiological and serological stu- dies on swine influenza are presented in this report and an alternative hypothesis is proposed to account for the survival of swine influenzavirus between epi- zootics. MATERIALS AND) METHODS Epizootiological studies Naturally occurring outbreaks of swine influenza were studied on farms in south-central Wisconsin, USA (Dane, Green, Rock, and Lafayette counties) and in Knox County, Nebraska. On visits to these 2920 - 481 - R. M. NAKAMURA ET AL. farms epizootiological information was obtained, clinical examinations of animals were made, blood and nasal swab specimens were obtained, and hus- bandry practices were noted. Virus isolation Applicator sticks tipped with cotton-wool were used to swab the nasal passages of swine suspected of having influenza. The swabs were placed in small vials containing 2 ml of brain-heart infusion broth. The broth was then filtered through membrane filters with an average pore diameter of 0.22 ,um. The fil- trate was inoculated by the amniotic and allantoic routes into 10-day embryonated chicken eggs imme- diately or stored at - 60°C until eggs were available. After incubation for 72 hours at 35°C, the egg fluids were collected and tested for the presence of virus by haemagglutination (HA) tests. A specimen was considered negative when virus was not detected after 3 passages. Serological studies Serum was obtained from swine involved in natu- rally occurring outbreaks of swine influenza and from swine being slaughtered at a local abattoir. Serological studies were also conducted over a period of 6 months on animals purchased from a farm where a natural outbreak of swine influenza occurred. These animals, designated XI, X3, X5, X9, and X1O, were approximately 2 weeks old when the outbreak occurred, and were purchased at 6 weeks of age and held in semi-isolation. Seven pigs, designated K1-K7, were exposed at 5 months of age, by intranasal swabbing, to virus (A/swine/Wisconsin/1/66(HswlNl)) isolated from a natural outbreak of swine influenza (Table 1). Treatment of sera All sera for testing were treated to decrease non- specific inhibitor substances. The sera were heated to 56°C for 30 minutes and treated with 0.8% trypsin and with M/90 potassium periodate (Jensen, 1961). In addition, the sera were absorbed with washed and packed chicken erythrocytes to remove the non- specific agglutinins that occur in some swine sera. Physiological saline was added to the treated serum to adjust the volume to a final dilution of 1: 10. The haemagglutination-inhibition (HI) test The 1 3theggpassage ofswine/Iowa/15/30(HswlNI) virus (Shope, 1931), propagated in embryonated chicken eggs, was concentrated and partially purified by 3 cycles of adsorption and elution with washed chicken erythrocytes (Lief& Henle, 1956). This anti- gen was used in HI tests according to standardized methods (Expert Committee on Respiratory Virus Diseases, 1959) in plastic disposable trays. Statistical analysis Statistical analyses were made by calculating the chi-square for 2 x 4 contingency tables. In all cases, the results of the HI tests of sera from one group of animals were compared with those of another group. The sera from each group were placed in 1 of 4 subsets according to the determined HI titre (titres: 0-20, 40-80, 160-320, or 640-1 280). The calculated chi-square values were then compared with a table of chi-square values to determine the probability that the 2 groups were different. RESULTS Epidemiological studies Virus was isolated most consistently from swine during the early febrile stage of the disease and less frequently thereafter. Virus was isolated on 9 occa- sions from outbreaks of swine influenza during the period 1965-68. Some of the virus isolations were made in the autumn, some in January, and some as late as March (Table 1). Serological testing con- firmed the clinical diagnosis of swine influenza on six additional farms. The disease was suspected on 4 other farms based only on clinical signs. On farms R, E, D, and Ba, virus was isolated from sows or suckling pigs in the farrowing pens (Tables 1 and 2). With the exception of farm Ba, there were no overt signs of disease in the sows. Outbreaks of swine influenza occurred on farms S, BI, and H in young fattening swine where no