Brucella Ovis Infection In Rams Research Articles

First evidence of Brucella ovis infection in rams in the Pirot Municipality, Serbia.

This paper describes a research on Brucella ovis infection in rams in the Pirot Municipality of South Serbia. A positive result with indirect immunoenzyme test (i-ELISA) was confirmed in 67 (29.8%) and suspicious in 31 (13.8%) out of 225 tested rams. Complement fixation test (CFT) was used as a confirmation test on 67 ELISA positive sera and gave positive reaction in 41 (61.2%) ram serum samples. Rams originated from 113 flocks with 4751 sheep, from 28 villages in the Pirot Municipality of southern Serbia. Clinical examination was performed on epididymis and testes of 12 rams from 7 seropositive flocks by inspection and palpation. The examination showed scrotum asymmetry and unilateral increase of the epididymistail in 5 (41.7%) out of 12 seropositive rams. Pathomorphological examination of testes and epididymis confirmed pathological changes in 7 (58.3%) of the 12 examined rams. Onesided epididymitis with pronounced hypertrophy of the epididymitis was also confirmed. Twelve rams were tested for the presence of bacteria, i.e. 21 epididymis, testes and lymph nodes samples. We isolated 20 Brucella strains from 11 (91.7%) of the 12 examined animals. All isolates were identified with bacteriological and molecular techniques as B. ovis. This is the first evidence of ovine epididymitis (B. ovis) in Republic of Serbia.

Indirect ELISA for diagnosis of Brucella ovis infection in rams

Brucella ovis is a major cause of epididymitis in sexually mature rams, resulting in subfertility, infertility, and economic losses for the sheep industry worldwide. The aim of this study was to develop an indirect ELISA (iELISA) using recombinant proteins, namely rBoP59 and rBP26, as antigens for serological diagnosis of B. ovisinfection. The BoP59 and BP26 recombinant proteins were expressed in E. coli and purified by affinity chromatography. Antigenicity was tested by Western blot and iELISA. Standardization of iELISA was performed with 500ng and 1µg BoP59 and rBP26 per well, testing serum from uninfected and experimentally infected rams. rBP26 was effective in distinguishing positive from negative rams. The rBP26 iELISA developed in this study is the first to use a completely purified rBP26 as antigen resulting in high sensitivity (100%) and specificity (90.2%), and an overall accuracy equal to 1.0.

Species-specific nested PCR as a diagnostic tool for Brucella ovis infection in rams

The aim of the present study was to evaluate a species-specific nested PCR based on a previously described species-specific PCR for detection of B. ovis in semen and urine samples of experimentally infected rams. The performance of the species-specific nested PCR was compared with the results of a genus-specific PCR. Fourteen rams were experimentally infected with the Brucella ovis REO 198 strain and samples of semen and urine were collected every week up to 180 days post infection. Out of 83 semen samples collected, 42 (50.6%) were positive for the species-specific nested PCR, and 23 (27.7%) were positive for the genus-specific PCR. Out of 75 urine samples, 49 (65.3%) were positive for the species-specific nested PCR, whereas 11 (14.6%) were genus-specific PCR positive. Species-specific nested PCR was significantly more sensitive (P<0.001) than the genus-specific PCR in semen and urine from experimentally infected rams. In conclusion, the species-specific nested PCR developed in this study may be used as a diagnostic tool for the detection of B. ovis in semen and urine samples from suspected rams.

Assessment of the diagnostic sensitivity and specificity of an indirect ELISA kit for the diagnosis of Brucella ovis infection in rams

BackgroundBrucella ovis causes an infectious disease responsible for infertility and subsequent economic losses in sheep production. The standard serological test to detect B. ovis infection in rams is the complement fixation test (CFT), which has imperfect sensitivity and specificity in addition to technical drawbacks. Other available tests include the indirect enzyme-linked immunosorbent assays (I-ELISA) but no I-ELISA kit has been fully evaluated.The study aimed to compare an I-ELISA kit and the standard CFT. Our study was carried out on serum samples from 4599 rams from the South of France where the disease is enzootic. A Bayesian approach was used to estimate tests characteristics (diagnostic sensitivity, Se and diagnostic specificity, Sp). The tests were then studied together in order to optimise testing strategies to detect B. ovis.ResultsAfter optimising the cut-off values in order to avoid doubtful results without deteriorating the concordance between the results of the two tests, the I-ELISA appeared to be slightly more sensitive than CFT (Se I-ELISA = 0.917 [0.822; 0.992], 95% Credibility Interval (CrI) compared to Se CFT = 0.860 [0.740; 0.967], 95% CrI). However, CFT was slightly more specific than I-ELISA (Sp CFT = 0.988 [0.947; 1.0], 95% CrI) compared to Sp I-ELISA =0.952 [0.901; 1.0], 95% CrI).The tests were then associated with two different interpretation schemes. The series association increased the specificity of screening and could be used for pre-movement testing in rams from uninfected flocks. The parallel association increased sequence sensitivity, thus appearing more suitable for eradicating the disease in infected flocks.ConclusionsThe high sensitivity and acceptable specificity of this I-ELISA kit support its potential interest to avoid the limitations of CFT. The two tests could also be used together or combined with other diagnostic methods such as semen culture to improve the testing strategy. The choice of test sequence and interpretation criteria depends on the epidemiological context, screening objectives and the financial and practical constraints.

