Diagnosis of human and animal brucellosis by the indirect haemagglutination test

Bull. Org. mond. Sante 1974, 51, 191-197 Bull. Wid Hith Org.J Diagnosis of human and animal brucellosis by the indirect haemagglutination test P. A. VER9ILOVA,1 M. I. eERNYgEVA,2 R. G. ASLANJAN,2 & I. N. KNJAZEVA2 This paper describes the results of many years' study of the indirect haemagglutination (IHA) test for brucellosis. It is shown that the use of sheep erythrocytes sensitized with a specific lipopolysaccharide antigen in the IHA test provides a specific method, which is more sensitive than the agglutination test, for the diagnosis of brucellosis in man and farm animals. The agglutination test (tube and plate methods) and the intradermal test are at present the main methods for the diagnosis of brucellosis in man and animals. However, since chronic and latent brucel- losis infections cannot always be diagnosed by these serologic reactions, the development of new and more sensitive methods became necessary. For the past several years, therefore, the appli- cation of the indirect haemagglutination (IHA) test to the serological diagnosis of brucellosis has been studied (1-7, 9-17; P. A. Versilova et al., unpub- lished report to FAO and WHO, 1970).G Antigens obtained by different methods were used by pre- vious workers to sensitize the red cells, e.g., bacterial extracts obtained at different temperature and pH levels, or antigens extracted from brucellas with trichloroacetic acid (Boivin antigen), or with acetic acid and heat (White's method), or with water and ether (Foster's method). The supernatant fluid of a chilled suspension of brucellas or of brucellas dis- rupted by ultrasonic treatment, and polysaccharide fractions isolated from cultures of brucellas of various species have also been used as sensitins. Although it is usual to employ fresh sheep red cells in the IHA test, some workers have also used formol- treated erythrocytes. These investigations have shown that erythrocytes could be sensitized by antigens isolated from bru- cellas by the most varied methods. It should be noted that the majority of the antigens studied were proteins and that the red cells used in the IHA test I Head, Brucellosis Laboratory, Gamaleja Institute of Epidemiology & Microbiology, Moscow, USSR. ' Scientific Officer in the Brucellosis Laboratory. a Isenko, N. I. (1964) Thesis, University of Odessa. had previously been treated with tannic acid. The preparation of red cells sensitized with protein anti- gens requires pretreatment with tannic acid and also the use of buffered solutions, which considerably complicate the carrying out of the IHA test. The development of a highly active sensitin capable of being adsorbed on the surface of untreated red cells therefore became necessary. The demonstration that the serologic specificity of brucellas was carried by surface antigens (8) led to the development in our laboratory of a method for obtaining a lipopolysaccharide which was subse- quently used as a sensitin. Twenty batches of lipo- polysaccharide isolated from the vaccine strains Brucella abortus 19 and B. abortus l9BA, and from virulent strains of B. melitensis and B. abortus were prepared, the antigens obtained from virulent cul- tures of the smooth forms of B. melitensis and B. abortus proving to be the most active and those obtained from the vaccine strains quite active in the IHA test. The presence ofcells of the rough form in the culture appreciably reduced the activity of the lipo- polysaccharide, the dose of which for sensitizing the red cells had to be increased from 0.25-0.5 mg/ml to 1-2 mg/ml. This paper presents the results of studies, carried out in cooperation with other workers in the period 1967-73, on the diagnostic value of the IHA test with lipopolysaccharide-sensitized red cells in the examination of sera from persons with chronic bru- cellosis, vaccinated persons and others examined in foci of infection, and brucellosis-infected and vac- cinated animals (cattle and reindeer). The results are also given of a comparative study of the sen- sitivity of the IHA test and the agglutination test (tube method). 