breed- ing stock were kept (Tables 1 and 2). Virus was isolated from swine on farm Bi in October 1966, and again in late January 1968, but the disease was not noticed in the autumn of 1967. The outbreak on farm H occurred in March 1968 in fattening pigs shortly after they were transported from Missouri to Nebraska. Virus was isolated from swine during an outbreak of swine influenza on farm R and clinical and sero- logical evidence (Nakamura & Easterday, 1967) indicated that swine influenza had occurred on farm K in the autumn of 1965 (Tables 1 and 2). However, influenza was not observed in swine on these farms (R and K) in the autumn or winter of 1966-67, in spite of the fact that many outbreaks 482 EPIZOOTIOLOGICAL AND SEROLOGICAL STUDIES OF SWINE INFLUENZA Table 1. The isolation of swine influenzaviruses during natural outbreaks of influenza on farms from 1965 to 1968 Farm Date of virus isolation Type of animal Isolate designation a R 7 Oct. 1965 sows A/swine/Wisconsin/i /1965 S 29 Sept. 1966 fattening swine A/swine/Wisconsin/i /1966 Bi 4 Oct. 1966 fattening swine A/swine/Wisconsin/2/1966 E 4 Oct. 1966 suckling pigs A/swine/Wisconsin/3/1966 P 7 Jan. 1967 fattening swine A/swine/Wisconsin/i /1967 D 4 Jan. 1968 suckling pigs A/swine/Wisconsin/i /1 968 Ba 9 Jan. 1968 sows and suckling pigs A/swine/Wisconsin/2/1968 Bi 26 Jan. 1968 fattening swine A/swine/Wisconsin/3/1968 H 26 March 1968 fattening swine A/swine/Nebraska/i /1968 a All these strains have the same antigenic designation -Hswl Ni. Table 2. Summary of epizootiological studies Epizootiological observations Farms influenza among fattening pigs-no sows kept S, B1, H initial focus of infection in farrowing pens R, E, Ba, D absence of clinical influenza on farms where influenza was known to have occurred in previous years R, K, Ma - atypical " influenza D, A, Pmr, Bi influenza-like disease F, Z, M, J, W, S, C of swine influenza occurred on many other farms in the area. Negative serological tests of swine sera from these farms in the spring of 1967 confirmed that influenza had not occurred on these farms. The farm owners reported that swine influenza had occurred annually on their farms in the past. One explanation offered for the absence of influenza in the autumn of 1966 on farm R was that all breeding stock were sold in the spring of 1966. The breeding stock on farm K were immunized during the summer of 1966 with inactivated swine influenza virus, which resulted in marked anamnestic antibody responses with very high levels of antibody (Nakamura & Easterday, 1967). There was complete depopulation of swine on one university-owned farm (Ma) in 1965 and replace- ment with specific-pathogen-free swine. Since then, swine influenza has not occurred in clinical form and could not be detected by serological testing. Although influenza was known to have occurred on that farm in the past, there was no clear history of swine influ- enza in the years immediately prior to 1965. On another farm owned by the university (farm A, Tables 2 and 3) influenza has continued to occur annually. Swine influenza that differed from the usual clas- sical form was observed on farms D, A, Pmr, and Bi (Table 2). The disease in swine on farms D, A, and Pmr was less severe, involved fewer animals, but persisted for longer periods in the herd. The common feature of these three farms was that the swine were confined indoors. On farm Bi, virus was isolated (Table 1) from a few swine that had clinical signs of influenza. The clinical disease did not progress beyond the few animals initially in- volved. However, tests of convalescent sera indi- cated that most animals developed antibody follow- ing the disease (Table 2). Outbreaks of respiratory disease with all the char- acteristics of classical acute swine influenza were investigated on farms C and S (March), M (April), F (May), Z (June), and J and W (October) (Table 2). Attempts to isolate the causative factor in embryon- ated eggs and monkey kidney tissue culture failed. Antibodies were not detected in convalescent sera from the swine involved on these farms except from those on farm J. There had been an episode of res- piratory disease on farm J three weeks previously and this probably accounted for the low levels of antibody in the acute phase sera and