Some observations on the necessity for serological testing of rodent sera for Pasteurella pestis antibody in a plague control programme.

Notes Some Observations on the Necessity for Serological Testing of Rodent Sera for Pasteurella pestis Antibody in a Plague Control Programme by D. C. CAVANAUGH,1 P. J. DEORAS,2 D. H. HUNTER,8 J. D. MARSHALL, JR,4 DO-VAN-QUY,5 J. H. RUST, JR,1 SITHIBUN PURNAVEJA 6 & P. E. WINTER 3 There appears to be general agreement that the passive haemagglutination test developed by Chen & Meyer (1966) to detect antibody to the Fraction 1 antigen of Pasteurella pestis (Yersinia pestis) is valuable in detecting rodent plague foci (WHO Expert Committee on Plague, 1970). Since 1960, several thousand wild commensal or peridomestic rodent sera have been tested for haemagglutinating antibody to the Fraction 1 antigen of P. pestis. The rodents from which the sera were obtained were trapped in actual or former plague foci. The plague epidemic that is occurring in the Republic of Viet-Nam has provided a unique oppor- tunity for a large-scale evaluation of the haemag- glutination test as a supplementary tool to follow the course of events in the commensal rodents during the epidemic and subsequent control period. The results of the serological tests demonstrate that serum surveys for plague antibody should be included in every plague surveillance and control programme. The survey data which led to this conclusion are presented below. In the United States ofAmerica, sera from Citellus, Eutamius, Cynomys, Dipodomys, Neotoma, Sigmo- don, Microtus, Onychomys, Peromyscus, Sylvilagus and Lepus were collected in plague foci in New Mexico and Utah and tested for haemagglutinating antibody to the Fraction 1 antigen of P. pestis. The data obtained in these studies showed that while the isolation of P. pestis is seasonal and rather rare, the haemagglutinating antibody can be detected 1 Walter Reed Army Institute of Research, Washington, D.C., USA. 2 Director, Department of Entomology, Haffkine Insti- tute, Bombay, India. 3Office of the Surgeon General, Washington, D.C., USA. ' United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Md., USA. 5 Director, Department of Entomology, Institut Pasteur, Saigon, Viet-Nam. ' Director of Research, Thai Component, SEATO Medi- cal Research Laboratory, Bangkok, Thailand. frequently and over long periods in rodents surviv- ing infection. The seasonal aspects of enzootic plague were particularly emphasized in the Utah study (Cavanaugh et al., 1965) since P. pestis could be isolated from the rodent population, or from the rodent fleas, in only 5 of the 21 months the focus was studied. By contrast, haemagglutinating antibody to the Fraction 1 antigen of P. pestis was detected in 19 of the 21 months. In New Mexico, there was sero- logical evidence that plague had persisted in areas where it was epizootic. Further confirmation has been obtained by infrequent isolations of P. pestis, and by collecting and testing large numbers of pools of rodent tissues or fleas over a period of years. The frequency with which P. pestis antibody can be detected in the sera of rodents trapped in enzootic plague foci in comparison with the rare isolations of P. pestis indicated that serological testing should be incorporated into routine plague survey measures. Materials and methods The P. pestis Fraction 1 antigen used in these studies was obtained from Dr K. F. Meyer.7 Prior to utilizing the antigen in serological tests, it was studied for conformity to the published descriptions of Baker et al. (1947, 1952). In standard potency tests, 50% of test mice receiving a total of 25 jig of the Fraction 1 antigen in 2 equally divided doses, survived when challenged with 1500 LD50 of virulent P. pestis. In Ouchterlony tests (Chen & Meyer, 1955) the Frac- tion 1 antigen in a 100 jig/ml concentration produced a single line when diffused against a 1:10 dilution of P. pestis polyvalent antiserum (Lederle) at a distance of 5 mm. Of 1056 well-studied