THE INFLUENCE OF COXIELLA BURNETII PHASE I AND PHASE II ANTIGENS ON THE SEROLOGICAL DIAGNOSIS OF Q FEVER IN CATTLE

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1 Slov Vet Res 2003; 40 (3/4): UDC The influence of coxiella burnetii phase I and phase II antigens... THE INFLUENCE OF COXIELLA BURNETII PHASE I AND PHASE II ANTIGENS ON THE SEROLOGICAL DIAGNOSIS OF Q FEVER IN CATTLE B. KRT Key words: Q fever; serological diagnosis; cattle The aim of our work was to study the influence of C. burnetii phase I and phase II antigens when used in the serological diagnosis of Q fever in cattle. Samples were obtained from a cattle herd with an outbreak of Q fever and examined, using the phase I and phase II antigens (together and separately), in ELISA and complement fixation tests (CFT). The CFT indicated that 11 sera (39 %) were positive when both antigens were used, 3 (11 %) were positive using only the phase I antigen and 2 sera (7 %) were positive to phase II. The ELISA examinations revealed that 16 sera (57 %) were positive when both antigens were used and 3 (11 %) were positive using only the phase I antigen. No positive sera were found using the phase II antigen only. In milk testing there were 9 (32 %) ELISA-positive samples using both phases and 5 (18 %) were positive when the phase I antigen was used separately. The phase II antigen only demonstrated one positive milk sample (4 %). It was concluded that both antigens should be used simultaneously in CFT, whereas the phase I antigen could be sufficient for herd-level ELISA tests. VPLIV UPORABE ANTIGENOV FAZE I IN FAZE II COXIELLA BURNETII NA SEROLO[KO DIAGNOSTIKO MRZLICE Q PRI GOVEDU Klju~ne besede: mrzlica Q; serolo{ka diagnostika; govedo Namen na{ega dela je bil ugotoviti vpliv uporabe antigenov faze I in faze II C. burnetii na serolo{ko diagnostiko mrzlice Q pri govedu. V raziskavi smo uporabili vzorce iz ~rede, kjer je bila ugotovljena mrzlica Q. Preiskovali smo jih z metodama reakcije vezanja komplementa (RVK) in ELISA, v katerih smo uporabili antigene faze I in faze II (skupaj in lo~eno). V RVK je pri uporabi antigenov obeh faz reagiralo pozitivno 11 (39 %) vzorcev, 3 (11 %) vzorci so bili pozitivni samo na fazo I, 2 (7 %) pa le na fazo II. V testu ELISA je bilo pri uporabi antigenov obeh faz pozitivnih 16 (57 %) in pri uporabi faze I 3 (11 %) serumi, medtem ko nobeden ni bil Received: June 30, Address of author: Dr. Brane Krt, Assist. Prof., Institute of Microbiology and Parasitology, Veterinary Faculty, Gerbi~eva 60, 1000 Ljubljana, Slovenia. B. Krt

