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The kit for serological diagnosis of glanders in complement fixation test

The glanders is a dangerous zooanthroponotic disease which is caused by Gram-negative bacterium Burkholderia mallei. This disease strikes mainly horses, donkeys and mules. Today, the detection of infected animals by serological methods and their isolation are crucial to preventing the spread of glanders, including its entry into the territory of Ukraine. At the same time the diagnosis of glanders can be established only on the basis of comprehensive studies. The main role in this matter occupies laboratory diagnosis. There are several methods of laboratory diagnosis of glanders, but the only method recognized by the OIE for international trade is the complement fixation test (CFT). Unfortunately, Ukraine lacks its own production of diagnostic tests to identify glanders.

That is why the main purpose of our study was to develop the laboratory CFT kit for the diagnosis of glanders, which consists of the antigen and positive control serum, to determine the diagnostic sensitivity and specificity of obtained antigen and to establish the activity of the obtained positive serum.

The development of the CFT kit for glanders was performed in two stages. In the first stage the antigen was produced and its diagnostic sensitivity and specificity was determined. During the second stage a positive control serum was received.

Three strains of B. mallei Bogor, Mukteswar and Zagreb were used for manufacturing the series of experimental antigen. Cultures were plated on blood agar with the addition of 3% glycerol and incubated at 37 °C for 72 hours. Then each strain subcultured on Brucella-agar. After incubating for 5 days the cultures were collected and washed with normal saline. Finally, a 2% suspension was prepared with normal saline and 0,5 % phenol. The suspension was inactivated at 80 °C for 3 hours. The obtained antigen was plated on blood agar with 3 % glycerol and incubated for 10 days. In the absence of microbial growth during this period we concluded that the antigen was inactivated and sterile. To determine the working concentration of the antigen successive antigen dilutions of 1:10, 1:20, 1:40, 1:80, 1: 160, 1: 320 were prepared and CFT with a positive serum (, Germany) with a known titer 1:160 (± 1 dilution) was performed. Definition of diagnostic sensitivity and specificity was performed according to Martin et al. (1977). The panel of the negative serum samples was n = 400 and positive serum samples panel was n = 92.

During the second phase of research we aimed to obtain a positive glanders serum. To develop the serum we conducted immunization of rabbits with the glanders antigen that was received by the previously described method.

It was found that the diagnostic sensitivity of the experimental antigen was 79.3 % and diagnostic specificity ‒ 98.97 %. Thus, the antigen received by the method described is comparable to commercially available foreign analogues.

To evaluate the activity of the experimental positive serum CFT was performed according to the Guidelines for the diagnosis of glanders, approved by the State Committee of Veterinary Medicine of Ukraine № 214 from 11.06.2010. According to the results of CFT it was found that the activity of the obtained serum was +++ at a dilution of 1:320. In our opinion, the serum can be used as a positive control during serological tests for the diagnosis of glanders in horses in Ukraine.

Thus, as a result of our studies a kit for the diagnosis of glanders in CFT was developed consisting of a trivalent antigen and a positive control serum. It was established that the diagnostic sensitivity of obtained antigen was 79.3 % and specificity - 98.97 %, which is not inferior to commercial counterparts. It was established that glanders positive control serum has high activity in CFT and can be used as a positive control.

We believe that an important area of future research is to study the effectiveness of monoclonal antibodies against specific lipopolysaccharide antigens of B. mallei. Identification of new protein antigens as well as novel lipopolysaccharide and capsular polysaccharide epitopes will significantly increase the sensitivity and specificity of serological diagnostic methods of glanders.

Key words: glanders, horses, serological diagnostics, complement fixation test.

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