P. pestis strains isolat- ed in the Republic of Viet-Nam (RVN) 14 were also tested Ouchterlony tests against Lederle antiserum. Strains isolated from 10 suspected plague cases, 7 George Williams Hooper Foundation for Medical Research, University of California Medical Center, San Francisco, Calif., USA. 2488A - 451 452 NOTES 2 strains isolated from fleas on Rattus norvegicus, 1 strain isolated from the spleen of R. norvegicus, and 1 strain isolated from fleas on Suncus murinus all produced lines of identity with the Fraction 1 antigen. No lines of identity were observed when Fraction 1 was compared with P. pseudotuberculosis, type I-type V, in similar Ouchterlony tests. Sera were tested for haemagglutinating antibody to the Fraction 1 antigen of P. pestis by the methods outlined by Cavanaugh et al. (1965). Microtitration equipment' was used throughout. The actual test pro- cedures are given in an appendix to the report of the WHO Expert Committee on Plague (1970). As a control on the actual testing technique, the develop- ment of the haemagglutinating antibody response in plague-infected human beings was studied. Paired sera and clinical material suitable for the isolation of P. pestis were collected from patients with suspected plague. Circumstances dictated that only 2 speci- mens of sera could be obtained from each patient. The first specimen was collected on the first day in hospital. The second specimen ofserum was obtained on the day the patient left the hospital. As a negative control in these studies, sera obtained from 1332 patients with fevers of undetermined origin seen in neighbouring plague-free Thailand were included in the tests. Rodents were collected in cage traps. Trapped rodents were placed in cotton bags and bled from the heart, through the bag. Fleas were removed from the rodents which were then autopsied. Flea and tissue pools were handled as outlined by Balta- zard et al. (1956) and by the WHO Expert Com- mittee on Plague (1959); they were sent to the labora- tory and inoculated into susceptible white laboratory mice. The principles of laboratory animal care as promulgated by the National Society for Medical Research were observed. Pasteurella pestis isolated from the mice dying of plague were identified by the criteria of Baltazard et al. (1956) and Marshall et al. (unpublished data). Sera were tested for haemag- glutinating antibody to the Fraction 1 antigen of P. pestis described above. Results While no difficulties were experienced with tests of human sera, the testing of sera from a multi- plicity of small mammalian species presented an early problem. 1 Cooke Engineering Co., Alexandria, Va., USA. Non-specific haemagglutination. It was observed that occasional sera produced haemagglutination in a control series of serum dilutions which was included in the testing of each particular serum. The antigen in the control series of serum dilutions was tanned erythrocytes which were not sensitized with Fraction 1 antigen, but which were otherwise prepared in an identical way. This non-specific haemagglutination was observed in sera which were previously absorbed with sheep erythrocytes as recommended by Chen & Meyer (1954) and subse- quent additional absorptions often failed to remove this non-specific haemagglutinin which often reacted to the same degree as the reaction observed in the dilutions of serum tested against tanned erythrocytes which had been sensitized with Fraction 1 antigen. In the results given below, sera showing such non- specific haemagglutinations are simply recorded as " negative " under the conditions of the test. The number of such non-specific reactions, unfortunately, was not recorded, but they were observed in all species of mammalian sera tested with the exception of those collected from human beings. Apparently, not all sheep are suitable sources. Further, erythrocytes from individual sheep were suitable when collected at one time and not suitable when collected at another. Most of the unsuitable erythrocytes