Received in 9/10/2017 Accepted in 13/11/2017

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1 Evaluation of some serological tests for diagnosis of brucellosis in imported camel Mahmoud, H. Abdel-Halim and Rania, I. Ismail Brucellosis Department Animal Health Research Institute Dokki, Giza ISSN: Received in 9/10/2017 Accepted in 13/11/2017 Abstract Brucellosis remains a major worldwide zoonosis. Some countries have been successful in the eradication of the disease, while, others especially in the developing world, still live with a huge disease burden in the form of severe health risk to the consumer of dairy products. Little information is known especially on camel brucellosis and its impact on human health. For surveillance and control of the disease, sensitive and reliable detection methods are needed. Although serological tests are the mainstay of diagnosis in camel brucellosis, these tests have been directly transposed from cattle without adequate validation. Therefore, this study was performed to evaluate the florescence polarization assay (FPA) in camel in comparison with conventional serological tests taking complement fixation test (CFT) as a gold standard test. A total of 118 serum samples collected from apparently healthy imported Sudanese camels were examined by different serological tests namely; the buffered acidified plate antigen test (BAPA),s erum Agglutination Test (SAT), Rivanol test (Riv.T) and complement fixation test (CFT). Findings: Our findings revealed that that the percent positivity between different serological tests was 39.83%,32.2%,29.66%,35.59%and 35.59% in following tests BAPA, msat, Riv.T, FPA and CFT respectively. We concluded that using of BAPA as a presumptive test to exclude negative cases and FPA for confirmation of positive samples as it is highly sensitive and highly specific test and verification of these results by CFT for evaluation of FPA results to estimate its efficiency to be recommended for diagnosis of brucellosis in camels in the field. Key words: Brucella Camel- FPA- CFT- BAPA. Introduction Brucellosis is one of the most important zoonosis worldwide affecting livestock and humans OIE (2016). Brucellae are facultative intracellular, Gram-negative coccobacilli that lack capsules, flagellae, and endospores. The genus Brucella is composed of nine recognized species, six of which are the "classical" members (B. abortus, B. suis, B. melitensis, B. canis, B. ovis, and B. neotomae) (Cutler et al., 2005). Recently, the species B. ceti. pinnipedialis, and B. microti have been described Foster et al., (2007) and the species B. inopinata has been proposed (Scholz et al., 2009) and Whatmore et al., (2014). B. melitensis, B. suis and B. abortus are of major impact by causing significant economic losses to animal owners and by provoking severe human disease. Brucella spp. are also a focus of interest as they are categorized as biological agents due to their high contagiousness and their impact on human and animal health. In countries of the Near East region, brucellosis was reported in almost all domestic animals, particularly cattle, camels, sheep, and goats. In Egypt, brucellosis has been reported 55

