Validation of a competitive ELISA for diagnosis of Brucella melitensis infection in sheep and goats

A competitive enzyme-linked immunosorbent assay (cELISA) was validated for the serodiagnosis of Brucella melitensis infection in small ruminants using 2108 positive and 2154 negative reference sera from sheep and goats. The optimum cut-off values, offering the highest diagnostic sensitivity (DSn) and diagnostic specificity (DSp), determined by receiver operating characteristic analysis, were at 23.6%, 21.8% and 25.0% inhibition of the conjugate control for sheep, goats and both species, respectively. The DSns of the cELISA for sheep, goats and both species at these cut-off values were 89.2% (95% confidence interval 87.1-91.1%), 74.0% (95% CI 71.4-76.5%) and 77.9% (95% CI 76.1-79.7%), whereas DSps were 96.4% (95% CI 95.2-97.4%), 92.9% (95% CI 91.1-94.3%) and 97.2% (95% CI 96.4-97.8%), respectively. Compared to cELISA, indirect ELISA and fluorescence polarisation assay have higher DSns and DSps. However, the results obtained with the cELISA were in good agreement with those of the complement fixation test (CFT) under field conditions using 5735 sheep and goat sera. The cELISA can be used as an alternative to the CFT for diagnosing B. melitensis infection in small ruminants.     

羊布鲁氏菌甲酰基转移酶的生物信息学研究

本研究旨在分析和预测羊布鲁氏菌甲酰基转移酶基因及其编码蛋白的结构与功能,用于指导其生物学功能的实验室研究。综合利用生物信息学软件(DNAMAN和Vector NTI)、数据库(prosite和PDB)和网络服务器对甲酰基转移酶进行基本性质分析、三维结构预测和功能预测,展示了甲酰基转移酶的氨基酸分布情况,二级结构中形成螺旋、折叠、卷曲的氨基酸区段及结构域;预测并综合阐释了B细胞表位的柔韧性、表面可及性和亲水性参数。生物信息学预测和分析结果可为研究在布鲁氏菌致病及机体免疫应答中甲酰基转移酶的结构与功能的关系提供丰富资料。     

Protection against Brucellosis in Goats,Five Years after Vaccination with Reduced-Dose Brucella melitensis Rev 1 Vaccine

The protection conferred by the reduced-dose Rev 1 Brucella melitensis vaccine in goats that had been immunized 5 years previously was evaluated. Sixteen goats vaccinated 5 years before with Rev 1 (1 x 10(5) cfu) and 5 non-vaccinated goats were challenged with B. melitensis 16M (4 x 10(5) cfu) using the conjunctival route. After giving birth or aborting, the goats were sacrificed and tissue samples were taken for bacteriological study. The challenge strain was recovered in 12%, of the animals from the vaccinated group, and in (80% of the control group. It is concluded, therefore that the use of reduced-dose Rev 1 protects goats vaccinated in endemic areas for at least 5 years after immunization.     

Comparative Evaluation of a Field-based Dot-ELISA Kit with Three Other Serological Tests for the Detection of Brucella Antibodies in Goats

A dot-ELISA (d-ELISA) test was evaluated and compared with the serum agglutination test (SAT), micro-complement fixation test (CFT) and a plate-ELISA (p-ELISA) for field use in screening herds of goats against brucellosis. During the standardization of the dot-ELISA kit on 1732 caprine serum samples, 1571 samples out of 1666 were found to be negative in d-ELISA, SAT and micro-CFT, while 59 were positive in different combinations. Of a further 66 serum samples, 34 were negative and 31 were positive in different combinations in d-ELISA, SAT, micro-CFT and p-ELISA. A total of 1584 goats belonging to different herds were then screened for brucellosis. Of the 694 serum samples screened in the first batch using d-ELISA, a positive reaction was observed in 26 cases. Further screening of these cases revealed 13 and 21 goats as positive reactors in SAT and CFT, respectively. In a second batch of 890 goats there were 109 positive reactors in d-ELISA. Among these 109 goats, 34, 40 and 80 goats were positive reactors in SAT, CFT and p-ELISA, respectively. The results of d-ELISA correlated well with those of p-ELISA. Dot-ELISA was found to be a more suitable and rapid test for screening large numbers of goats in the field.     

Induction of immune response in mice with a DNA vaccine encoding outer membrane protein (omp31) of Brucella melitensis 16M

Brucellosis causes serious economic losses to goat farmers by way of reproductive losses in the form of abortions and stillbirths. Nucleic acid vaccines provide an exciting approach for antigen presentation to the immune system. In this study, we evaluated the ability of DNA vaccine encoding the omp31 protein of Brucella melitensis 16M to induce cellular and humoral immune responses in mice. We constructed eukaryotic expression vectors called pTargeTomp31, encoding outer membrane protein (omp31) of B. melitensis 16M. pTargeTomp31 was injected intramuscularly three times, at 3-week intervals in groups of mice 6 weeks of age. pTargeTomp31 induced good antibody response in ELISA . pTargeTomp31 elicited a T-cell-proliferative response and also induced a strong gamma interferon production upon restimulation with either the omp31 antigen or B. melitensis 16M extract. We also demonstrate that animals immunized with this plasmid elicited a strong and long-lived memory immune response. Furthermore, pTargeTomp31 elicited a typical T-helper 1-dominated immune response in mice, as determined by immunoglobulin G isotype analysis. This vaccine also provided the moderate degree of protection to the mice. This study for the first time focuses on DNA immunization of a gene from B. melitensis. These results may lead to the development of a DNA-based vaccine for the control of brucellosis in goats.     