Factors associated with failure in breeding soundness examination of Western USA rams

Breeding-soundness examination (BSE) and eradication of Brucella ovis infection in rams are critical components of flock-health programs. The aims of this retrospective, cross-sectional study were to describe the results of BSE in a large sample of rams in the Western USA and to determine the association between BSE outcome and the semen collection method (penis manually extended vs. retained in the preputial cavity), ram body-condition score (BCS), the presence of ulcerative posthitis, and the size of the flock of origin. We evaluated the first BSE in a given year for rams from Colorado, Wyoming, and Utah, USA, from 2000 through 2007. Breeding-soundness examination consisted of physical examination, scrotal circumference and BCS measurement, semen collection by electroejaculation, and microscopic examination of semen motility, morphology, and leukocyte concentration. We assigned a reason for failure to each failed BSE and used multivariable logistic and Poisson regressions to measure associations between ram and flock variables and the risk or reason for failure on BSE. A non-random, owner-selected subset of rams was tested for antibodies to B. ovis by serum indirect ELISA (iELISA). The Rogan–Gladen corrected B. ovis seroprevalence was measured.Of the 14,667 BSEs performed on 11,804 rams, 29.0% were classified as “failed;” the most common reason for failure was substandard semen parameters (43.8%). Breeding-soundness examinations were more likely to have been categorized as failure for inflammatory causes when performed on rams from medium-sized flocks (OR 1.6; 95% CI 1.1, 2.3) and large flocks (OR 1.4; 95% CI 1.0, 1.9) (P=0.02), suggesting that larger flocks are at higher risk of contagious diseases. The adjusted seroprevalence of B. ovis antibodies among tested rams in this study was 10.0%. Of 233 rams seropositive to B. ovis, 125 (53.6%) were subclinical, a finding that supports the importance of this test in ram BSE. We found that emaciation in rams was associated with an increased risk of BSE failure from substandard semen parameters (P<0.001), but ulcerative posthitis and the semen collection method were not (P=0.09 and 0.34, respectively). However, collection of semen with the penis retained in the preputial cavity resulted in greater odds of leukospermia relative to semen collection with the penis extended (OR 4.1; 95% CI 2.9, 5.9; P<0.001), presumably from contamination of the semen sample with preputial leukocytes. For ram BSE, therefore, semen collection with the penis manually extended from the sheath is recommended to limit leukocyte contamination of the sample.

Rapid serum agglutination and agar gel immunodiffusion tests associated to clinical signs in rams experimentally infected with Brucella ovis

The purpose of this study was to evaluate the agar gel immunodiffusion and the rapid serum agglutination tests associated to clinical signs in rams experimentally infected with Brucella ovis. The serological profile during the 12 months of infection showed a large fluctuation of antibodies that favors the failure in the diagnostic. The evaluation of tests after the experimental infection allowed to suggest that none of the tests were able to detect the infection throughout the period of study. The study reinforces the importance of considering the clinical signs to support the diagnosis of Brucella ovis infection in rams.

Development and evaluation of a species-specific PCR assay for the detection of Brucella ovis infection in rams

Brucella ovis infection is a major cause of epididymitis and infertility in rams, resulting in reproductive failure and significant economic losses worldwide. The goal of this study was to develop a PCR test targeting specific B. ovis genomic sequences. Specific primer pairs were designed targeting 12 of those ORFs. Samples of blood, serum, semen, urine, and preputial wash were collected from experimentally infected rams ( n = 9) every other week up to 180 days post infection (dpi), when tissue samples were obtained. Blood, serum, semen, urine, and preputial wash samples were obtained, in weekly intervals for 1 month, from eight rams belonging to a B. ovis-free flock. Semen samples were also obtained from rams belonging to naturally infected flocks ( n = 40). The limit of detection of this PCR protocol was 100, 10, and 1 CFU/mL for semen, urine and prepucial wash samples, respectively. Sensitivity and specificity values obtained with this PCR method were similar to that of bacteriology when evaluating biological samples. Agreement between PCR and bacteriology results was greater than 90%. These results clearly indicate that this species-specific PCR method is highly efficient for the diagnosis of B. ovis infection in semen, urine, preputial wash and tissue samples from infected rams.

