Non specific serological reactions in BPA and FPA tests following immunization with inactivated Gram negative bacteria in the mouse model

Abstract

Most serological test in diagnosis of bovine brucellosis are based on detection of antibodies to the O-chain (OPS) epitopes of LPS (2). Thus, non specific serological reactions are observed between Brucella spp. and other Gram (-) bacteria that share antigenic determinants in the OPS molecule (1,3). The aim of this study was to determine cross reacting antibodies resulting from the administration of different inactivated Gram (-) bacteria in the mouse model. Female BALB/c mice 6-8 weeks old were randomly distributed into five groups. Following bacteria were used: (I) Campylobacter fetus fetus (n=4), (II) Haemophilus somnus (Histophilus somni) (n=4), (III) Moraxella bovis (n=5), (IV) Pasteurella haemolytica (Mannhemia haemolytica) (n=4) and (V) Pasteurella multocida (n=5). Bacteria were cultured, inactivated, formulated in Incomplete Freund Adjuvant and employed as immunogens. Booster was carried out at day 25. A positive control group infected with B. abortus S544 was also included. Sera samples were obtained before immunization and at weeks 2, 4, 5, 6, 7, 9, 12, 15, 18, 21, 24 and 27 post vaccination. Buffered Plate Antigen (BPA) and Fluorescence Polarization Assay (FPA) were performed on all specimens. All mice had negative results in both tests before vaccination. Hundred percent of animals gave positive result at week 2 (group V), week 4 (groups II, III and IV) and at week 6 (group I) to BPA. At the end of the experiment, all animals in group V (P. multocida) remained positive to this test while in the other groups only one mouse per group reacted to BPA. In contrast, only sera from groups IV (Pasteurella haemolytica) and V (Pasteurella multocida) were positive to FPA. The highest number and most persistent reactions were found in mice immunized with P. multocida (from 80% reactors at week 4 to 20% at the end of trial). Mice from positive control group remained positive to both tests throughout the experiment. These results showed that Gram (-) bacteria included in the experiment induced cross reacting antibodies with B. abortus. However, differences in cross reactivity between bacterial genera were observed. Although false positive reactions were found in both tests, FPA was more reliable. In conclusion, Gram (-) bacteria can be a potential source of confusion in serological diagnosis of bovine brucellosis.