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Ovine brucellosis caused by Brucella ovis is considered one of the most important reproductive diseases of rams worldwide. This study aimed to characterize the kinetics of infection of a ΔabcAB B. ovis mutant strain in rams. Twelve 1-year-old crossbred rams were used. Six rams were challenged with 2mL of a suspension containing 1.2×109 CFU/mL of B. ovis strain ATCC25840 (wild type) by intraprepucial inoculation and additional 50μL in each conjunctival sac of a suspension containing 1.2×1010 CFU/mL of the same strain. The other six rams were challenged with an equivalent number of CFU of the mutant strain ΔabcAB B. ovis through the same routes. Serum samples for serology and semen and urine samples for bacteriologic culture and PCR were collected weekly during 24 weeks. At 24 weeks post infection, tissue samples were collected for bacteriologic culture and PCR. All rams inoculated with wild type or the ΔabcAB strain seroconverted at the fourth week post infection, remaining positive up to the 16th week post infection. PCR and bacteriology demonstrated that only rams inoculated with the wild type strain shed the organism in semen and urine. Lymphocytes from rams inoculated with wild type or ΔabcAB B. ovis had significantly higher proliferation in response to B. ovis antigens when compared with unstimulated controls. Tissue bacteriology and PCR detected B. ovis in all rams challenged with the wild type strain, whereas only one ΔabcAB-infected ram had a positive iliac lymph node sample by PCR. [ABSTRACT FROM AUTHOR]

The article discusses as study that investigated the pathogenesis of Brucella ovis infection in bighorn sheep (BHS). Findings show that BHS experimentally infected with B. ovis became antibody and culture positive, and showed clinical signs of infection including abortion and epididymal and testicular swelling. BHS lesions were consistent with those observed in domestic sheep.

Summary: This paper describes a study on Brucella ovis infection of sheep flocks in southern Serbia. Using serological testing, positive reactions were confirmed in 67 (29.8%) and suspicion in 31 (13.8%) out of the total 225 tested sheep sera samples. Rams originated from 113 flocks with 4751 sheep from 28 settlements in the Pirot region of southern Serbia. Pathological changes indicative of Brucella ovis infection were confirmed by macroscopic examination in the testes of 7 (58.3%) out of 12 examined rams. In 7 (58.7%) animals, unilateral epididymitis with pronounced hypertrophy of the tail, body and head of epididymis was confirmed. Using bacteriological and molecular techniques, the presence of B. ovis was confirmed in the samples (testes, epididymis, lymph nodes) in 11 (91.7%) out of 12 rams. The clinical manifestation of ovine epididymitis arose following the procurement of rams and mating season. The disease was more pronounced in flocks in which it occurred for the first time. On the basis of the results presented here, it can be concluded that B. ovis infection causes substantial economic losses in sheep production, and is manifested in reduced conception (24.85%), miscarriages (5.38%), reduced number of lambs (0.74 lambs per ewe) and increased perinatal mortality (7.78%). [ABSTRACT FROM AUTHOR]

Brucella ovis is an important cause of epididymitis in rams, which results in impaired fertility and economic losses. This study demonstrated the role of TLR during the acute phase of infection in the mouse model. C57BL/6 wild type and TLR2-/-, TLR4-/-, TLR9-/-, and MyD88-/- mice were infected with B. ovis and bacteriology, histopathology, and pro-inflammatory gene expression were evaluated at seven days post-infection. MyD88-/- mice had higher bacterial loads in the spleen when compared to wild type mice. This enhanced susceptibility was associated with decreased inflammatory response in the liver. TLR9-/- mice also had higher bacterial loads when compared to wild type mice, but, surprisingly, they developed stronger inflammatory response. TLR2-/- and TLR4-/- mice were as susceptible as wild type mice to B. ovis infection. Therefore, MyD88 and TLR9 are required for controlling B. ovis multiplication during the early stages of infection. [ABSTRACT FROM AUTHOR]