Evaluation of particulate acellular vaccines against Brucella ovis infection in rams

The attenuated Brucella melitensis Rev 1 vaccine, used against brucellosis infection, interferes with serological diagnosis tests, may induce abortions in pregnant animals, and may infect humans. In order to overcome these drawbacks, we developed acellular vaccines based on a Brucella ovis antigenic complex (HS) containing outer membrane proteins and R-LPS entrapped in poly(anhydride) conventional and mannosylated nanoparticles (NP-HS and MAN-NP-HS) or in poly(ɛ-caprolactone) microparticles (HS-PEC) as antigen delivery systems and immunoadjuvants. Brucellosis free rams were vaccinated subcutaneously with a single dose of particles containing 3 mg of HS, and challenged 6 months thereafter. Protection was evaluated by clinical, bacteriological and serological examinations, in comparison with non-vaccinated control rams. HS-PEC vaccine induced protection (7 out of 13 animals were infected) equivalent to that induced by the reference Rev 1 vaccine (8/14). In contrast, animals immunized with NP-HS were not protected, showing similar results to that obtained in the control unvaccinated rams. Furthermore HS-PEC vaccine did not interfere against B. melitensis serodiagnostic tests. In summary, HS-PEC microparticles could be used as a safe and effective vaccine against brucellosis in rams.

The polymeric antigen BLSOmp31 confers protection against Brucella ovis infection in rams

We have engineered the polymeric vaccine BLSOmp31 by decorating the highly immunogenic and decameric Brucella lumazine synthase with an exposed loop of the Brucella outer membrane protein Omp31. In the present study, we have immunized different groups of rams with the recombinant chimera rBLSOmp31 in two different adjuvants (Incomplete Freund Adjuvant—IFA and QUIL A) and with the plasmid pCIBLSOmp31 administered either by i.m. injection alone or by using electroporation. In addition, we have used a heterologous prime-boost strategy consisting of repeated pCIBLSOmp31 electroporation priming followed by a single protein boost. Both, chimera rBLSOmp31 in IFA and the prime-boost strategy induced the highest IgG specific antibodies with bacteriolytic activity. While electroporation-enhanced humoral immune responses as compared to pCIBLSOmp31 injection alone, the highest levels of specific IFN-γ and protection against bacterial challenge were achieved with prime-boost (76%) and chimera rBLSOmp31 in IFA (63%). Taken together these results strongly support the usefulness of the chimera BLSOmp31 as a vaccine against Brucella ovis in ovine brucellosis.

Association of Maedi Visna virus with Brucella ovis infection in rams.

Maedi Visna Virus (MVV) is the etiological agent of a systemic disease of sheep, which causes lesions in lungs, the central nervous system, joints, and mammary glands. It has been speculated that the association with Brucella ovis may lead to the venereal shedding of the virus. In this work, samples of epididymis from ten rams positive for MVV and infected experimentally with Brucella ovis, were subjected to liquid-phase PCR, immunohistochemistry (IHC) and in situ PCR tests, aimed at identifying the pathogens in a tissue context. IHC was carried out using a monoclonal antibody raised against p28 MVV protein and a polyclonal antibody to B. ovis. Liquid phase- and in situ PCR were designed to amplify a portion of MVV proviral DNA Pol sequence. In the animals showing B. ovis-related histopathological changes, IHC clearly demonstrated a positivity for B. ovis and MVV in interstitial and epithelial ductal cells. In situ PCR assessed the presence of MVV proviral DNA in macrophages and elements inside the epithelium. The unaffected and reagent control samples constantly gave negative results. Taken together, these data demonstrate that MVV may affect ovine epididymis, apparently taking advantage of the concurrent infection by B. ovis. The tropism of MVV for the epididymal epithelial cells, may be responsible for its excretion with the semen.