Seeking a niche: putative contributions of the hfq and bacA gene products to the successful adaptation of the brucellae to their intracellular home

Long-term residence of the brucellae in the phagosomal compartment of host macrophages is essential to their ability to produce disease in both natural and experimental hosts. Correspondingly, the Brucella spp. appear to be well adapted to resist the multiple environmental stresses they encounter in their intracellular home. This brief review will focus on the contributions of the hfq and bacA gene products to this adaptation. Studies with Brucella hfq mutants suggest that stationary phase physiology is critical for successful long-term residence in host macrophages. Analysis of Brucella bacA mutants, on the other hand, reveal very striking parallels between the strategies employed by the rhizobia to establish and maintain protracted intracellular residence in their plant host and those used by the brucellae during their long-term survival in the phagosomal compartment of host macrophages.     

The genome of Brucella melitensis

The genome of Brucella melitensis strain 16M was sequenced and contained 3,294,931 bp distributed over two circular chromosomes. Chromosome I was composed of 2,117,144 bp and chromosome II has 1,177,787 bp. A total of 3198 ORFs were predicted. The origins of replication of the chromosomes are similar to each other and to those of other -proteobacteria. Housekeeping genes such as those that encode for DNA replication, protein synthesis, core metabolism, and cell-wall biosynthesis were found on both chromosomes. Genes encoding adhesins, invasins, and hemolysins were also identified.     

Characterization of pathogen-specific expression of host immune response genes in Anaplasma and Mycobacterium species infected ruminants

Anaplasma and Mycobacterium species are among the most prevalent bacterial pathogens in European red deer (Cervus elaphus) in south-central Spain and are known to modify gene expression in ruminants. In this study, we used microarray hybridization and real-time RT-PCR analyses to characterize global gene expression profiles in red deer in response to Anaplasma ovis and A. ovis/Mycobacterium bovis/Mycobacterium avium sub. paratuberculosis (MAP) infections, compare the expression of immune response genes between red deer infected with A. ovis, M. bovis and A. ovis/M. bovis/MAP, and characterize the differential expression of immune response genes identified in red deer in cattle infected with M. bovis and Anaplasma marginale. Global gene differential expression in A. ovis- and A. ovis/M. bovis/MAP-infected deer resulted in the modification of common and pathogen-specific cellular biological processes. The differential expression of host immune response genes showed pathogen and host-specific signatures and the effect of infection with multiple pathogens on deer immune response. These results suggested that intracellular bacteria from Anaplasma and Mycobacterium genera produce similar genes expression patterns in infected ruminants. However, pathogen and host-specific differences could contribute to disease diagnosis and treatment in ruminants.     

Use of an Immunobinding Test on Nitrocellulose Paper to Diagnose Caprine Brucellosis

Veterinary Research Communications -     

Diagnosis of canine brucellosis by ELISA using an antigen obtained from wild Brucella canis

An indirect ELISA test was developed for the diagnosis of Brucella canis infection in dogs. A bacterial whole cell extract was used as a solid phase antigen, using B. canis isolated from an infected animal. Sera from culture-positive and healthy negative animals were used as internal reference controls. The cut-off point was determined by a mathematical formula for a statistically valid value, which defined the upper prediction limit, based on the upper tail of the t-distribution of 21 negative control sera readings, for the confidence level of 99.5%. The sensitivity and specificity of the ELISA test were 95% and 91%, respectively. The ELISA test showed a significant concordance index (K=0.84) with the agar gel immunodiffusion test. The reliability of the ELISA for the detection of infected animals was established by a double blind study testing 280 sera provided by serum banks from different diagnostic and research institutions and analyzed by ROC Curve.     

Prevalence of antibodies to Brucella spp. and individual risk Factors of Infection in Traditional Cattle, Goats and Sheep Reared in Livestock–Wildlife Interface Areas of Zambia

A cross-sectional study was performed in the livestock–wildlife interface areas of Lochinvar and Blue Lagoon National Parks and the non-interface area of Kazungula to determine the prevalence of antibodies to Brucella spp. in domestic ruminants and identify individual animal risk factors of infection. A total of 1245 cattle from 124 herds and 280 goats and sheep from 29 flocks were tested sequentially for Brucella antibodies using the Rose Bengal test (RBT) and competitive ELISA. In cattle, individual seroprevalence ranged from 14.1% to 28.1%, while herd sero–prevalence ranged from 46.2% to 74.0% in the three study areas. No goat or sheep tested positive for Brucella antibodies. Three types of cattle grazing strategies were encountered: locally grazed herds (LGH), transhumantly grazed herds (TGH) and river flood plain grazed herds (FGH). Brucella seroprevalence was seen to vary according to area and grazing strategy: Lochinvar and transhumant grazed herds recorded the highest figures, respectively. Age, sex and history of abortion were found to have independent effects on individual seroprevalence. This study establishes that brucellosis is endemic in domestic animals in the livestock–wildlife interface areas of Blue Lagoon and Lochinvar national parks and the disease is also present in Kazungula. We observed that type of grazing strategy had significant impact on cattle Brucella seroprevalence and that transhumant herds were at high risk of being infected.     

Use of Brucella abortus vaccine strain RB51 in pregnant cows after calfhood vaccination with strain 19 in Argentina

One hundred and seven pregnant cows, which had been calfhood vaccinated with Brucella abortus strain 19 (S-19) were revaccinated with either S-19 or strain RB51 (S-RB51). All S-19-revaccinated animals seroconverted, while none of the RB51-revaccinated animals seroconverted. Two out of 25 (8%) S-19-revaccinated animals aborted, while none of the 57 RB51-revaccinated group aborted. Four of the S-19-revaccinated animals shed S-19 in the milk for at least 7 days, while only 1 cow shed S-RB51 for at least 3 days (but <7 days) post-parturition. Revaccination of strain 19 calfhood-vaccinated, pregnant cattle with S-RB51 appears to be a safe procedure with no diagnostically negative consequences.