2 First International Conference of Animal Health Research Institute Mahmoud and Rania also in buffaloes, equines, and swine. Brucellosis has been recognized during the last 20 years to be a serious zoonotic disease in most countries of the region. The disease has been reported in animals in most of the countries in the region, which depend on import ation of animals, particularly from outside the region for both slaughter and breeding. With the intensification of animals by importation and the establishment of big farms in the last decades, the incidence of brucellosis rose sharply in many countries, both in man and animals. Camel is an even-toed ungulate within the genus Camelus, bearing distinctive fatty deposits known as humps on its back. There are two recognized species of camels (Dromedarius and Bactrianus). The dromedary or Arabian camel has a single hump, and the Bactrian camel has two humps. They are native to the dry desert areas of West Asia, Central, and East Asia, respectively. Both species are domesticated to provide milk and meat (Wilson, 1984). The world population of camels is about 20 millions mainly in arid zones. Of which, 15 millions camels live in Africa and 5 million in Asia (GLIPHA, 2006). In 2001, the total camel population was 19 million. Of which, 17 million were dromedaries and 2 million were Bactrian (Farah and Fischer 2004). In most countries, the camel population increased after a period of decreasing number due to the introduction of modern transport facilities. Camels are not known to be primary hosts of Brucella, but they are susceptible to both B. abortus and B. melitensis (Cooper, 1991). Consequently, the prevalence depends upon the infection rate in primary hosts being in contact with them. Brucellosis may spread from camels to humans, especially via milk. Therefore, the zoonotic risks from camel milk must be considered in view of the traditional African and Arabian preference for raw milk consumption. Accurate diagnosis is the key to prevent the spread of infection and to control brucellosis. However, diagnosis of brucellosis is frequently difficult to establish. This is not only because the disease can mimic many infectious and non -infectious diseases, but also because the established diagnostic methods are not always sensitive enough. Although serological tests have been used as diagnostic tool for screening of camel brucellosis, they are neither adequately sensitive nor specific due to an insufficient immune status of the host or serological crossreactivity (Morgan and Mackinnon, 1979 and Farina, 1985), as well as most tests have been directly transposed from cattle, without validation for camels. The present study was aimed to evaluate various serological tests as a diagnostic tool for camel brucellosis at the same time the usage of florescence polarization assay as a recent technique for diagnosis of camel brucellosis. Materials and Methods Samples. A total of 118 target samples out of them; selected 48 positive samples and 70 random samples were collected from apparently healthy camels which were imported from Sudan at the end of Antigens. Antigens used for BAPA test was supplied from APHIS (USDA). Serum Agglutination Test and Complement Fixation Test antigen were provided by Institute Pourquier, France. In addition Positive and negative control sera were national reference sera standardized according to OIE (2009). Control positive sera contain 421 I.U/ml for SAT and 595 ICFTU/ml for CFT. Reagents for CFT Complement, haemolysin or amboceptor's and were supplied by Virion /Serion GmbH, Germany. Veronal buffer tablets were purchased from Oxoid Company. Sheep RBCs were collected from adult male sheep in our institute on sterilized glass beads under an aseptic condition and was kept in re- 56

3 frigerator till usage. Methods Buffered Acidified Plate Agglutination Test (BAPA): It is a qualitative plate agglutination test that uses a coloured acidified antigen (ph 3.8 ± 0.05 cell volume 11%) to inhibit non-specific reactions due to IgM and enhance the agglutination ability of specific IgG 1. This test is done and interpreted according to Angus and Burton (1984) The standard microplate agglutination test (msat) It is a quantitative plate agglutination test that uses white unstained antigen. Its advantage is that it measures the amount of antibody as international units per milliliter of sample. It is aslow quantitative test.this test was done according to OIE (2016) The Rivanol test (Riv.T): This test was applied according to Alton et al.,(1988) It is a supplementary serological test that uses rivanol solution (2 ethoxy-6-9 diamino acridine lactate) that precipitate IgM immunoglobulin which then removed by centrifugation, and the s upernatant is used in the analysis. It is a rapid, specific semi-quantitative confirmatory test. This could be useful especially when facilities for performing CFT are not available. The Complement fixation test (CFT): Titration of reagents and performing of the test was done according to Alton et al., (1988). It is an accurate quantitative plate (micro-titer) or tube test that uses white antigen of SAT or a soluble antigen to fix Guinea pig complement. The reaction is made visible by an indicator System. Fluorescent polarization assay (FPA). The test was applied according to OIE, (2016) The assay was done, and results were interpreted according to the instructions of the manufacture (Diachemix LLC, 223 North Water Street, suit 500 Mil Wankee, WI LCC, November 2015 product code B 1002 Kit size 250 tests, USA). FPA was conducted in 96well flat-bottom black polystyrene microtiter plate. Initially, 180 μl of dilution buffer was added to all wells. The dilution buffer was provided by the manufacturer in 25 concentrated form, and the working dilution was prepared using ultra clean, sterile water. In each microplate, positive and negative control sera of bovine origin, provided by the manufacturer. 20 μl of three negative controls were pipetted in the first three wells. 20 μl of one positive control was dispensed in the fourth well. 20 μl of test sera were dispensed in duplicate to the other next wells. Serum and buffer samples were mixed by setting for 2 min at room temperature in microplate on a rotating microplate shaker. After an initial mixing, the background reading was taken in a fluorescence polarization mode by a multi-mode microplate reader (Tecan Genios Pro, Switzerland) connected to a computer. Subsequently, 10 μl of conjugated antigen with Fluoresce in isothiocyanate (FITC) added to every well. After through mixing for 3 min. at room temperature, a second reading was made. The reader automatically subtracted the background reading and calculated the value for every sample in millipolarization units (mp). The results of each microplate measurement were interpreted as follow: < 10 mp considered as negative, = considered as suspected, > 20 considered as positive. Statistical analysis and Relative sensitivity and Specificity were done according to Altman and Bland (1994) Relative sensitivity = True positive / (True positive + False negative) X 100 Relative specificity = True negative / (True negative + False Positive) X