Abstract: Objective: To design a duplex PCR for rapid and simultaneous detection of Brucella species. in human blood samples. Methods: Fifty-two peripheral bloods samples were collected from suspicious patients with brucellosis. Following DNA extraction, PCR assay were performed, using three primers that could simultaneously identify and differentiate three major species of pathogenic Brucella in humans and animals. Results: Of the 52 peripheral bloods samples tested, 25 sample (48%) showed positive reactions in PCR. Twelve samples were positive for Brucella abortus 39 (B. abortus 39) (23%), 13 for Brucella melitensis 39 (B. melitensis 39) (25%) and 0 for Brucella ovis 39 (B. ovis 39) (0%). Conclusions: This work de=monstrates that in case where specific primers were utilized, duplex PCR has proved to be a simple, fast, and relatively inexpensive method for simultaneous detection of important species of Brucella in clinical samples. [Copyright &y& Elsevier]

Background: Infectious ovine epididymitis results in substantial economic losses worldwide due to reproductive failure and culling of breeders. The most common causative agents of these infections are Brucella ovis, Actinobacillus seminis, and Histophilus somni. The aim of this study was to develop a multiplex PCR assay for simultaneous detection of Brucella ovis, Actinobacillus seminis, and Histophilus somni with species-specific primers applied to biological samples for molecular diagnosis of these infections. Results: The multiplex assay was capable of detecting B. ovis, A. seminis, and H. somni DNA simultaneously from genomic bacterial DNA samples and pool of semen samples from experimentally infected rams. The method was highly specific since it did not amplify DNA from other bacterial species that can potentially cause epididymitis in rams as well as species phylogenetically related to B. ovis. All negative control samples were negative in PCR multiplex assay. Urine can be used as an alternative to semen samples. Conclusions: The species-specific multiplex PCR assay developed in this study can be successfully used for the detection of three of the most common bacterial causes of ovine epididymitis. [ABSTRACT FROM AUTHOR]

The present report describes an atypical Brucella ovis strain (Bo10) isolated from the epididymis and testis of an infected ram. Macroscopic and microscopic lesions characteristic for the infection, including positive Brucella immunostaining, were observed within lesions in the genital organs. Compared to other isolates, strain Bo10 required an additional day (a total of 96 hr) of incubation to form visible colonies, showed a distinct carbon source utilization profile, agglutinated only weakly with rough (R) serum, but showed a high capacity for autoagglutination. Isolate Bo10 failed to produce the 1,071-bp fragment in the outer membrane protein (omp) 31 gene–based part of the “Bruce-ladder” multiplex polymerase chain reaction system but did produce a 1,915-bp amplicon, thus presenting a profile similar to Brucella abortus. Sequence analysis of the 1,915-bp fragment revealed an 842-bp long insertion sequence (IS)711 transposon element inserted into the promoter region of the omp31 gene, immediately upstream from the ribosome binding site (-10 box/Pribnow box). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of a whole-cell lysate showed the absence in Bo10 of the approximately 31-kDa protein fragment associated with omp31. The results demonstrate a natural inactivation of omp31 and, consequently, the absence of the Omp31 protein in this B. ovis isolate. The novel location of IS711 within the genome of a naturally occurring B. ovis strain supports the hypothesis that IS711 could be an active transposon in this Brucella species. [ABSTRACT FROM PUBLISHER]

The article discusses research on the use of species-specific nested polymerase chain reaction (PCR) diagnostic tool in detecting Brucella ovis infection in semen and urine of rams. In addition to being contagious, it mentions how ovine brucellosis tend to lead to reproductive failure in infected rams. Brief details on the sample collection and experimentation process performed, frequencies of Brucella ovis positivity detected, and increased sensitivity of nested PCR observed are given.

