Melitensis Bv Research Articles

Real-time PCR for identification of Brucella spp.: A comparative study of IS 711, bcsp31 and per target genes

Culture is considered as the reference standard assay for diagnosis of Brucella spp. in humans and animals but it is time-consuming and hazardous. In this study, we evaluated the performances of newly designed real-time PCR assays using TaqMan probes and targeting the 3 following specific genes: (i) the insertion sequence IS711, (ii) bcsp31 and (iii) per genes for the detection of Brucella at genus level. The real-time PCR assays were compared to previously described conventional PCR assays targeting the same genes. The genus-specificity was evaluated on 26 Brucella strains, including all species and biovars. The analytical specificity was evaluated on a collection of 68 clinically relevant, phylogenetically related or serologically cross-reacting micro-organisms. The analytical sensitivity was assessed using decreasing DNA quantities of Brucella ovis, B. melitensis bv. 1, B. abortus bv. 1 and B. canis reference strains. Finally, intra-assay repeatability and inter-assay reproducibility were assessed. All Brucella species DNA were amplified in the three tests. However, the earliest signal was observed with the IS711 real-time PCR, where it varied according to the IS711 copy number. No cross-reactivity was observed in all three tests. Real-time PCR was always more sensitive than conventional PCR assays. The real-time PCR assay targeting IS711 presented an identical or a greater sensitivity than the two other tests. In all cases, the variability was very low. In conclusion, real-time PCR assays are easy-to-use, produce results faster than conventional PCR systems while reducing DNA contamination risks. The IS711-based real-time PCR assay is specific and highly sensitive and appears as an efficient and reproducible method for the rapid and safe detection of the genus Brucella.

Comparison between immune responses and resistance induced in BALB/c mice vaccinated with RB51 and Rev. 1 vaccines and challenged with Brucella melitensis bv. 3

BALB/c mice were immunized with live rough Brucella abortus RB51 or smooth Brucella melitensis Rev. 1 vaccines and challenged with a B. melitensis field strain. Protection was assessed by a variety of serological tests and recovery of vaccinal and challenge strains by culture. Mice vaccinated with RB51 gave negative results in the conventional serological tests prior to challenge, namely; standard tube agglutination test (SAT), Rose Bengal plate test (RBPT), buffered acidified plate antigen test (BAPAT), and mercaptoethanol test (MET). Sero-conversion took place to a whole-cell bacterial buffered RB51 antigen after vaccination and persisted for 7 weeks post-vaccination. Mice challenged with B. melitensis were assessed for bacterial load and immune response for 12 weeks after challenge. Protection units were showed that Rev. 1 vaccine was superior to RB51 vaccine in protection of mice against B. melitensis. However, RB51 vaccine has the advantage that it would not elicit antibodies to standard serological tests based on the LPS O antigen. RB51 vaccine could therefore be used for control of B. melitensis infection and avoid confusion in the use of standard sero-diagnostic tests.