were obtained from sheep which had recently received vermifuges of one type or another. This finding meant that erythrocytes from several sheep had to be tested with known positive and negative sera in a standard haemagglutination test to certify the reliability of the test. The test data described below represent the results of serum surveys undertaken with due regard to the mandatory precautions outlined above. For the sake of brevity, haemagglutinating antibody to the Frac- tion 1 antigen of P. pestis will, in the results and discussion, be simply referred to as plague HA antibody. Human sera. Sera from 1053 patients with fevers of undetermined origin, collected in plague-free Thailand, were negative for plague HA antibody. Among this group murine typhus was isolated from 38 patients and an additional group showed a rise in titre against this flea-borne disease. By contrast, Table 1 lists the results obtained when paired sera collected from bacteriologically confirmed Vietnamese plague patients were tested for plague HA antibody. These results are presented as the initial specimen of serum obtained from all patients SEROLOGICAL TESTING OF RODENT SERA IN A PLAGUE CONTROL PROGRAMME TABLE I HAEMAGGLUTINATING ANTIBODY TO P. PESTIS FRACTION I ANTIGEN OBSERVED IN CONVALESCENT SERA OF 34 BACTERIOLOGICALLY CONFIRMED BUBONIC PLAGUE PATIENTS IN SAIGON, VIET-NAM, 1964-1965 Titre No. of days in hospitalTitre 5-9 10-14 15-19 20-30 1:16 1:32 1 1:64 I 2 1:128 1 2 1 1 :256 3 2 1 2 1:512 2 4 1:1024 1 2 2 1 1:2048 1 2 1:4096 2 1 1:8192 1:1634 1 Totals 12 8 4 10 was negative for plague HA antibody and the plague HA antibody titre observed in the second specimen may, therefore, represent the antibody response to plague infection seen in human beings. Further support of this contention is provided by the fact that all the patients denied that they had been vaccinated against plague. In this series of patients, plague HA antibody was not detected prior to the fifth hospital day. The appearance of plague HA antibody on the fifth day of disease has been confirmed by all the sub-human primate studies (K. F. Meyer, personal communication). Unpurified Fraction 1 may contain any of the antigens which P. pestis shares with P. pseudotuber- culosis and may, therefore, react non-specifically with sera containing antibody against P. pseudotuber- culosis. Animal sera. Table 2 gives the details of the isola- tion of P. pestis from commensal mammals and their flea ectoparasites that were collected in the city of Saigon. Plague HA antibody has been demonstrated in the sera of all the species of mammals listed in Table 2, but the collection of sera from all animals except Rattus norvegicus has been rather sporadic. The data for testing the sera from 5192 R. norvegicus TABLE 2 NUMBER OF ISOLATIONS OF PASTEURELLA PESTIS PER QUARTER IN SAIGON, 1963-67 Year 1963 1965 Quarter 1St 2nd 1St 2nd 3rd a 4th a Source of isolations Mammalian host Spleen Flea Rattus norvegicus 0 2 Suncus murinus 0 1 R. norvegicus 1 0 R. norvegicus R. norvegicus R. norvegicus R. exulans R. norvegicus R. rattus R. exulans 2 0 0 5 1 4 2 2 1 0 0 1966 1st a R. norvegicus 4 0 R. exulans 5 1 S. murinus 2 0 2nd a R. norvegicus 1 0 S. murinus 0 2 3rd R. norvegicus 0 1 S. murinus 1 0 4th R. norvegicus 0 1 R. exulans 1 0 1967 1St R. norvegicus 1 0 R. exulans 1 0 a Data from Marshall et al. (1967). collected in Saigon is given in Table 3. In calculating the geometric mean titres shown in Table 3, an arbitrary value of 1.0 was assigned to the negative sera. Data on the incidence of human plague in the residents of Saigon are included in Table 3 and the flea indices (Xenopsylla cheopis) for R. norvegicus in Table 4. Note the cyclic seasonal nature of both the flea index and the percentage of positive sera. Table 5 presents the details of the isolation of P. pestis from small mammals or flea ectoparasites in the city of Nhatrang, Viet-Nam. Again, plague HA antibody has been demonstrated in the sera of all the species listed. Table 6 