the substantial 4 483 R. M. NAKAMURA ET AL. levels of antibody in the convalescent sera following the second disease outbreak. Continued surveillance throughout the years 1968-70 in southern Wisconsin supported the obser- vations reported above. Virus was isolated from several outbreaks of swine influenza during this period. Including the isolation of virus from one atypical outbreak at the end of May 1970, virus has been obtained in every month of the year except June. Six herds have been under special surveillance since 1968. Swine influenza virus had been isolated on 2 of these farms in 1968 and 1969. There has been no evidence of the disease on 5 of the 6 farms since 1968, but it has continued to occur annually on one farm. Two of the farms have not had overt signs of disease within the memory of the owners (5-10 years) and there has been no evidence of the disease during the study. Serological studies The results of HI tests of sera from swine after natural outbreaks of swine influenza are presented in Table 3. Higher levels of antibody were detected in sera from the breeder swine than in those from the feeder swine. However, sera were not always obtained from both age groups on all the farms studied. Sera from breeder and market swine were obtained at a local abattoir on 3 successive days in late November (approximately 2 months after the start of influenza outbreaks in Wisconsin) and again in May and August. Samples were collected from swine without regard to point of origin as they were being exsanguinated on the slaughter line and not according to any prearranged plan. The results of these tests are presented in Table 4. Higher levels of antibody were detected in serum samples from breeding animals than in those samples from " mar- ket" swine. This difference was found to be of high statistical significance in samples collected at the three different times of the year from these two age groups. The serum samples obtained from market pigs in May had significantly higher antibody levels than the market pig samples taken in Novem- ber. However, there was less difference between the antibody levels of samples from the breeder swine obtained at different times of the year. Serological studies were conducted on serum samples obtained from fattening swine on farm S at the time of the natural outbreak of swine influ- enza, and also 1 and 2 months after the outbreak (Fig. 1). Higher antibody levels were found in a significantly larger proportion of the sera obtained at 2 months after the outbreak than in those obtained at 1 month. The long-term serological responses to naturally and experimentally infected animals were studied. Two of the five naturally infected swine had higher levels of antibody at 3 and 4 months after infection than at 1 month, whereas the level in one pig was high at 1 month and gradually decreased. The level of antibody in the other two pigs remained constant throughout the observation period. There was a gradual rise in antibody in 6 of the 7 experimentally infected swine so that the levels at 6 months were higher than at 1 month after exposure. DISCUSSION In order to understand the natural history of influenza of human beings and other species it is important to be able to account for the survival and transmission of the viruses between epidemics or epizootics. Observations of outbreaks of swine influenza and other respiratory diseases of swine in Wisconsin provided an opportunity to accumulate data on the occurrence of the virus during these periods under natural conditions. Perhaps the most significant observation has been that the swine influ- enza virus is active between epizootics as is shown by the isolation of the virus during 11 of the 12 months of the year. It has been stated that the prevalence of swine influenza in the USA has been decreasing (Blood & Henderson, 1968). Although our data cover only a relatively short period they do not indicate a decreasing prevalence of the disease and are similar to the data reported by Young & Underdahl (1951, 1955). During the course of our observations it became obvious that swine influenza could not be diagnosed solely on the basis of clinical signs and that the disease did not always occur in a typical or classical form. The virus has been recovered