3253 -191- P. A. VER§ILOVA ET AL. MATERIALS AND METHODS Sheep red cells Fresh red cells or cells preserved in Alsever's solu- tion were used in the IHA test. The preserved cells were kept in the refrigerator at 4°C for use within 2-3 weeks. Before sensitization, the cells were centri- fuged for 5 minutes at 500 g, washed 3 times with physiological saline at pH 7, and diluted in the pro- portion of 1 ml cells to 40 ml physiological saline (1: 40) to give a 2.5 % suspension. Normal rabbit serum Serum from a healthy rabbit was examined by means of the agglutination test for the absence of specific antibodies, then dispensed into ampoules, and inactivated for 30 min at 50°C. A 1: 100 dilution of this serum in physiological saline at pH 7.0-7.2 was used for diluting the test sera. Specific brucellosis serum Rabbits weighing 3-4 kg were given an intra- venous injection of virulent cultures of B. melitensis and B. abortus strains in doses of 3 x 109 organisms per ml and then exsanguinated after 9-10 days. High serum antibody titres were required for the aggluti- nation test (at least 1: 3 200 to 1: 6 400) and for the IHA test (about 1: 25 000 to 1: 50 000). The lipopolysaccharide specific antigen The method of obtaining the lipopolysaccharide has been described earlier (P. A. Versilova, unpub- lished report to WHO, 1970; M. I. Cernyseva et al., unpublished report to FAO/WHO, 1973). Although the serologic specificity of the preparation is mainly determined by its polysaccharide complex (total amount of sugars up to 75 %), the lipopolysaccharide has also been found to contain about 11 % of lipids, about 5% of protein, and a total of 0.6% of phos- phorus. No substances relating to nuclei have been found. The yield of the lipopolysaccharide prepa- ration amounts to 0.5-0.6 kg per kg of the Boivin antigen complex, or 38-67 g per kg of the initial microbial mass. Method ofpreparing the sensitized red cells To a measured volume (e.g. 10 ml) of a 2.5% sus- pension of washed erythrocytes is added an equal volume of the antigen solution containing a pre- viously determined quantity of dried lipopolysac- charide (see next paragraph for details of this). The titrated dose of dried antigen is dissolved in 1 ml of physiological saline at pH 7.0-7.2 (e.g., 0.25 mg of dried lipopolysaccharide in 1 ml of physiological saline). The red cells are sensitized in the incubator at 37°C for 40-50 min with periodic shaking, after which the mixture is centrifuged, washed twice with physiological saline, and then centrifuged again. The red cells are then resuspended in physiological saline to make up the initial volume of the 2.5% suspension. The freshly prepared sensitized red cells remain fit for use in the IHA test for a period of 3 days. Titration schedule for the lipopolysaccharide tigen A weighed portion of dried lipopolysaccharide (e.g. 1 mg) is dissolved in 1 ml of physiological saline at pH 7.0-7.2. Twofold serial dilutions are then made 2 or 3 times so that the antigen concen- tration becomes 0.5, 0.25, and 0.125 mg per ml. To 1 ml of a 2.5% suspension of the red cells is added 1 ml of the antigen solution in the test con- centrations. The red cells are then sensitized as de- scribed above and their activity checked in the IHA test with specific brucellosis serum. The working dose of the antigen is the dose that produces a titre of at least 1: 25 000 to 1: 50 000 in the IHA test. Method ofperforming the IHA test The test is carried out in wells in transparent polystyrene plates, but may also be carriod out in test-tubes. Human test sera are inactivated at 56-C and reindeer and cattle test sera at 58-59'C for 30 min. Inactivated normal rabbit serum, previously diluted 1: 100, is used for diluting the test sera. The test is carried out in at least 6 to 8 dilutions (e.g. 1: 50, 1: 100, 1: 200, 1:400, 1: 800, 1: 1 600, 1: 3 200, 1: 6400), the serum being diluted in a volume of 0.5 ml. To each dilution of serum is added 0.05 ml of sensitized red cells. The whole is then stirred and left for 2 h at room temperature. The following controls were carried out when the test was performed: test sera in a dilution of 1: 50 with unsensitized red cells; normal serum in a dilu- tion of 1: 50, 1: 100, and 1: 200, all