gives the results obtained when sera collected from R. norvegicus were tested for plague HA antibody. The tissues of the R. norvegicus collected in March 1967 were examined individually and not by pooling methods for plague infection. Each rat was tested by culture and animal inoculation, and the fleas from each rat were inoculated on an individual basis into white mice. The results of these tests and the results of 453 TABLE 3 HAEMAGGLUTINATING ANTIBODY TO PASTEURELLA PESTIS FRACTION I ANTIGEN OF RATTUS NORVEGICUS SERA IN SAIGON, VIET-NAM, 1962-67 Year and quarter Titre 1962 1963 1964 1965 1966 1967 Total 4th 1st 4th 1st 2nd 3rd a 4th a lsta 2nd a 3rd 4th 1st 2nd 1:16 3 9 7 18 15 1 4 6 4 1:32 1 2 4 14 6 4 24 8 3 7 7 7 1:64 2 2 2 6 13 2 3 11 4 2 2 5 1 1:128 6 3 4 1 1 1 2 1 3 1 1 :256 5 5 1 2 2 1 1 1 1:512 1 2 2 1 1 1:1024 2 1 1 No. tested 252 152 279 488 701 607 713 415 315 282 311 265 313 5 093 No. positive 17 13 7 21 50 15 9 55 29 6 16 20 12 270 % Positive 6.7 8.55 2.51 4.30 7.13 2.47 1.3 13.4 9.21 2.1 5.6 7.5 3.8 5.3 Geometric mean titre 1.43 1.52 1.13 1.20 1.34 1.08 1.05 1.57 1.36 1.08 1.21 1.31 1.13 1.27 Human plague _b 2 4 89 285 32 0 0 1 1 0 2 0 416 a Data from Marshall, Currie & Quy (1968). b There were rumours of a few cases. the serological examinations are given in Table 7. In Nhatrang, sporadic cases of plague occur through- out the year and seasonal epidemics of plague have occurred since 1962. The attack rates for plague in the city range from 15-25 per 10000. The peaks of the annual epidemics occur in the month of March. The plague calendar for Nhatrang is pre- sented in Table 8, but these data pertain only to the specimens which have been collected from patients seeking medical attention. The actual number of cases is thought to be much higher. For the purposes of comparison, the data obtained in Nhatrang and Saigon are summarized in Table 9. The 3 laboratory indices for enzootic plague in the 2 cities are different. The ratio of tissue isolates is 2.8: 1, that of flea isolation is 13.9: 1, while the ratio of HA positive animals is 6.5 : 1. All 3 indices provide a relative measure of the extent of flea transmission in a given location. In association with the Haffkine Institute, a serum survey of the commensal rodents of Bombay, India, was conducted in 1960. The survey was timed to coincide with the end of the season when plague had been most prevalent in former epidemics. All rats were trapped in areas which had been highly infected in past epidemics. P. pestis was not isolated from the tissues of the rodents collected or from their fleas. Some 534 sera were obtained from the following species during the course of the survey: R. norvegicus, 108; R. rattus, 231; and Bandicota bengalesis, 195. All the rodent sera were negative for plague HA antibody. Ouchterlony double- diffusion tests against whole-cell extracts of P. pestis and P. pseudotuberculosis resulted in the demon- stration of antibody in several sera against antigens common to both, but not against the Fl antigen of P. pestis (Deoras, 1960). The Haffkine Institute examines many thousands of rats each year for plague infection. The records of the Haffkine Institute indicate that 44 rats in 1948, only 6 rats in 1949 and 3 rats in 1952 were found to be plague-infected in Bombay. No further plague-infected rats were found in Bombay until the time of survey. Until 1952, when extensive, integrated plague control measures were instituted employing extensive dusting with DDT, certain areas of Thailand were subject to sporadic outbreaks of plague. Since 1952, plague has not been recorded in Thailand and P. pestis has not been isolated from rodents or fleas collected in the extensive surveys undertaken 454 NOTES SEROLOGICAL TESTING OF RODENT SERA IN A PLAGUE CONTROL PROGRAMME TABLE 4 QUARTERLY XENOPSYLLA CHEOPIS INDICES FOR SMALL COMMENSAL MAMMALS, SAIGON, VIET-NAM, 1963-67 Host Year Quarter R. norvegicus R. exulans S. murinus 1963 1st 2nd 4.34 1.16 1.43 3rd 1.34 0.66 0.62 4th 0.93 0.68 0.47 