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In a previous paper an ELISA for detecting anti-Brucella antibodies in cattle sera was elaborated and evaluated. The aim of this study was to evaluate the ELISA for testing individual milk samples from cattle. Validation was performed by comparing the results of this test with those in the Rose Bengal Test (RBT), tube agglutination test (TAT), complement fixation test (CFT), Coombs anti-globulin test (Coombs), 2-mercaptoet- hanol test (2-ME) and serum ELISA. The examinations involved 79 milk samples from cows in which sera were positive in the RBT, 14 milk samples from cows in which sera were positive in the CFT, Coombs or 2-ME, 530 milk samples from cows considered free of brucellosis and 309 milk samples from cows from herds suspected of brucellosis. No correlation between the milk ELISA and RBT results was found - among 79 samples from cows in which sera reacted positively in the RBT only 22 were positive or doubtful in the ELISA, but all samples in which the presence of anti-Brucella antibodies was confirmed in the CFT, Coombs or/and 2-ME reacted positively in the ELISA (14 out of 14 samples). All 530 milk samples from cows free of brucellosis were negative in the ELISA. Among 309 milk samples from cows from herds suspected of brucellosis 11 positive and 20 doubtful results were obtained in the ELISA and this method proved to be more sensitive than traditional methods and serum ELISA, in which method only 3 samples from the same cows reacted positively and 2 doubtfully. The ELISA for testing individual milk samples proved to be a useful method for diagnosis of brucellosis in cattle.
The diagnosis of brucellosis is mainly based on serological tests. All animals classified as serologically positive are obligatorily slaughtered and subjected to bacteriological examination. B. abortus has not been reported in Poland since the eradication of bovine brucellosis in 1980. On the other hand, B. suis biovar 2 is sporadically isolated from cattle. In accordance with the instructions of the Chief Veterinary Officer, samples of feces from animals slaughtered following positive serological results for brucellosis have been examined for the presence of Yersinia enterocolitica O:9 since 2011. Because of the similarity of its O-polysaccharide component of S-LPS with that of Brucella, this microorganism is considered to be a major contributory factor of false positive serological reactions (FPSR). The paper presents the results of the bacteriological and molecular examination of feces from 26 cows seropositive for brucellosis and 30 healthy cows, negative for brucellosis, for the presence of Y. enterocolitica O:9. Y. enterocolitica O:9 was found in 7 of the positive cows, whereas all samples from cows negative for brucellosis were free from this bacteria. These results indicate that Y. enterocolitica O:9 may be responsible for some of the positive results for brucellosis in cattle.
Brucella abortus is a facultative intracellular gram-negative bacterial pathogen that causes abortion in pregnant cattle and undulant fever in humans. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of subunit vaccines against brucellosis. It has already been expressed in several bacteria and has been used as DNA vaccine. In order to construct yeast expressing vector for the tHSA-L7/L12 fusion protein, the 171112 ribosomal gene was amplified by PCR. The expression plasmid pYtHSA-L7/Ll 2 was constructed by inserting the L7/L12 gene into the pYHSA5 shuttle vector (containing inulinase signal sequence, HSA gene and Gal 10 promoter). The recombinant vector was transformed into S. cerevisiae and was then induced by galactose. The secreted recombinant fusion protein was detected in supernatant by SDS-PAGE and confirmed by western blot analysis using anti-HSA and anti-L7/L 12 antibodies. Fusion protein was purified by affinity chromatography and its amount was approximately 500 μg/liter.
The objective of the present study was to determine the prevalence of brucellosis in household animals of Mirpur, Azad Kashmir due to its geographic importance. A total of 360 blood samples of cattle, buffaloes, sheep and goats were initially screened through Rose Bengal Plate test (RBPT) and then positive samples were subjected to Enzyme Linked Immunosorbent Assay (ELISA) for confirmation and quantification of antibody titers. Molecular confirmation of serologically positive samples was performed by real time polymerase chain reaction (PCR). RBPT and ELISA showed a total of 8.6% and 6.87% positive samples respectively. The species wise seropositivity by RBPT was greater in cattle followed by buffaloes, goats and sheep. Similarly ELISA showed more seropositivity in cattle than buffaloes, while sheep and goats were negative for brucellosis by ELISA. RT-PCR revealed 100% samples positive for Brucella abortus by species specific PCR. This study revealed the presence of Brucella abortus in Mirpur for the first time. Since brucellosis is listed in transboundary diseases, its presence in this geographically important region could be a potential threat for neighboring countries.
The investigation aimed at preparing an ELISA kit for examination of bovine sera for brucellosis and evaluating the diagnostic properties of the test. Lipopolysaccharide extract (LPS) was used as the antigen for microplate coating, antibodies against IgG of bovine sera with horseradish peroxidase were used as the conjugate and ABTS with H202 as the substrate. A weak-positive serum prepared from the second International Standard of anti-Brucella abortus Serum (ISaBaS II) and a negative serum obtained by mixing sera from Brucella-free cows were used as controls. Evaluation of the diagnostic usefulness of the ELISA was performed by comparison of the results obtained with those in the rose bengal test (RBT) and complement fixation test (CFT). The examinations involved 212 bovine sera positive in the RBT, 60 sera positive in the CFT, 5 standard sera, and 1005 sera negative in the RBT. A high correlation was found between the ELISA and CFT results (55 sera out 60 sera) and no correlation between the ELISA and RBT results. Only 60 out of 212 sera positive in RBT gave positive results in the ELISA. All standard sera were positive in the ELISA and only 1 serum from those negative in the RBT was doubtful in the ELISA. The ELISA proved to be a suitable method for diagnosis of brucellosis in cattle.
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Brucellosis in humans is a zoonosis of greatly varied clinical image. It occurs on all inhabited continents. The course of the disease may be acute, sub-acute or chronic. The etiologic factors of brucellosis are small, aerobic Gram-negative rods of the genus Brucella, which currently contains ten species: B. abortus, B. suis, B. ovis, B. melitensis, B. canis, B. neotomae, B. pinnipedialis, B. ceti, B. microti and B. inopinata. In humans, the disease is caused mainly by: B. melitensis as the most pathogenic species, followed by B. suis, whereas B. abortus is considered as the mildest type of brucellosis. The natural reservoir of the germ and the source of infection in humans are infected domestic animals, primarily cattle, sheep, goats, as well as wild animals. Infection in humans occurs by penetration through damaged skin, conjunctiva, and more rarely via the alimentary route by the consumption of infected products. Especially exposed are: veterinarians, veterinary technicians, insemination service employees, zoo technicians, farmers working on multi-herd farms (production cooperatives), e.g. cattlemen, also private farmers, employees of slaughter houses and meat processing enterprises. A basis for diagnosing brucellosis are serologic tests which allow the detection of antibodies occurring in response to infection, performed with the use of the following methods: agglutination test, complement fixation test, Coombs test, 2-mercaptoethanol agglutination test, and Burnet’s intradermal allergy test which detects the state of hypersensitivity of the infected organism to Brucella abortus rods.
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