with sensitized red cells; specific brucellosis serum in a dilution from 1: 50 to 1: 200 000 with sensitized red cells and in a dilution of 1: 50 with unsensitized red cells. The results, based on the following criteria, are read after 2 h: ± + + +, when the erythrocytes cover the whole bottom of the well in a thin, even layer and a " parasol " is formed; ++ +, when the erythrocytes cover almost the whole bottom of the well and the " parasol " has a some- what smaller diameter than in the previous case; 192 IHA TEST FOR DIAGNOSING BRUCELLOSIS 193 Table 1. Results of serologic examination of persons with chronic brucellosis Agglutination test titres I HA test titres Grus personls ngtv :5 1 :100 mean :10 maGro,es e snegative 1:50 & over titre negative 1: 50 & over titre 1 190 - 54 136 1 :140 - 46 144 1:250 2 154 154 - - - - 74 80 1 :158 3 44 - 24 20 1:100 44 - - - 4 136 136 - - - 136 - - total 524 290 78 156 180 120 224 a Group 1: agglutination and IHA tests with titres of 1: 50 and over. Group 2: agglutination test negative, IHA test with titres of 1: 50 and over. Group 3: agglutination test with titres of 1: 50 and over, IHA test negative. Group 4: agglutination test and IHA test negative. + +, when a small agglutinate is present in the very centre of the well; +, when there are a few granules of agglutinate round the erythrocyte sediment in the very centre of the well; negative reaction, when the red cells settle on the bottom of the wells in the form of a " button " or a small " ringlet " with even, sharply outlined edges. The IHA test titre is considered to be the one with the smallest quantity of serum (i.e. the highest dilution) that produces a positive reading of at least + + +. RESULTS Specificity of the IHA test The specificity of the IHA test was studied in blood sera from 244 healthy persons, 345 persons suffering from communicable diseases other than brucellosis, 1 460 healthy cattle, and 200 reindeer (7; M. I. Cernyseva et al., unpublished report to FAO and WHO, 1973).a Normal haemagglutinins were found in 10.5% of the human sera in titres ranging from 1: 10 to 1: 40, and occasionally 1: 80 and 1: 160. Normal haemagglutinins were also found in the sera from cattle mainly in titres from 1: 10 to 1: 40, and in reindeer sera (but not from all the animals) in titres from 1: 5 to 1: 40. Taking into account the presence of normal anti- bodies, we consider that human serum and cattle serum should be used in the IHA test in dilutions beginning at 1: 50, and reindeer serum in dilutions beginning at 1: 25. Whenever agglutination is ob- a U. Hajdarov (1972) Thesis, University of Tashkent; E. I. Skarsevskaja (1971) Thesis, University of Moscow. served in the control sera with unsensitized red cells, such sera should be adsorbed with normal sheep red cells, after which they should be examined in the IHA test as described. It is recommended that the IHA reaction should be considered as positive in human and cattle sera in dilutions of 1:100, and in reindeer serum in dilutions from 1: 50. Titres of 1: 50 in man and cattle and of 1: 25 in reindeer are to be regarded as doubtful. Sensitivity of the IHA test with human sera A comparative study of the sensitivity of the IHA and agglutination tests was carried out in serologic examinations of 3 519 persons. Of these, 524 were suffering from chronic brucellosis, 249 had been inoculated with live B. abortus l9BA vaccine, and 2 646 were living in various foci of brucellosis infection. The results of the tests on sera from persons with chronic brucellosis (Table 1) show that a concord- ance of positive and doubtful results in both tests was observed in 190 cases (36.2%), and a concord- ance of negative results in 136 cases (26.0%). Of 154 patients with a negative result in the aggluti- nation test, 80 (52%) were found to have haemag- glutinins in diagnostic titres. It was only in 20 cases that the agglutination test produced positive results when the IHA test was negative. Thus, of 524 persons with chronic brucellosis, 156 (29.8%) had antibodies in diagnostic titres dis- covered by the agglutination test, and 224 (42.7%) by the IHA test. In some patients the IHA titre reached 1: 12 800, whereas