from pigs on at least 5 farms where there was respiratory disease not typical of swine influenza. The disease may be typical in individual animals but it is atypical on a herd basis, e.g., the disease spreads slowly and only 25-40% of the animals may have overt signs of disease. Within such outbreaks there will be a small proportion of animals with typical signs, a simi- lar proportion with mild respiratory disease, and a much larger proportion with no overt signs of dis- ease. The failure to recover virus and to demon- 484 EPIZOOTIOLOGICAL AND SEROLOGICAL STUDIES OF SWINE INFLUENZA Table 3. Amounts of haemagglutination-inhibiting antibody in convalescent sera from breeding and fattening swine on Wisconsin farms a No. of samples with following HI titres: Farm 10 20 40 80 160 320 640 >1280 Fattening stock 1 0 2 4 3 1 0 0 0 2 0 0 3 5 1 4 0 2 3 0 0 0 2 2 2 0 0 4 0 2 2 4 1 1 0 0 5 2 9 17 8 2 0 0 0 total 2 13 26 22 7 7 0 2 Breeding stock 6 0 0 1 1 3 3 2 0 7 0 0 0 0 6 9 8 0 8 0 0 0 0 0 4 1 0 9 0 0 0 0 0 0 2 2 4 0 0 0 1 4 7 2 5 10 0 0 0 1 5 2 3 0 total 0 0 1 3 18 25 18 7 a Swine/lowa/i 5/30 virus used as antigen. Table 4. Results of haemagglutination inhibition tests of sera obtained from a Wisconsin abattoir in November, May, and August a No. of samples with the following HI titres: b <10 10 20 40 80 160 320 640 >1 280 Breeder pigs November 36 116 74 20 13 19 30 22 52(9.4) (30.4) (19,4) (5.2) (3.4) (5.0) (7.9) (5.8) (13.6) May 26 46 25 11 17 13 10 18 6 (15.1) (26.7) (14.5) (6.7) (9.9) (7.6) (5.8) (10.5) (3.5) August 35 40 21 8 9 17 10 19 27 (18.8) (21.5) (11.3) (4.3) (4.8) (9.1) (5.4) (10.2) (14.51) Market pigs November 24 85 27 10 9 3 0 0 0(15.2) (53.8) (17.1) (6.3) (5.7) (1.9) May 59 68 15 3 2 9 6 3 0(35.8) (41.2) (9.1) (1.8) (1.2) (5.5) (3.6) (1.8) August 67 70 21 9 4 5 1 1 1 (37.4) (39.1) (11.7) (5.0) (2.2) (2.8) (0.6) (0.6) (0.6) a Breeder pigs were mostly in the age range of 1-2 years and the market pigs were 5-7 months old. Swine/lowa/i 5/30 virus was used as antigen. b Percentage distribution of titres shown in parentheses. 485 R. M. NAKAMURA ET AL. I a a I eu.uumiimiAcute-30 Sept. 1MM I Nov. -001 S Dec. M 0 a 0 a~~~~ M 0 M , 0 M~~~ , * M s : a a *M am a U U U U U M U M Mu-~~~~ I CIO 10 20 40 80 160 320 Reciprocals of Hi titres Fig. 1. Levels of haemagglutination-inhibiting antibody in swine at 1 and 2 months swine influenza. strate antibody in outbreaks of what appeared to be classical swine influenza further emphasize the need for laboratory confirmation of suspected influ- enza outbreaks in any species. The observations made in this study, and by others, suggest that there are means other than, or in addition to, the masked virus-lungworm mechanism proposed by Shope to account for the survival of the swine influenzavirus between epi- zootics. While none of the observations excludes Shope's hypothesis, collectively they suggest that virus may be maintained by alternative means. The isolation of the virus in every month of the year except June indicates that the virus is circulat- ing among pigs throughout the year. The increased prevalence in the autumn and early winter in the north central areas of the USA may be explained by certain features of current swine management practice. A very small percentage of the animals that live through one influenza season are alive 1 year later. Most of the pigs are slaughtered when they are 5-7 months old. Thus at the time of the next " season " there is again a very high proportion of young nonimmune pigs in the herd. In October there are marked climatic changes and changes in management. The fact that the disease occurs first in the far- rowing pens among suckling pigs is difficult to explain by the lungworm mechanism. It is postu- lated that the sows infected during the previous year retain their infection and thus infect the suck- following an outbreak of ling pig. Pigs suckling immune sows are susceptible and may show signs of disease (Easterday, 1972). It is unlikely that the disease would have dis- appeared on 3 farms in our study if lungworms were important in the maintenance of the disease; lung- worm larvae are known to remain infective for up to 3 years in earthworms. On one farm (R) all of the breeding stock were sold. On the second farm (K) all of the breeding stock