2 204 B. Krt pozitiven samo na fazo II. Pri mle~nih vzorcih smo v testu ELISA pri uporabi antigenov obeh faz ugotovili 9 (32 %) pozitivnih vzorcev, pri uporabi antigenov faze I 5 (18 %) in pri fazi II 1 (4 %). Ugotovili smo, da je potrebno pri RVK uporabljati antigene obeh faz hkrati, medtem ko bi za testiranje ~rede v testu ELISA lahko zado{~ala uporaba antigenov faze I. Introduction Q fever is a zoonosis of worldwide distribution. Man usually becomes infected following the inhalation of rickettsia Coxiella burnetii. The desiccation-resistant microorganism is shed from infected animals, usually cattle, sheep, goats or cats, especially at the time of parturition or abortion. Cows shed C. burnetii in milk for up to 32 months, while sheep shed the organism in faeces for 11 to 18 days postpartum (1). In some regions cows excreting the organism are of major importance in the epidemiology of Q fever (2). Coxiella burnetii occurs in two forms: the pathogenic phase I, isolated from animals and humans, and the less pathogenic phase II, obtained by repeated passages in embryonated eggs or tissue cultures. Antibodies against C. burnetii are highly specific and cross-reactions with other microorganisms are unknown (3). Two distinct types of antibodies are produced. Phase II antibodies appear early in the infection, while phase I antibodies arise later (4). The agent can be visualised by microscopy of Stamp-stained smears, which are usually prepared from an aborted foetus, placenta or vaginal discharges soon after an abortion. This method is not specific because C. burnetii can be confused with other abortion-causing bacteria. The isolation of C. burnetii is possible using cell cultures or embryonated chicken-yolk sacs or laboratory animals (guinea pig, mouse, hamster). Immunological and molecular methods (PCR) can also demonstrate the agent in samples. Since the isolation and identification of C. burnetii is difficult and laboratory infections of personnel are frequent, diagnosis of Q fever is usually based on the results of serological tests. Indirect fluorescent antibody tests, complement fixation tests (CFT) and enzyme-linked immunosorbent assays (ELISA) are the most often used serological methods in the diagnosis of Q fever. The influence of the phase I and phase II antigens on the results of CFT and ELISA examinations were evaluated in the present study. Material and methods Samples: Twenty-eight blood samples were taken from a herd of cattle with an outbreak of Q fever. Forty days later a second sampling was carried out where milk samples were also taken. Eight cows in the herd had aborted between 6 and 180 days before the samples were taken. The CFT was only used to test sera. Commercial antigens (phase I and phase II), complement and an amboceptor (Virion-Serion, Switzerland-Germany) were used. The tests were performed in accordance with the manufacturer's instructions with some modifications. Briefly, all dilutions were made in saline containing 0.1 g/litre MgSO 4 x7h 2 O. The sera were diluted 1:10 and inactivated for 30 minutes at 58 C. For the test twofold dilutions from 1:10 to 1:640 were prepared. After adding the

3 The influence of coxiella burnetii phase I and phase II antigens complement and antigen (phase I or phase II) the micro-plates were incubated at 5 C overnight. The next day a mixture of amboceptor and 1 % sheep erythrocyte was added. The results were interpreted in accordance with the OIEManual (5). ELISA kits were used to test both sera and milk samples. Three different tests with respect to the antigens were carried out. Micro-plates sensitised with either the phase I or phase II antigen (Virion-Serion) were used in two of them. The third test was performed using ELISA kits (Dr. Bommeli AG, Switzerland) where the micro-plates were coated with both antigens. The kits, with the exception of the coated micro-plates, the test procedures and the interpretation of the results, which was done in accordance with the kit manufacturer's instructions, were the same in all three tests. Samples found to be ambiguous in ELISA were considered to be positive because of their origin (infected herd). Results As the results of the first and second samplings were similar only the results of the second sampling (when the milk was also examined) are shown below. Table 1: Results of the CFT and ELISA evaluations of the different antigens No. of CFT (titre 1:) ELISA (optical density)* sample Serum Serum Milk (*=abortion) Ph I Ph II Ph I Ph II kit Ph I Ph II kit neg. 1 neg neg neg neg neg. 3 neg neg. neg. neg. neg. neg. neg. neg neg. neg. neg. 13* neg neg. neg. neg. 14* 20 neg. 3 neg. 2 neg. neg. neg. 15* neg. neg. 3 neg. 2 neg. neg. neg. 16* neg. neg neg. 1 17* neg neg. 1 neg. neg. 18* * neg. neg * neg. 2 Legend: Ph I = phase I antigen ELISA *(optical density with respect Ph II = phase II antigen to the positive control serum) kit = ELISA kit (phase I and phase II antigens) 1 = % (ambiguous) CFT 1:10, 1:20, 1:40 = latent infection 2 = % (positive) 1:80, 1:160, 1:320 = active infection 3 = % (positive) 4 = >200 % (positive)