in the agglutination test P. A. VER§ILOVA ET AL. antibodies were found in titres of 1: 100 to 1: 200, and only on very rare occasions in titres of 1: 400 and 1: 800. Consequently, in patients with chronic brucellosis, haemagglutinins were detected 1.4 times as often as agglutinins and the haemagglutinin titres were 1.5 times higher. The sensitivity of the IHA test was studied in 249 persons at various intervals after inoculation with live B. abortus 19BA vaccine. The results (Table 2) show that, in the first 3 months after injecting the vaccine, haemagglutinins were detected 2-3 times as often as agglutinins. After 6-12 months, however, the intensity of both reactions tended to diminish. These results indicate that the IHA test is highly sensitive in detecting antibodies in inocu- lated persons when immunity is developing. Table 2. Results of serologic examination of persons inoculated with live B. abortus 1 9BA vaccine Agglutination test IHA test Period since vaccination no. positive/ mean no. positive/ mean no. examined titre no. examined titre 2 weeks 13/48 (27.0) a 1: 85 26/39 (66.6) a 1: 97 1 month 13/45 (28.8) 1: 80 32/34 (94.1) 1:116 3 months 12/39 (30.8) 1:133 23/39 (59.0) 1:106 6 months 15/87 (17.3) 1: 57 7/87 (8.0) 1: 55 12 months 3/30 (10.0) 1: 50 2/30 (6.6) 1: 50 a Figures in parentheses are percentages. The IHA test was also used to study the immuno- logical status of the population in 3 zones with differing epizootic situations. A total of 2 646 per- sons in groups that were occupationally exposed and unexposed to brucellosis were examined. The first zone was characterized by the fact that there was active brucellosis infection among collec- tively-owned cattle. No brucellosis was recorded among individually-owned cattle. The second zone was characterized by the fact that brucellosis infection had been widespread among sheep and cattle in the past. During the period of this study, no acute epizootics were recorded. The third zone was characterized by active infec- tion among sheep and cattle, some abortions being recorded. In addition, brucellosis was widespread in this zone among individually-owned cattle. The results of serologic examinations of the popu- lation in these different foci of brucellosis infection (Table 3) show that in all 3 zones the proportion of reactors was greater with the IHA test than with the agglutination test. Thus, in 3 different foci in the third zone, haemagglutinins were present in 39.6%, 22.2%, and 12% of the population and agglutinins in 21.7 %, 11.1 %, and 4.8 %, respectively. In addition, the data indicate a connexion between the proportion of serologically positive reactors among human subjects and the intensity of brucel- losis infection among the various species of animal. Thus, the percentage of positive reactors was con- siderably higher in an active focus of infection among sheep and goats in zone 3 (39.6% in the IHA test) than on farms where the infection was recorded Table 3. Results of serologic examination of the population in various foci of brucellosis infection a No. of Agglutination test IHA test Zone Type of focus persons 1 .100 1 .100 examined negative 1 : 50 & over negative 1 : 50 & over first cattle (active foci) 806 790 (98.0) 10 (1.2) 6 (0.8) 776 (96.3) 17 (2.1) 13 (1.6) second cattle, sheep, and goats 1 291 b 1 276 (98.9) 5 (0.4) 10 (0.7) 975 (95.4) 35 (3.4) 12 (1.2) (cleared foci) third sheep and goats (active foci) 106 73 (68.9) 10 (9.4) 23 (21.7) 43 (40.5) 21 (19.9) 42 (39.6) cattle (active foci) 72 54 (75.0) 10 (13.9) 8 (11.1) 41 (56.9) 15 (20.9) 16 (22.2) cattle (foci of chronic brucellosis) 371 332 (89.5) 21 (5.7) 18 (4.8) 278 (74.9) 50 (13.1) 43 (12.0) a Figures in parentheses are percentages. b 1 022 persons were examined by means of the IHA test. 194 IHA TEST FOR DIAGNOSING BRUCELLOSIS 195 Table 4. Results of examining the sera of cattle No. of Agglutination test Complement fixation IHA test Type of focus animals test examined % positive mean titre % positive mean titre % positive mean titre active infection 140 40.0 1: 300 61.4 1:143 90.7 1: 960 chronic infection 279 13.3 1: 260 43.0 1: 17 55.9 1:430 Table 5. Results of examining the sera of vaccinated