were hyperimmunized by inoculation of inactivated virus and on the third farm there was complete depopulation and replace- ment with specific-pathogen-free swine. The results of the serological studies suggest that the virus may persist following initial infection. The consistently higher levels of antibody in breeding stock may result from a continuous antigenic stimu- lus from persisting virus. The increasing and per- sisting levels of antibody in experimentally and naturally infected pigs also suggest the persistence of the virus. In pigs the TGE antibody has been found to reach a peak at 17 days after exposure, to remain level for about 10 weeks, and to decline gradually to barely detectable levels 11 months after infection (Witte & Easterday, 1968). It has been suggested that advance seeding of influenzavirus in swine (Shope, 1964) may explain the outbreaks among swine assembled on farms where no breeding stock is kept. In such cases swine from several farms may be mixed and trans- ported many miles to the fattening farms. The idea of advance seeding has also been discussed in rela- 'C a saj5C E co E x @2 0 02 CL 1 42 0~ Iu ,1 I 486 EPIZOOTIOLOGICAL AND SEROLOGICAL STUDIES OF SWINE INFLUENZA 487 tion to epidemics of human influenza (Andrewes, 1964). Blaskovi6 et al. (1970) reported experiments in which they considered that virus shedding and trans- mission of swine influenzavirus had occurred some time during the 4-month period following experi- mental infection. No lungworms were found on necropsy of the pigs and they reported that " pos- sible sources of technical error such as laboratory contamination could be almost certainly excluded." On the basis of all the above observations it seems reasonable to propose that upon convalescence from influenza some swine may be persistently infected with the virus. Continued investigation of the natural history of swine influenza may be expected to provide further useful information for the understanding of all influ- enza infections. ACKNOWLEDGEMENTS This work was supported in part by a fellowship from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md., USA (5-F3-Al-24, 485-03), by research grant 5-ROI A110215 from the same agency, by Research grant 5 ROI HD02397 from the National Institute of Child Health and Human Development, and by the World Health Organization. RISUMt GRIPPE PORCINE: tTUDES EPIZOOTIOLOGIQ UES ET SEROLOGIQUES Des enquetes ont ete menees durant des 6pizooties survenues dans des 6levages de porcs au Wisconsin (Etats-Unis d'Am6rique) et completees par des etudes virologiques et serologiques. La grippe est fr6quente chez le porc et rev8t des formes legeres aussi bien que des formes classiques. On observe aussi des pouss6es d'affections des voies respiratoires d'etiologie inconnue mais de symptomatologie clinique semblable a celle de la grippe. Des virus de la grippe porcine ont 6te isoles pendant 11 mois de l'annee. Dans certains elevages, 1'6pizootie a atteint en premier lieu les porcelets nouveau-n6s. Dans d'autres, out des poussees de grippe avaient et6 observees les ann6es pr&c& dentes, les animaux sont restes indemnes de l'infection. Le fait est attribue au remplacement ou A l'immunisation des animaux reproducteurs. Les etudes s6rologiques ont montr6 que chez les porcs infect6s naturellement ou exp6rimentalement la hausse des titres d'anticorps se poursuivait pendant plusieurs mois. Les s6rums de porcs reproducteurs ag6s renfermaient davantage d'anticorps que les serums des jeunes porcs A 1'engraissement. Pour les auteurs, la survie et le maintien de la trans- mission des virus grippaux dans l'intervalle des epi- zooties, chez le porc, s'explique par leur persistance chez certains animaux convalescents de l'infection qui jouent le role de r6servoir de virus. REFERENCE S Andrewes, C. H. et al. (1935) Brit. J. exp. Path., 16, 566-582 Andrewes, C. H. (1964) Science, 146, 1274-1278 Blaskovid, D. et al. (1970) Bull. Wld Hlth Org., 42, 767-770 Blood, D. C. & Henderson, J. A. (1968) Veterinary medi- cine, 3rd ed. Baltimore, Md., Williams and Wilkins Company, pp. 501-503 Davenport, F. M. et al. (1953) J. exp. Med., 98, 614-656 Dorset, M. et al. (1922) J. Amer. vet. med. Ass., 62, 162-171 Easterday, B. C. 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