Characterization of possible correlates of protective response against Brucella ovis infection in rams immunized with the B. melitensis Rev 1 vaccine

Vaccination with the live attenuated Brucella melitensis Rev 1 vaccine is used to control ovine brucellosis caused by Brucella ovis in sheep. The objective of this study was to identify possible correlates of protective response to B. ovis infection through the characterization by microarray hybridization and real-time RT-PCR of inflammatory and immune response genes differentially expressed in rams previously immunized with B. melitensis Rev 1 and experimentally challenged with B. ovis. Gene expression profiles were compared before and after challenge with B. ovis between rams protected and those vaccinated but found infected after challenge. The TLR10, Bak and ANXI genes were expressed at higher levels in vaccinated and protected rams. These genes provide possible correlates of protective response to B. ovis infection in rams immunized with the B. melitensis Rev 1 vaccine.

Inquérito soro-epidemiológico e fatores de risco associados à infecção por Brucella ovis em carneiros deslanados do semi-árido da Paraíba

Foi realizado um levantamento soro-epidemiológico da brucelose por Brucella ovis em reprodutores ovinos deslanados na Paraíba com os objetivos de verificar a prevalência e distribuição da infecção por B. ovis em propriedades rurais e analisar os possíveis fatores de risco associados à infecção. Foram investigadas 283 propriedades criadoras de ovinos, das quais foram colhidas 498 amostras de soro sanguíneo de carneiros, a partir de oito meses de idade, nas mesorregiões do Sertão Paraibano e Borborema. Todos os soros foram examinados pela IDGA (teste de triagem) e RFC (teste confirmatório). De acordo com as análises, 8,59% (I.C.95% = 5,83%-12,48%) das propriedades apresentaram evidência sorológica de infecção por B. ovis, com uma prevalência de 5,57% (I.C.95% = 3,86%-7,97%) de reprodutores sororreagentes. Nas propriedades que higienizavam suas instalações com maior freqüência, a soropositividade foi estatisticamente inferior (p &lt; 0,05).

Evaluation of a PCR test for the diagnosis of Brucella ovis infection in semen samples from rams

The sensitivity and specificity of a PCR assay with primers derived from the insertion sequence IS 6501 was compared with that of bacteriological culture and serological tests for the diagnosis of Brucella ovis infection in rams. No amplifications were detected with DNAs from the strains phylogenetically related to Brucella and from the seven bacterial species considered as the main etiologic agents of epididymitis in rams. In addition, the specificity of the PCR was 100% when testing semen samples from Brucella-free rams. The comparison of the semen culture and PCR results from 192 semen samples tested, showed a proportion of agreement of 0.91 between both tests. The PCR-based test described has sensitivity similar to that of semen culture and could be used as a complementary test for the direct diagnosis of Brucella ovis in semen samples of rams.

Single-step purification and evaluation of recombinant BP26 protein for serological diagnosis of Brucellaovis infection in rams

To investigate the value of the BP26 protein in the serological diagnosis of ovine brucellosis caused by Brucella ovis, recombinant BP26 protein was produced in Echerichia coli and purified for use in an indirect enzyme-linked immunosorbent assay (I-ELISA). The majority of the recombinant protein was recovered from the supernatant of sonicated recombinant E. coli cells in a soluble form. This facilitated the purification of the recombinant BP26 protein which was achieved by using ion-exchange chromatography. After one step of purification, the purity of the recombinant BP26 protein was analyzed by using SDS-PAGE, Coomassie blue staining, and Western blot with a monoclonal antibody (MAb) directed against the BP26 protein. The degree of purity appeared satisfactory so that it could be directly used in I-ELISA. Although the recombinant BP26-ELISA appeared less useful than I-ELISA using the B. ovis hot saline (HS) extract as antigen, the high number of sera from B. ovis infected rams found positive (90%) in the recombinant BP26-I-ELISA indicated that the BP26 protein may be an additional suitable antigen for serological diagnosis of B. ovis infection in rams.

Performance of an enzyme-linked immunosorbent assay for the diagnosis of Brucella ovis infection in rams

Aim. To describe the performance characteristics (sensitivity and specificity) of an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Brucella ovis infection in rams. Methods. Sera from a negative (n = 2535) and a positive (n = 589) reference population were tested in an ELISA for anti-B. ovis antibodies and cut-off values calculated from the raw, log10-transformed and fitted data. Statistical methods were used to fit curves to the frequency distribution of the data and receiver-operated characteristics analysis used to optimise the cut-off values. Results. Analysis of the frequency distribution of the positive ELISA values suggested a normal distribution of the data, whereas, in the case of the negative population, a Pearson type IV curve appeared to best fit the data. The cutoff values calculated as the mean plus 1.96 standard deviations (s.d.) from the raw, log-transformed and fitted ELISA data did not differ markedly. The differences were much greater at the mean plus 3.09 s.d. cut-off, with the cut-off value calculated from the log-transformed data giving a much better estimate of specificity. Optimisation (minimisation of classification error) of the cut-off calculated from the fitted curves suggested varying cut-off values, depending on the prevalence of B. ovis infection. Discussion. Calculation of cut-off values from curves that were fitted from the observed data give more accurate estimations of the performance characteristics of an assay than traditional calculations from observed values. They also allow the calculation of optimal cut-off values taking into account the prevalence of B. ovis infection and give additional information about the performance of the assay at cut-off values varied according to the epidemiological situation.