4 First International Conference of Animal Health Research Institute Mahmoud and Rania Results Table (1). Results of various serological tests used camel sera. Sample n =118 BAPA msat Riv.T CFT FPA Total n = Percent % BAPA: Buffered acidified plate antigen test msat: Serum Agglutination Test Riv.T: Rivanol test CFT: complement fixation test Table (2). Correlation between different serological tests Taking CFT AS gold standard Test Sample BAPA Riv.T SAT FPA Pos. Neg. Pos. Neg. Pos. Neg. Pos. Neg. CFT BAPA RIV msat Pos. (n = 42) Neg. (n = 76) Pos. (n = 47) Neg. (n = 71) Pos. (n = 35) Neg. (n = 83) Pos. (n = 38) 34 4 Neg. (n = 80) 8 72 Pos.: Positive. Neg.: Negative 58

5 Table (3). Relative sensitivity and Relative specificity of different serological tests taking CFT as a gold standard. BAPA SAT Riv.T FPA Relative Sensitivity Relative Specificity 41/42 X 100 = 97.62% 28/42 X 100 = 66.67% 30/42 X 100 = 71.43% 41/42 X 100 = 97.62% 70/76 X 100 = 92.11% 66/76 X 100 = 86.84% 71/76 X 100 = 93.42% 75/76 X 100 = 98.68% Relative sensitivity = True positive / (True positive + False negative) X 100 Relative specificity = True negative / (True negative + False Positive) X 100 Discussion Although Brucella isolation is the golden mark for diagnosis, it is of low sensitivity Alton et al. (1988). Its application is impractically in this study as the samples collected are only blood for serological tests. So CFT is chosen as a gold standard due to its high specificity and sensitivity Kabat and Mayer (1961), Alton et al., (1988) and OIE (2016). Also, application of complement fixation test (CFT) offered an equilibrium between sensitivity and specificity of 97.62% and 98.68% respectively. While, the other serological tests recorded lower sensitivities and specificities in comparison to CFT. And this may be attributed to the detection of a little quantity of IgG1 specific of infection which is the ideal test has been used to confirm results (Sayour et al., 2015) Most of the previous studies on camel brucellosis depend mainly on the application of serological tests samples collected mainly from the blood. In the present work, the results displayed in Table 1 revealed that the percent positivity between different serological tests was 39.83%, 32.2%, 29.66%, 35.59% and 35.59% in BAPA, msat, Riv.T, FPA and CFT respectively. Serum agglutination test (SAT neutral ph) which is 7. It is a truly quantitative test detects mainly IgM specific of early infection due to Brucella and other gram negative microorganisms. Resulted in poor relative specificity (86.84%) comparing to other serological tests when used as serotest in camel. While not recognized as a prescribed or alternative test, the SAT has been previously used with success for many years in surveillance and control programmes for bovine brucellosis. Its specificity is significantly improved with the addition of EDTA to the antigen, (OIE, 2009). Meanwhile applying buffered acidified Plate antigen test (BAPA acidic ph) It is a quick, easy presumptive test to start with to exclude negative samples from further serological testing which has high relative sensitivity and specificity 97.62% and 92.11% respectively due to detection of IgG1 mainly for excluding negative cases for further testing. Angus and Barton (1984). The application of Rivanol test on serum of studied camels showed high relative specificity (93.42%) of the Rivanol test. This may be ascribed to complete removal of IgM using rivanol solution (2 ethoxy-6-9 diamino acridine lactate) for promotion of IgG2 reactivity resulting in improving its diagnostic relative specificity 93.42% which was higher than that obtained by Sayuor et al, (2015) which was 88.9%while their sensitivity was 87.4% that was higher compared to our finding % to confirm positive samples of both SAT and BAPA (Alton et al., 1988). Table 2 revealed that correlation between dif- 59