1964 1 st 1.54 0.76 0.71 2nd 3.31 1.46 1.89 3rd 1.31 4.63 0.65 4th 1.24 0.74 0.41 1965 1 st 4.31 0.78 0.76 2nd 2.96 1.35 0.95 3rd 1.41 1.07 0.81 4th 0.98 0.85 0.42 1966 1 st 2.0 0.99 0.71 2nd 1.39 0.98 1.08 3rd 0.79 0.42 0.34 4th 1.12 0.38 0.42 1967 1 st 2.02 0.55 0.30 2nd 3.61 1.47 0.88 TABLE 5 NUMBER OF PASTEURELLA PESTIS ISOLATIONS FROM SMALL MAMMALS, NHATRANG, VIET-NAM, 1966-67 Source of isolation Date Rattus R. exulans Surinus of collection norvegkcusmuis Spleen IFlea ISpleen Flea ISpleen Flea April 1966 0 0 0 0 1 1 May 1966 0 2 0 0 1 2 October 1966 1 1 0 1 0 1 March 1967 6 4 0 1 0 2 TABLE 6 HAEMAGGLUTINATING ANTIBODY TO PASTEURELLA PESTIS FRACTION I ANTIGEN IN SERA OF RATTUS NORVEGICUS COLLECTED IN NHATRANG, VIET-NAM, 1966-67 Date of collection Titre April May October March 1966 1966 1966 1967 1:8 4 1 1:16 5 6 10 1:32 7 3 7 1:64 2 2 1 13 1:128 1 7 6 9 1:256 2 7 1 7 1:512 1 4 1:1024 1 1 1:2048 1 1 No. tested 26 113 66 117 No. positive 7 33 18 52 % Positive 16.9 29.2 27.3 44.4 Geometric mean titre 4.22 3.30 2.80 6.58 X. cheopis index 8.48 7.14 6.43 6.3 TABLE 7 HAEMAGGLUTINATING ANTIBODY TO PASTEURELLA PESTIS FRACTION I ANTIGEN IN SERA OF RATTUS NORVEGICUS WHICH WERE EITHER PLAGUE-INFECTED OR INFESTED WITH PLAGUE-INFECTED FLEAS COLLECTED IN AN EPIDEMIC PLAGUE FOCUS Non-plague-infected HA antibody titre Plague-infected i nfested withR. norvegicus plague-infected fleas Non-reactive 1 2 1:16 1 1:64 1 1:128 1 1: 256 1 455 456 NOTES TABLE 8 PLAGUE IN HUMAN BEINGS, NHATRANG AND VICINITY, SEPTEMBER 1962-DECEMBER 1966a 1962 1963 1964 1965 1966 Totals No. of No.'of No. of No. of No. of No. of suspect P.pestis suspect P.pestis suspect P.pestis suspect P.pestis suspect P.pestis suspect P. pestis cases isolated cases isolated cases isolated cases isolated cases isolated cases isolated tested tested tested tested tested tested _ January 7 2 29 15 32 14 18 12 86 143 February 14 8 20 14 27 17 39 20 100 59 March 9 4 102 71 68 38 26 10 205 123 April 54 26 98 52 36 19 21 7 209 104 May 32 15 16 7 28 16 19 11 95 49 June 15 0 20 11 14 5 9 3 58 19 July 7 2 15 3 11 5 11 2 44 12 August 16 6 15 3 7 0 14 5 52 14 September N.A.b 2 10 6 12 1 10 2 15 5 47 16 October N.A.b 0 1 1 19 5 1 0 31 17 52 23 November N.A.b 1 0 0 20 9 9 4 39 19 68 33 December N.A.b 4 21 15 32 17 15 1 67 37 135 74 Totals [ 7 186 [85 398 j208 258 121 309 148 1 151 569 a Data from Cavanaugh et al. (1968). b N.A. = not available. by the Government of Thailand. With the advent mensal rodents were negative for plague HA of plague in neighbouring Viet-Nam, further surveys antibody. were undertaken in formerly endemic areas. A total of 321 tissue pools collected from commensal rodents Discussion and approximately 100 pools of flea ectoparasites A programme of testing sera from commensal from these rodents were negative for P. pestis when rodents trapped in actual plague foci for haemag- tested by animal inoculation. Sera from 576 com- glutinating antibody to the Fraction 1 antigen of TABLE 9 PASTEURELLA PESTIS ISOLATIONS (1965-67) FROM ALL SMALL COMMENSAL MAMMALS COLLECTED IN TWO CITIES IN VIET-NAMa Isolations P. pestis isolations per 1 000 mammals antibodYCity tested irantibody Spleen Flea Spleen Flea (% positive) Nhatrang-epidemic area with annual plague epidemics 9 15 7.5 12.5 34.2 Saigon/Cholon-endemic area with occasional plague epidemics 30 10 2.7 0.9 5.3 a Tissues and flea ectoparasite tested by pooling technique. b Rattus norvegicus only. SEROLOGICAL TESTING OF RODENT SERA IN A PLAGUE CONTROL PROGRAMME Pasteurella pestis (plague HA antibody) has been in progress for several years in the Republic of Viet-Nam. It has been possible to include control studies based on collections made in former epidemic plague foci in neighbouring countries. Sufficient data have been accumulated to conclude that testing commensal or peridomestic rodent sera for plague HA antibody is a necessary feature of any modem, integrated, plague control or sur- veillance programme. Over 80% of the sera dis- cussed in this report were