calves Brucellosis-free farm Brucellosis-infected farm Time since agglutination complement IHA test agglutination complement IHA test vacci- test fixation test test fixation test nation (months) % mean % mean % mean % mean % mean % mean positive titre positive titre positive titre positive titre positive titre positive titre 3 12.6 1:120 13.2 1:12 18.4 1:190 25.2 1:130 29.3 1:10 39.0 1:200 6 1.0 1:100 1.0 1:10 3.7 1:130 2.0 1:100 2.0 1:20 11.8 1:130 9 4.2 1 :100 1.0 1 :10 negative not tested not tested not tested 12 negative 0.5 1 :10 negative 0.7 1 :100 0.7 1 :100 6.3 1 :110 only among cattle or had not been recorded at all in the last few years (zones 1 and 2: IHA test reactors, 1.6% and 1.2%, respectively). Thus, for establishing the immunological status of the population in various foci of infection the IHA test was shown to be more sensitive than the agglutination test. Sensitivity of the IHA test in animals The sensitivity of the IHA test in cattle, in com- parison with those of the agglutination and comple- ment fixation tests was studied by Skarsevskaja.a The animals examined were in herds with acute or chronic brucellosis, for both of which the IHA test proved to be the most sensitive (Table 4). The next most sensitive was the complement fixation test, although the IHA test detected about 30% more positive reactors from among the cattle with acute brucellosis. The sensitivity of the IHA test was also studied by examining the sera of calves vaccinated at the age of 6-8 months with live B. abortus 19 strain vaccine.a A total of 190 calves were inoculated on a E. I. Skarsevskaja (1971) Thesis, University of Moscow. a farm where brucellosis was absent and 246 on a farm where it was present. The sera from all these calves were examined 3, 6, 9, and 12 months after vaccination and the results of these investigations are given in Table 5. In the vaccinated animals from the brucellosis-free farm, the immunological reac- tions diminished rapidly so that after 9-12 months the IHA test was negative and antibodies to the agglutination and complement fixation tests were discovered only in occasional animals. The calves in the farm where brucellosis was present had post- vaccination reactions that persisted for a longer period. Both the number of reactors and the titres were greater in every case in the IHA test than in the complement fixation and agglutination tests; this shows that the IHA test is more sensitive. The long persistence of haemagglutinins in the blood of vac- cinated calves in the farm where brucellosis was present suggests that the calves might have become infected as a result of contact with sick animals. These results enable us to recommend the IHA test for examining animals even at long intervals after vaccination. The sensitivity of the IHA test was also studied by examining the sera of 407 reindeer from brucellosis- infected farms and of271 reindeer inoculated with live P. A. VER§ILOVA ET AL. Table 6. Results of examining the sera from reindeer on brucellosis-infected farms Agglutination test b IHA test b Group no.of mean meananimals negative 1: 25 1:50 & over titre negative 1: 25 1:50 & over me 1 237 - 19 (8.0) 218 (92.0) 1:125 - - 237 (100.0) 1:400 2 141 141 (100.0) - - - - - 141 (100.0) 1:200 3 6 - - 6 (100.0) 1:40 6 (100.0) - _ - 4 23 23 (100.0) - - - 23 (100.0) - - - total 407 164 (40.3) 19 (4.7) 224 (55.0) 29 (7.1) - 378 (92.9) * Group 1: agglutination and IHA tests positive. Group 2: agglutination test negative, IHA test positive. Group 3: agglutination test positive, IHA test negative. Group 4: agglutination test and IHA test negative. b Figures in parentheses are percentages. B. abortus 19 strain vaccine. A comparison of the sen- sitivity of the agglutination and IHA tests for examin- ing sera from reindeer showed that the two tests were in agreement in 237 cases (58.2%) of positive results and in 23 cases (5.6%) of negative results (Table 6). Haemagglutinins were found in 141 animals (34.6%) when the agglutination test was negative, and in only 6 cases (1.4%) was the IHA test negative when the agglutination test was positive. Thus, antibodies were found in 92.9% of sera by the IHA test as against 55.0% of