Immunoblot studies in the differential diagnosis of porcine brucellosis: an immunodominant 62 kDa protein is related to the mycobacterial 65 kDa heat shock protein (HSP-65)

Abstract

Smooth Brucella spp. share certain lipopolysaccharide antigens with other bacteria, resulting in serological cross-reactions which can prevent the definitive diagnosis of brucellosis. To identify other antigens with serodiagnostic potential, immunoblot studies following sodium dodecyl sulphate-polyacrylamide gel electrophoresis were carried out. Sera from pigs experimentally infected with Brucella suis and naturally infected feral pigs, sera from pigs from a farm with a known history of Yersinia enterocolitica 0:9 infection, Brucella Complement Fixation Test (CFT) reactor pigs (aetiology unknown) and pigs from consistently Brucella CFT negative farms were examined. Although B. suis infected pigs recognized a total of nine B. melitensis antigens, individual pigs rarely recognized more than three antigens in the range. A 62 kDa antigen was recognized by the majority (73%) of the Brucella infected pigs, but only by 10 to 23% of pigs from the other groups. This antigen was shown to be the Brucella homologue of the ubiquitous 65 kDa heat shock protein (HSP-65) family by immunoblot studies with 14 monoclonal antibodies to the Mycobacterium leprae HSP-65. Only four of these monoclones (Y1.2, ML-30, D7C and IIIC8) identified the B. melitensis 62 kDa protein suggesting that unshared, potentially Brucella specific, regions exist. Sera from Y. enterocolitica 0:9 infected pigs, CFT reactor pigs (aetiology unknown), CFT negative pigs and hyperimmune pig serum raised to Y. enterocolitica 0:9 also recognized B. melitensis antigens, most notably a 17 kDa protein. This antigen appears to be a common cross-reactive protein.

A Serological Survey on Swine Brucellosis Using Standard Procedures, Dot Blot, and Western Blot in Finisher Pigs in Central-North Italy

“…WB assay resulted useful to confirm and support traditional serology, as also reported previously by other authors [17,18,19]. However, WB execution requires specialized and trained personnel, it is time consuming, and previous work has showed that its sensitivity is low [28].…”

“…Y. enterocolitica O:9 is particularly widespread in swine populations [16] and represents a frequent cause of false positive serological reactions (FPSR) [13,14]. For these reasons, several authors developed different immunoblotting methods in order to improve sensitivity and specificity of the serological diagnosis [17,18,19].…”

Serological survey for Brucella antibodies in feral pigs from eastern Australia

“…2 However, serological cross-reactions may occur in Brucella s e rology as a result of infection with Yersinia entero c o l i t i c a s e rotype 0:9. 8 We are unsure of the extent of Y entero c o l i t i c a infection in feral pigs in Australia, but suspect the positive reactor was in fact due to B suis as the sample was re c ove re d from the enzootic area. No other reactors were recovered, which we would have expected if Y enterocolitica was a common source of infection in feral pig populations in Australia.…”

Differentiation between serological responses to Brucella suis and Yersinia enterocolitica serotype O[ratio ]9 after natural or experimental infection in pigs

“…Brucella LPS contain Brucella spp. Attempts to overcome the LPS crossreactions with the use of Brucella protein antigens is hampered by the strong humoral immunodominance of smooth LPS in Brucella infections [5,6]. However, although the exact structures of the various epitopes remain unclear, it appears that LPS epitopes span 2-5 overlapping polysaccharide units and that antibodies generated against the common A-or C/Y-epitopes are able to block the specific binding to adjacent M-and C-epitopes in competitive ELISAs [3,4].…”