collected, tested and analysed as a routine procedure in an actual plague control programme. Examination of rodent sera for HA antibodies furnishes the following information. (1) The method, provided it is continuously and carefully controlled, is a reliable procedure to detect active plague foci. (2) It permits thorough evaluation of enzootic and epizootic plague in commensal rodent populations. (3) It is valuable in estimating the efficacy of control measures. (4) It is useful in the elucidation and prognosis of epidemiological features peculiar to the particular geographical areas under study. (5) The rate of HA positive sera serves as an indicator of the degree of inter-host transmission of P. pestis. Plague HA antibody was readily demonstrated in the sera of small commensal rodents and other small mammals collected in foci where attempts to isolate P. pestis from human beings and the tissues and fleas of the small mammals were consistently suc- cessful. The P. pestis isolated in these plague foci had the Fraction 1 antigen. Plague-infected humans living in these foci also responded to infection by producing the same plague HA antibody during the course of the disease. The patients reported in this study denied having been vaccinated and P. pestis was isolated from every case. By contrast to the results obtained in proven plague foci, attempts to isolate P. pestis from the tissues and fleas of commensal rodents collected in former epidemic plague foci in India and Thailand were unsuccessful. Plague HA antibody was not detected in 1110 sera collected from commensal rodents in these 2 areas (1110/5514 is a 20% control sample) and plague HA antibody was not detected in the sera of the 1332 fever patients seen in Thailand. Thus, testing the sera of commensal rodents for plague HA antibody appears to be reliable for detecting rodent plague. In the evaluation of the results obtained in this programme of testing the sera of commensal rodent sera for plague HA antibody, some concern was felt that co-existing infections with another rat pathogen, P. pseudotuberculosis, might cause some non-specific positive results. P. pestis and P. pseudo- tuberculosis have common antigens (Lawton, Fukui & Surgalla, 1960). The Fraction 1 antigen used in these tests did not cross-react with P. pseudotuber- culosis type I-type V in Ouchterlony tests. About 40 % of the R. norvegicus sera collected in the pro- gramme discussed above were tested for P. pseudo- tuberculosis antibody. While antibody to P. pseudo- tuberculosis was detected, it did not interfere with an evaluation of the serological tests for plague HA antibody. Geographical and seasonal patterns of rat sera positive for plague and pseudotuberculosis antibody were quite distinct Marshall, Currie & Quy, (1968). It is also noteworthy that none of the sera collected from rodents trapped in India or Thailand and none of the sera collected from human beings in Thailand were positive in the serological tests for plague HA antibody, whereas there is strong indication that rodents in India are exposed to P. pseudotuberculosis. Sera from Thailand have not as yet been tested for P. pseudotuberculosis antibody. In addition to detecting plague foci in commensal rodent populations, chronological serum surveys provide data that are of extreme importance to a control programme. The data from the city of Saigon are illustrative. The data from Table 3 are presented graphically in the accompanying figure. This figure is a chronological illustration of the per- centages of R. norvegicus tested which were positive for plague HA antibody, and the geometric means of the titres of the sera tested. Both curves show annual fluctuations that coincide exactly with what is known about the seasonal fluctuations of the flea index of R. norvegicus in Saigon. Historically, plague epi- demics occur in the first 2 quarters of the year in Saigon and during the period represented, did in fact occur during the peaks of 1963 and 1965, but not of 1966 or 