sera by the agglutination test, i.e., in 37.9 Y. more sera. The mean antibody titre in the IHA test was 6 times higher than in the agglutination test. Inoculated reindeer maintained in brucellosis- infected herds were examined 1-3 years after vac- cination. Antibodies were found in the sera of these animals by both the agglutination and the IHA tests in 10.3% and 44.5% of cases, respectively, one year after vaccination (Table 7). Thus, in reindeer, as in cattle, the IHA test proved to be more sensitive than the agglutination test. It was also established that while, one year after vaccination, antibodies were discovered by the IHA test in 44.5% of cases in a titre of 1 : 100, after 3 years (provided that the inoculated animals had been kept with brucellosis- infected herds) the number of reactors in this test had increased by 19% and the titre by twofold. Consequently, with the IHA test it was also possible to identify those inoculated animals that had sub- sequently become infected. CONCLUSIONS The IHA test examination of 2249 sera from healthy persons and animals indicated that the diag- nostic titre in human subjects and cattle was a serum dilution of 1: 100 and in reindeer one of 1: 50. The examination of 3 519 sera from persons with chronic brucellosis, persons inoculated with live vaccine, and persons living in areas where there was brucellosis infection among animals, showed that Table 7. Results of examining the sera of vaccinated reindeer a Time Agglutination test IHA test since No. of (years) negative 1: 25 1: 50 & over mean negative 1: 25 1 :50 & over mttren 1 164 131 (80.0) 16 (9.7) 17 (10.3) 1 :100 91 (55.5) - 73 (44.5) 1: 100 3 107 61 (57.0) - 46 (43.0) 1 :100 39 (36.5) - 68 (63.5) 1:200 a Figures in parentheses are percentages. 196 IHA TEST FOR DIAGNOSING BRUCELLOSIS 197 antibodies were detected in a greater number of cases (in 13-18% more cases) by the IHA test than by the agglutination test. Examination of 1 533 ani- mals (cattle and reindeer) in brucellosis-infected herds showed that a greater number of infected animals (20-30% more) were detected with the IHA test than with the agglutination test. The examination of animals inoculated with live vaccine showed that the haemagglutinins disappeared earlier than the agglutinins and complement-fixing antibodies. However, in vaccinated animals in bru- cellosis-infected herds, antibodies were detected over a longer period by the IHA test. These data suggest that positive results in the IHA test at long intervals after vaccination might indicate infection of the inoculated animals. Thus, the data obtained show that the IHA test is more specific and sensitive than the agglutination test, and justify its use in the diagnosis of human brucellosis, the study of the immunological status of the population, and the examination of animals for brucellosis. RtSUMt DIAGNOSTIC DE LA BRUCELLOSE HUMAINE ET ANIMALE PAR L'EPREUVE D'HEMAGGLUTINATION INDIRECTE Les auteurs exposent les resultats obtenus dans le diag- nostic de la brucellose par une epreuve d'hemagglutina- tion indirecte (IHA) utilisant des 6rythrocytes de mouton sensibilises avec un antigene lipopolysaccharidique spe- cifique. L'examen de 2249 serums de sujets sains et d'animaux a conduit a considerer comme titre diagnostique une dilution de 1: 100 chez l'homme et les bovins et de 1:50 chez les rennes. L'6tude de 3519 serums preleves chez des sujets atteints de brucellose chronique, ou vac- cines par un vaccin vivant, ou habitant dans des regions ofi l'affection est enzootique, a montre que l'epreuve IHA decelait les anticorps dans un plus grand nombre de cas (13-18% de plus) que l'epreuve d'agglutination. Chez 1533 animaux (bovins et rennes) infectes, 1'effica- cite diagnostique de l'epreuve IHA a e de 20 i 30% sup6rieure a celle de l'epreuve d'agglutination. Chez les animaux vaccines a l'aide d'un vaccin vivant, les hemagglutinines disparaissent plus precocement que les agglutinines et les anticorps fixant le complement. Cependant, lorsque les animaux vaccines sont maintenus dans des troupeaux infectes, 1'epreuve IHA d6cle les anticorps pendant une periode plus longue. 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