6 First International Conference of Animal Health Research Institute Mahmoud and Rania ferent serological tests in comparison with CFT, six samples were positive to BAPA and negative to CFT this may be attributed to nonspecific reaction of BAPA, Angus and Barton (1984).While five samples were positive to Riv.T and negative to CFT, this may be due to its detection of IgG2. While ten samples were positive to msat and negative to CFT, this may be due to its detection of IgM of recent infection while 1, 12 and 14 samples were positive to CFT and negative to BAPA, msat and Riv.T respectively is attributed to CFT detects mainly a little amount of IgG1 not detected by BAPA, msat and Riv.T. Alton et al, (1988). The results of FPA were similar to CFT as 42 were positive and 76 were negative, and this may be attributed to high relative sensitivity and relative specificity of FPA which may be due to using of low molecular weight fluorescein isothiocyanate conjugated LPS antigen (Nielsen and Gall 2001). In this study the percent positivity, relative sensitivity and relative specificity of FPA were 35.59%, 97.62%, and 98.68% respectively and were superior in comparison to the other serological tests. These results were compatible with that of Gewada (2010) and more than the results of Ayman El Behiry (2014) which were 84.87%, % and 77.56% in FPA, CFT and SAT respectively. Nielsen and Gall (2001) revealed that FPA prepared as well as celisa and ielisa and better than CFT and BAPA with bovine sera.it was shown previously that FPA was considerably more specific than ielisa and celisa when testing vaccinated animals. They suggested that the FPA is homogenous assay which may be performed in the field with accuracy approaching that of the laboratory-based assays. On the other hand, the FPA has the advantage that it can be applied to whole blood and milk both from individual animal or bulk tank samples, thus the FPA can detect antibody to smooth Brucellae species in all domesticated animals very rapidly without need for transportation of the samples to the central laboratory especially in quarantine stations. Application of this test will allow much higher progress in the control of brucellosis in Egypt. Conclusion We concluded that BAPA should be used as a presumptive test to exclude negative cases followed by FPA as it is highly sensitive and highly specific test for confirmation of positive samples. CFT must be applied as a confirmation of these results till final evaluation of FPA and to estimate its efficiency to be recommended for diagnosis of brucellosis in camels in the field. References Altman, D.G. and Bland, J.M. (1994). Diagnostic tests: 1: sensitivity and specificity BMJ. 308: Angus, R.D. and Barton, C.E. (1984). The production and evaluation of a buffered plate antigen for use in presumptive test for brucellosis. Dev. Biol. Stand., 56: Alton, G.G.; Jones, L.M.; Angus, R.D. and Verger, J.M. (1988). Techniques for the brucellosis laboratory. Institute National de la Recherché Agronomique, Paris, France Ayman, El Behiry (2014). Evaluation of Diagnostic Techniques and Antimicrobial Resistance of Brucella spp. Isolated from Blood Serum of Camels and Camel Ranchers, Journal of Microbiology Research, 4(2): Cooper, C.W. (1991). The epidemiology of human brucellosis in a well-defined urban population in Saudi Arabia. Journal of Tropical Medicine and Hygiene, 94: Cutler, J.S.; Whatmore, A.M. and Commander, N.J. (2005). Brucellosis new aspects of an old disease. Journal of Applied Microbiology. 98: Farah, Z. and Fisher, A. (2004). Milk and Meat from the Camel. Handbook on Products and Processing. ISBN X. Farina, R. (1985). Current Serological Methods in B. melitensis Diagnosis. In Brucella melitensis, Verger, J.M. and Plommet M. leds. Martinus Nijhoff, Dorderchi 1985,

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