1967. Comparison of the data given in Table 2 with the accompanying figure shows that a persistent reservoir of plague is present in Saigon. An extensive flea control programme, utilizingDDT dust, was undertaken in Saigon in the latter portion of the second quarter of 1965. It can be seen in the figure however, that this measure did not control plague in the R. norvegicus population. It is now known that the major flea vector (Xenopsylla cheopis) in Saigon and Nhatrang is resistant toDDT (Cavanaugh 457 1.5 _ i _L l NOTES PREVALENCE OF HAEMAGGLUTINATING ANTIBODY TO THE FRACTION I ANTIGEN OF PASTEURELLA PESTIS IN RATTUS NORVEGICUS SERA COLLECTED IN SAIGON 1.6 15 bt _aX 0 10 OC co E 1.3 2= C 1.2 1.1 _ 2nd 3rd 1966 WHO 91808 0' 4 th lst 1962 1963 4th 1964 cm 1st 2nd 3rd 4th 1st 1965 Year and quarter o----o Percentage of positive sera - 0 Geometric mean titre 4th 1st 2 nd 1967 et al., 1968). More than 10 000 kg of DDT were expended in the areas from which the R. norvegicus sera were collected in the second quarter of 1965. The epizootic, however, in the same areas, reached maximal intensity in late 1965 and early 1966 as shown in Table 2 and in the figure. It is evident that the flea control measures with DDT were unsuccessful. Further, epizootic disease persisted in Saigon for the entire period covered by this report. Data forthcoming from serum surveys for plague HA antibody reflect transmission rates. The data presented in Table 9 show that the isolation rates for P. pestis from the tissues of small mammals in Nhatrang are 2.8 times greater than those of Saigon. Isolation rates for flea ectoparasites are much higher in Nhatrang (12.5) than in Saigon (0.9). With more infected fleas, Nhatrang should have more trans- mission of plague and this is clearly shown by the positivity rates for plague HA antibody in R. nor- vegicus sera collected in Nhatrang (34.2 %) as opposed to Saigon (5.3 %). Of further interest is the fact that annual plague epidemics occur in Nhatrang. By con- trast, Saigon has experienced only 2 epidemics during the period 1963-67. Plague control measures for both cities were similar. The populations of both cities were vac- cinated to the maximum extent possible with a living (EV) plague vaccine. Extensive flea control with DDT dust was undertaken in both cities. But, as noted above, the X. cheopis in both cities is resistant to DDT. During the period covered by this report, suitable climatic conditions for the transmission of plague by fleas, as outlined by Hirst (1953) and Pollitzer (1954) were present each year in Nhatrang. By con- trast, ideal conditions for the transmission of plague by fleas were present in only 2 of the plague seasons observed in Saigon, in the first quarter of 1963, when the first cases of plague since 1955 were recorded, and again in the first 2 quarters of 1965 when a more extensive epidemic was observed. Con- ditions of microclimate in the rat habitats of Saigon, however, were adequate to support the extensive epizootic recorded in late 1965 and early 1966, and to maintain enzootic plague during intra-epizootic periods. It would appear that the city of Saigon is afforded a certain territorial immunity by virtue of its gener- 458 SEROLOGICAL TESTING OF RODENT SERA IN A PLAGUE CONTROL PROGRAMME 459 ally unsuitable climate. The adverse climatic factors, however, are not so absolute that plague infection is eradicated from the rodent population. A dan- gerous level of plague infection persists in the rodent population. With the advent of more favourable climatic conditions a hazardous situation would again exist in Saigon. A word of caution is required. Although the test for haemagglutinating antibody to the Fraction 1 antigen of P. pestis is simple and reliable, it is man- datory that the Fraction 1 antigen be carefully pre- pared and standardized, and all reagents must be of the highest quality. Proper testing of sera requires that the pitfalls of non-specific agglutination and unsuitable sheep erythrocytes be kept in