4 206 B. Krt Twenty out of the 28 animals tested were positive in one or more of the tests, the results of which are shown in Table 1. From the 28 animals tested, 8 were negative (29 %) and 7 were positive (25 %) in all tests (serum and milk). The CFT indicated that 11 sera (39 %) were positive when both antigens were used, 3 sera (11 %) were positive using only the phase I antigen and 2 sera (7 %) were positive to phase II. The ELISA demonstrated that 16 sera (57 %) were positive when both antigens were used and 3 (11 %) were positive using only the phase I antigen. No positive sera were revealed using only the phase II antigen. In the milk testing there were 9 (32 %) ELISA-positive samples using both phases and 5 (18 %) were positive when phase I antigen was used separately. The phase II antigen only demonstrated one positive milk sample (4 %). There were big differences in the percentages of positive sera in CFT and ELISA when the phase I and phase II antigens were used separately (Fig. 1). Figure 1: Percentage of positive samples in CFT and ELISA Discussion The CFT and ELISA are widely used in the diagnosis of Q fever in animals. The CFT has been used in the serodiagnosis of Q fever for a long time, whereas ELISA is becoming more important in the last time. It is a more rapid and sensitive method than the CFT and should be considered as its replacement (3). The CFT usually only employs the phase II antigen, whereas the micro-plates in the ELISA kits (e.g. Dr. Bommeli AG, Switzerland) are coated with an antigen of both phases. The detection of antibodies against the phase I or phase II antigens can be useful in distinguishing acute and chronic cases. In humans, the anti-phase I IgG levels appear to be the most important parameter for the diagnosis of chronic Q fever (6). The results of the first and second samplings were so similar that only the results of the second sampling (when the milk was also tested) are shown. Actually, there

5 The influence of coxiella burnetii phase I and phase II antigens were no significant differences, as might be expected, between the results with respect to the antigen used. Samples found to be ambiguous in ELISA (after repeated testing) were considered positive, as their epizootiological background was already known (infected herd). The period between abortions and sampling was rather long and therefore it was expected that there would be a higher number of phase I positive samples. Actually, in practice this is often the case as samples usually only come to the laboratory after a series of abortions. In the ELISA only antibodies against phase I were found, except in one milk sample (where only antibodies against phase II were found), especially in cows that had had abortions. In these animals the CFT, using the phase I as well as the phase II antigens, were mostly negative. This could be due to the lower sensitivity of this method. In our study a very small number of sera were positive when the phase II antigen was used separately. This might be due to the class of antibodies. The CFT could detect IgM, whereas in the ELISA we only used anti-igg conjugate. On the other hand, Nielsen and Duncan (7) found that bovine IgM does not fix guinea pig complement. The higher sensitivity of ELISA in comparison with CFT could be due to the detection of the IgG 2 subclass of antibodies, which are not able to bind the guinea pig complement. The results of the milk ELISA were often different to those of the serum ELISA. It is known that many factors influence the results of milk testing and can lead to both false positive and false negative results. However, there were also differences between the phase I and phase II ELISA milk-tests. It was concluded from our study that if the CFT is used for diagnosing Q fever in cattle then the phase I and phase II antigens should be used simultaneously. When an ELISA is used then the phase I antigen could be sufficient for testing at the herd level. References 1. Marrie TJ. Q fever a review. Can Vet J 1990; 31: Kramer M. Zum Vorkommen, zur Verbreitung und zur Epidemiologie des Q-Fiebers in den neuen Bundeslaendern der Bundesrepublik Deutschland. Tierarztl Umschau 1991; 46: Cowley R, Fernandez F, Freemantle W, Rutter D. Enzyme immunoassay for Q fever: comparison with complement fixation and immunofluorescence tests and dot immunoblotting. J Clin Microbiol 1992; 30: Peter O, Dupuis G, Burgdorfer W, Peacock M. Evaluation of the complement fixation and indirect immunofluorescence tests in the early diagnosis of primary Q fever. Eur J Clin Microbiol 1985; 4: Q fever. In: OIEmanual of standards for diagnostic tests and vaccines. 4th ed. Paris: Office international des epizooties, 2000: Peter O, Dupuis G, Bee D, Luthy R, Nicolet J, Burgdorfer W. Enzyme-linked immunosorbent assay for diagnosis of chronic Q fever. J Clin Microbiol 1988; 26: Nielsen K, Duncan JR. Further evidence that bovine IgM does not fix guinea pig complement. Vet Immunol Immunopathol 1988; 19: