BACKGROUND: Brucellosis is one of the most common zoonosis in Middle East and Iran. OBJECTIVES: The purpose of this study was genomic detection of Brucella spp. in sero-positive dairy cattle. METHODS: We have collected 28,519 blood samples from cows during 2012-2013. Samples were screened by Slide and tube agglutination and 2-Mercaptoethanol tests. Samples with anti-Brucella antibodies titer ≥ 1:80 and ≥1:40 in tube agglutination and 2-ME tests were considered as positive respectively. Tissue samples include: lymph nodes, liver, testicle and kidney from 122 samples of slaughtered cows were collected. The Sero-positive samples were examined by a collection of specific primers for Brucella abortus, Brucella melitensis, vaccinal strains included RB51 and Rev1 using PCR tests. RESULTS: Results showed that 450 samples were positive in slide agglutination test and 447 samples had anti-brucella antibodies titer equal to or more than 1:80. So they were positive by tube agglutination test. Three hundred eighty nine samples were positive by 2- mercaptoethanol test. PCR test results showed that 46 samples (37.7%) out of 122 samples had a specific sequence of Brucella or otherwise they have an active infection with Brucella species, whereas 62.3% of samples were negative. The PCR results showed that 2 samples (4.35%) were infected by B. melitensis, 2 samples (4.35%) infected by Rev1 strain and 42 samples (91.3%) were infected by B. abortus. CONCLUSIONS: The results showed that, as we had expected, the majority of cows were infected by B. abortus. Animals who infected by B. melitensis and Rev1 strain may be a result of contact with sheep or goats. We couldn’t find Brucella genome in 76 samples (62.3%) of sero-positive cows. It may be caused by cross reaction of sera with Brucella species in tests or activation of immune system response and elimination of organism from internal organs.

Twenty-four rams inoculated with Brucella ovis by conjunctival and preputial routes were treated with a long-acting oxytetracycline alone or in combination with dihydrostreptomycin sulfate. The combined treatment eliminated Brucella ovis from 11 of 12 (91.6%) treated rams. Only 4 of 12 (33.3%) rams treated with oxytetracycline alone were bacteriologically negative. Neither treatment resolved clinical epididymitis in 2 rams affected before treatment. Many rams had pathologic lesions in the epididymis and ampullae, which limited the efficacy of antibiotic treatment.

The plate counting method widely used at present to discern viable from non-viable Brucella in the host or cell is time-consuming and laborious. Therefore, it is necessary to establish a rapid, simple method for detecting and counting viable Brucella organisms.

Abortions in domestic ruminants cause significant economic losses to farmers. Determining the cause of an abortion is important for control efforts, but it can be challenging. All available diagnostic methods in the bacteriology laboratory should be employed in every case due to the many limiting factors (autolysis, lack of history, range of samples) that complicate the investigation process. The purpose of this study was to determine whether the recovery of diagnostically significant isolates from domestic ruminant abortion cases could be increased through the use of a combination of the existing aerobic culture and Brucella selective method with methods that are commonly recommended in the literature reporting abortion investigations. These methods are examination of wet preparations and impression smears stained by the modified Ziehl–Neelsen method, anaerobic, microaerophilic, Leptospira, Mycoplasma and fungal culture. Samples of placenta and aborted foetuses from 135 routine clinical abortion cases of cattle (n = 88), sheep (n = 25) and goats (n = 22) were analysed by the new combination of methods. In 46 cases, bacteria were identified as aetiological agents and in one case a fungus. Isolation of Brucella species increased to 7.4% over two years compared with the previous 10 years (7.3%), as well as Campylobacter jejuni (n = 2) and Rhizopus species (n = 1). Salmonella species (5.9%) and Trueperella pyogenes (4.4%) were also isolated more often. In conclusion, the approach was effective in removing test selection bias in the bacteriology laboratory. The importance of performing an in-depth study on the products of abortion by means of an extensive, combination of conventional culture methods was emphasised by increased isolation of Brucella abortus and isolation of C. jejuni. The combination of methods that yielded the most clinically relevant isolates was aerobic, microaerophilic, Brucella and fungal cultures.

kills Brucella cells by causing lysis of the membrane, so the phenol-heat killed brucella antigen may lake specificity as a result of destruction the majority of proteins in the cell wall. Accordingly, attention was directed to produce antigen using binary ethylene imine as an inactivator. The produced antigen showed high specificity in detecting Brucella abortus and Brucella melitensis-infected animals, but sensitivity was not affected in comparison with the standard Rose Bengal antigen. In Enzyme immunotransfer blot (EITB), phenol–heat killed brucella cells showed only 3 bands (37.375, 23.47 and 7.83 kDa) that denotes denaturation for at least 6 bands whereas binary inactivated brucella cells showed similarity with non-treated ones

In this study field application of RB51 vaccine combined with the policy of test and slaughter as well as application of hygienic measures for control of bovine brucellosis were carried out and evaluated in a dairy herd of cattle for two years. Serological examination of 1280 cattle using tube agglutination, buffered acidified plate antigen, Rose Bengal plate antigen and Rivanol tests revealed 240 (18.75%) positive animals with a previous history of abortion of 12 cows. Brucella melitensis biovar 3 could be isolated from tissue specimens of slaughtered cows. Animals that tested negative in the first examination were vaccinated with RB 51 vaccine with periodical examination every three weeks and slaughtering of positive cases. New positive cows continued to develop up to the 5th examination then three successive sero-negative tests were obtained with release of the farm from quarantine. Examination of animals 6,12,18 and 24 months post release of quarantine revealed 2, 3, 0 and one positive cases respectively the matter which clarified that the control of the outbreak using RB51 vaccine associated with policy of test and slaughter and application of hygienic measures showed some limitations.

Evaluation of the real-time PCR, rose bengal test (RBT), competitive ELISA, and complement fixation test (CFT) was done on 335 camels sera. Real-time PCR, classified 335 camel serum samples to 268 (80%) as positive and 67 (20%) as negative. Real-time PCR, using species specific primers, distinguished 94/104 serum samples due to B. abortus, 4/104 samples due to B. melitensis and 6/104 due to mixed infection. The results of serological tests revealed that modified mRBT75 using 75 µl of serum, detected the highest number of positive samples 271 (80.9%), while 262 (78.2%), 257 (76.7%), 253 (75.5%) and 245 (73.1%) samples were found to be positive for brucellosis using CFT, cELISA, mRBT50, and RBT25, respectively. Compared to other serological tests, the CFT proved to have the best results in the criteria of test validations, namely; specificity (88%), PPV (96.9%), NPV (80.8%), PLR (7.9), NLR (0.06) and DOR (133.8). The Kappa (K) statistic agreements values between real-time PCR and rose bengal (RBT25), modified (mRBT50), (mRBT75), cELISA and CFT was 0.562 (± 0.053), 0.613 (± 0.052), 0.725 (± 0.048), 0.710 (± 0.047) and 0.801 (± 0.041), respectively. The authors recommend the use of real-time PCR on camel sera to confirm the disease.

The plate counting method widely used at present to discern viable from non-viable Brucella in the host or cell is time-consuming and laborious. Therefore, it is necessary to establish a rapid, simple method for detecting and counting viable Brucella organisms. Using propidium monoazide (PMA) to inhibit amplification of DNA from dead Brucella, a novel, rapid PMA-quantitative PCR (PMA-qPCR) detection method for counting viable Brucella was established. The standard recombinant plasmid with the target BCSP31 gene fragment inserted was constructed for drawing a standard curve. The reaction conditions were optimised, and the sensitivity, specificity, and repeatability were analysed. The optimal exposure time and working concentration of PMA were 10 min and 15 μg/mL, respectively. The correlation coefficient (R²) of the standard curve was 0.999. The sensitivity of the method was 10³ CFU/mL, moreover, its specificity and repeatability also met the requirements. The concentration of B. suis measured by the PMA-qPCR did not differ significantly from that measured by the plate counting method, and the concentrations of viable bacteria in infected cells determined by the two methods were of the same order of magnitude. In this study, a rapid and simple PMA-qPCR counting method for viable Brucella was established, which will facilitate related research.

Introduction: Peroxiredoxin 6 (Prdx6) is a bifunctional protein with glutathione peroxidase activity and phospholipase A2 activity. Previous studies have shown a significant positive correlation between the intracellular survival ability of Brucella and Prdx6. Here, the Prdx6 enzyme with a single activity was constructed to facilitate study of the relationship between the single function of Prdx6 and Brucella infection. Material and Methods: The target open reading frame (ORF) DNAs of Prdx6 with a single active centre were prepared using gene splicing by overlap extension PCR (SOE-PCR), and the recombinant eukaryotic expression plasmids inserted by Prdx6 with the single activity centre were constructed and transfected into murine Raw264.7 macrophages. The glutathione peroxidase activity and phospholipase A2 activity of the constructed Prdx6 were examined. Results: The core centres (Ser³² and Cys⁴⁷) of Prdx6 were successfully mutated by changing the 94ᵗʰ nucleotide from T to G and the 140ᵗʰ nucleotide from G to C in the two enzyme activity cores, respectively. The constructed recombinant plasmids of Prdx6 with the single active centre were transfected into murine macrophages showing the expected single functional enzyme activity, which MJ33 or mercaptosuccinate inhibitors were able to inhibit. Conclusion: The constructed mutants of Prdx6 with the single activity cores will be a benefit to further study of the biological function of Prdx6 with different enzyme activity.

Objective: Brucellosis is a highly contagious zoonosis with global distribution. The disease remains endemic in many countries including Iran, while its seroprevalence in endemic area is not well documented. We aimed to determine the seroprevalence of brucellosis in sheep and goats in Hamedan province, west of Iran. Material and methods: A total of 3,250 blood samples from 2,550 sheep and 700 goats were collected randomly. All samples were analyzed for the presence of Brucella antibodies using Rose Bengal, Wright standard tube agglutination and 2-mercaptoethanol agglutination tests. Results: The seroprevalence rate of brucellosis in animals and flock level were found in 4.6% and 13.6% of goats and 3% and 27.9% of sheep, respectively. No evidence of correlation between gender and Brucella infection rate were found in animals (P>0.05). Statistical significant differences was seen between age groups and infection rate in goats (P=0.033, OR=2.1); unlike to sheep (P=0.373). Also, the infection rate in nomads population of sheep was higher than fix location animals (P=0.003; OR=1.9); unlike to goats (P=0.195). In animals with history of abortion and vaccination against brucellosis, seroprevalence rate was significantly lower than other (P<0.05). Conclusion: This is the first report of brucellosis in sheep and goats in Hamedan province. The design of a comprehensive control program including vaccination, screening, and culling of brucellosis-positive animals is recommended. [J Adv Vet Anim Res 2016; 3(4.000): 399-405]