mind. Dr Bruce Hudson (personal communication) has shown that further specificity may be given to the tests by adding a solution of Fraction 1 antigen to a separate dilution series of test sera prior to adding tanned, sensitized erythrocytes. The Fraction 1 suppresses the agglutination of erythrocytes by posi- tive sera and confirms the positive reactions observed in the normal test dilutions. Unless the above-mentioned precautions are strin- gently observed, problems of non-specificity may arise. Conversely, when careful attention is given to the proper performance and control of the test for plague haemagglutinating antibodies, cross- reactions present no problem. In every instance where a programme of serological testing indicated that rodents in a particular area were hosts or reservoirs of plague infection, P. pestis was isolated from the tissues or fleas of those rodents. By con- trast, where serological testing indicated the absence of plague infection in the rodent population, P. pestis could not be isolated. In some circumstances, several years of intensive effort were required to prove that P. pestis could, in fact, be isolated from a rodent population. The only technique that has proved to be dependable is that of animal inoculation. When cultural methods were compared with animal inocula- tion, the ratio of success was 1: 10 in favour of ani- mal inoculation. Basing the Saigon survey on cul- tural techniques alone, 11 000 individual rodents would have had to be cultured to obtain confirmation of the epizootic. If surveys in remote areas are contemplated, facilities for maintaining and holding large numbers of experimental animals will be necessary if isolation is the criterion of an existing plague focus. If, how- ever, serological evidence is adequate, serum samples can be shipped to a designated laboratory for testing. To obviate reliance upon unpredictable sources of erythrocytes and Fraction 1 antigen, research on the use of formolinized erythrocutes as carriers for the Fraction 1 antigen may show that the complete anti- gen could be prepared and standardized in a central reference laboratory. Such certified antigens could then be dispensed to smaller laboratories as required. REFERENCES Baker, E. E., Sommer, H., Foster, L. E., Meyer, E. & Meyer, K. F. (1947) Proc. Soc. exp. Biol. (N.Y.), 64, 139-141 Baker, E. E., Sommer, H., Foster, L. E., Meyer, E. & Meyer, K. F. (1952) J. Immunol., 68, 131-145 Baltazard, M., Davis, D. H. S., Devignat, R., Girard, G., Gohar, M. A., Kartman, L., Meyer, K. F., Parker, M. T., Pollitzer, R., Prince, F. M., Quan, S. F. & Wagle, P. (1956) Bull. Wld Hlth Org., 14, 457-509 Cavanaugh, D. C., Dangerfield, H. G., Hunter, D. H., Joy, R. J. T., Marshall, J. D., Quy, D. V., Vivona, S. & Winter, P. E. (1968) Amer. J. publ. Hlth, 58, 742-752 Cavanaugh, D. C., Thorpe, B. D., Bushman, J. B., Nicholes, P. S. & Rust, J. H., Jr (1965) Bull. Wld Hlth Org., 32, 197-203 Chen, T. H. & Meyer, K. F. (1954) J. Immunol., 72, 282- 298 Chen, T. H. & Meyer, K. F. (1955) J. Immunol., 74, 501- 507 Chen, T. H. & Meyer, K.F. (1966) Bull. WldHlth Org., 34, 911-918 Deoras, P. J. (1960) Curr. Sci., 29, 475-476 Hirst, L. F. (1953) The conquest ofplague: A study of the evolution of epidemiology, Oxford, Clarendon Press Lawton, W. D., Fukui, G. M. & Surgalla, M. J. (1960) J. Immunol., 84, 475-479 Marshall, J. D., Jr, Currie, J. A. & Quy, D. V. (1968) Serological survey of small mammals of Vietnam for antibody against P. pestis and P. pseudotuberculosis. In: International Symposium on Pseudotuberculosis, Paris, 1967, Basel, Karger, pp. 309-318 (Symposia Series in Immunological Standardization, vol. 9) Marshall, J. D., Quy, D. V. Gibson, F. L., Dung, T. C. & Cavanaugh, D. C. (1967) Milit. med., 132, 896-903 Pollitzer, R. (1954) Plague, Geneva (World Health Organization: Monograph Series, No. 22) WHO Expert Committee on Plague (1959) Wld Hlth Org. techn. Rep. Ser., No. 165 WHO Expert Committee on Plague (1970) Wld Hlth Org. techn. Rep. Ser. (in press)