Diagnosis and epidemiology of Brucella ovis infection in rams

The relationship between clinical, serological and bacteriological diagnostic criteria have been determined in Brucella ovis culture-positive and Brucella free control rams and also in 110 field rams belonging to 13 different flocks, in which an epidemiological survey was conducted. In control rams, a direct relationship was found among the existence of positive bacteriological results and the presence of a positive serological response in the gel diffusion (GD) and complement fixation (CF) tests for B. ovis. In field rams, the presence of macroscopic testicular alterations (MTA) caused by B. ovis was correlated with bacteriological and serological results. The percentage of reactors and the percentage of rams showing MTA was not related to the size of flocks nor to the ratios of ram/ewes. The prevalence of B. ovis infection was significantly higher in rams from imported breeds than in those from local ones. Moreover, the younger and the oldest rams showed the lowest prevalence of B. ovis infection, but the age of rams did not influence the prevalence of MTA not caused by B. ovis.

Comparison of subcutaneous and conjunctival routes of Rev 1 vaccination for the prophylaxis of Brucella ovis infection in rams

The serological response and protection conferred against Brucella ovis by the Rev 1 vaccine was evaluated in both adult (experiment 1) and young rams (experiment 2) vaccinated either subcutaneously or conjunctivally. In experiment 1 the Rev 1 vaccine protected 55.5 per cent and 100 per cent, respectively, of subcutaneously and conjunctivally vaccinated rams against three consecutive challenges that infected 100 per cent of unvaccinated controls. In experiment 2, Rev 1 protected 100 per cent of rams vaccinated subcutaneously and 70 per cent of those vaccinated conjunctivally against a challenge dose able to infect all the unvaccinated controls. The serological response after vaccination was significantly lower in rams vaccinated conjunctivally than in those vaccinated subcutaneously.

The Complement Fixation Test forBrucella Ovis

Abstract Sir:- A project to investigate the efficacy of the complement fixation test (CFT) for the diagnosis of Brucella ovis infection in rams has recently been completed. The investigation was undertaken by field and laboratory staff of the Division of Animal Health and several practitioners. The results of CFT's done with 3 antigens by waim and cold fixation methods (WCFT and CCFT) were correlated with results of cultures done of semen from the same rams. It is our aim to publish a full report in this Journal at a later date. In view of the widespread interest in the subject some of the important findings are summarised below: • Semen and blood samples were collected from 541 rams in 40 flocks. • The type of antigen which has been used for a number of years in New Zealand was unsatisfactory. The CCFT using this antigen had a sensitivity of only 85% in 124 rams which gave positive semen cultures. These rams came from 16 infected flocks. • The CCFT using the best of the 3 antigens proved to be a remarkab...

SENSITIVITY AND SPECIFICITY OF TWO MICROTITRE COMPLEMENT FIXATION TESTS FOR THE DIAGNOSIS OF BRUCELLA OVIS INFECTION IN RAMS

An investigation was made into microtitre complement fixation test (CFT) procedures suitable for the serological diagnosis of naturally occurring Brucella ovis infection in rams. A procedure similar to the Australian standard procedure for bovine brucellosis was unsatisfactory when applied to sheep. Modification of the procedure by use of an initial serum and anticomplementary control dilution of 1:8 and increasing complement fixation time to 60 minutes at 37 degrees C, greatly improved the efficiency of the test. A sensitivity of 100% was recorded for 59 serums from known infected rams and a specificity of 99.9% for 1593 serums from rams known or believed to be free of infection. Some aspects of applying CF tests to sheep serums are discussed.

Characteristics of semen changes during Brucella ovis infection in rams.

The effect of brucellosis on semen quality in rams was studied by means of artificially infecting five rams with a strain of Brucella ovis isolated in Kenya. Infection resulted in reduced semen quality including reduced total sperm output, poor motility and a high percentage of morphological abnormalities. Variation in semen quality both between and within rams appeared to be related to progress of the disease in the epididymis as well as to the distribution and severity of lesions in the reproductive tract.