Abstract: The ovine brucellosis caused by Brucella ovis has tropism for reproductive tissues but until now the mechanism of bacterial persistence is not understood. Cytokine expression profiles were studied for 8 months in rams after being experimentally infected with the rough virulent strain of B. ovis (R-B. ovis) to study the pathogenesis of B. ovis and immune mechanism possibly associated to bacteria tropism and persistence. The messenger RNA (mRNA) expression levels of interleukin-1α (IL-1α), IL-1β, IL-6, IL-10, IL-12, interferon-γ (INF-γ) and tumour necrosis factor-α (TNF-α) cytokines were quantified by real-time quantitative RT-PCR (qRT-PCR) in reproductive tissues (epididymus, testicles, ampolae, vesicular glands and bulbourethral glands), and non-reproductive (liver, spleen and kidneys) tissues at 30, 60, 120 and 240 days post infection (dpi). During the acute phase of infection at 30dpi, the host immune response was most notable demonstrating an up-regulation of several cytokines in reproductive tissues, including the epididymus (IL-6, IL-1β and IL-1α), testicles (INF-γ and IL-12), bulbourethral glands (IL-6 and TNF-α) and ampolae (INF-γ, IL-10, IL-1β and IL-1α). During the development of infection, cytokine gene expression levels decreased, providing evidence of immunosuppression and evidence of immune evasion that favoured persistence of chronic R-B. ovis infection. During the chronic phase of R-B. ovis infection (120 and 240dpi), cytokine production was down-regulated in the epididymus (IL-1β and IL-1α), testicles (INF-γ and IL-12), and ampolae (INF-γ, IL-10, IL-1β and IL-1α), with the exception of the bulbourethral glands (IL-6 and TNF-α) and epididymus (IL-6); in these tissues, R-B. ovis infection resulted in up-regulation of the pro-inflammatory cytokine IL-6. Herein, we report cytokine expression profiles in tissues of rams experimentally infected with the rough strain of B. ovis, which are associated with bacterial persistence and macrophage activation. [Copyright &y& Elsevier]

Epididymitis due to Brucella ovis infection lead to a disease with great importance in Mexican sheep industry. In the affected rams produces a low productivity due to a decreased fertility. The objective of the study was to determine the relevance of different possible risk factors (production system, density of sires, population of ewes, ewes:ram ratio, mating system and sires breed) upon B. ovis prevalence in Zacatecas, México. A sample of 544 rams was obtained in 153 flocks from four production systems. The serological test was determined by double immunodiffusion. The positive sampled rams were 18.6 % (101/544) and 10.5 % (16/153) of the sampled flocks had at least one positive ram. The semi-intensive production system showed a major prevalence (P<0.05) with 86.1 % (87/101) of the positive rams, the extensive system with 11.9 % (12/101), the backyard with 2.0 % (2/101) and the intensive did not register positives. The prevalence of B. ovis was higher in larger flocks, with the largest number of ewes and rams. Katahdin rams showed a major prevalence (30.8 %) (24/78) than Rambouillet (14.0 %) (18/129), Dorper (13.8 %) (31/224) and Suffolk rams (13.8 %) (8/58); (P<0.05). The prevalence was associated more with the production system used, than the ratio ewes:ram or mating system. The results suggest that the amount of sires within each flock is the most important factor in the serological prevalence of B. ovis (OR= 17.38, 95% IC 7.76-38.94), although this potential factor could be subject to the production system. [ABSTRACT FROM AUTHOR]

Abstract: Brucella spp. are gram-negative intracellular bacterial pathogens that cause chronic infections. Brucella virulence factors include a type IV secretion system (T4SS) and its lipopolysaccharide (LPS), which are essential for persistence. However, the role of the virB-encoded T4SS has not been investigated in naturally rough Brucella species such as Brucella ovis. In this study, male 6-week old BALBc mice were infected with B. ovis, Brucella abortus, and their respective ΔvirB2 mutant strains. During early infection, B. ovis and B. abortus wild type strains were similarly recovered from spleen. Interestingly, in contrast to ΔvirB2 B. abortus that was recovered at similar levels when compared to the wild type strain, the ΔvirB2 B. ovis was markedly attenuated as early as 24h post infection (hpi). The ΔvirB2 B. ovis was unable to survive and multiply in murine peritoneal macrophages and extracellularly within the peritoneal cavity at 12 and 24hpi with lower splenic colonization than the parental strain at 6, 12 and 24hpi. In contrast, wild type B. abortus and ΔvirB2 B. abortus had a similar kinetics of infection in this model. As expected, the T4SS was essential for intracellular replication of smooth and rough strains in RAW macrophages at 48hpi. These results suggest that T4SS is important for survival of B. ovis in murine model, and that a T4SS deficient B. ovis strain is cleared at earlier stages of infection when compared to a similar B. abortus mutant. [Copyright &y& Elsevier]

The article discusses the reliability of a method to diagnose Brucella (B.) ovis infection in urine samples of naturally infected rams using serology and polymerase chain reaction (PCR). Results of the study showed that despite low agreement between serology and PCR, the method can be used as a suitable complementary diagnostic tool for identifying B. ovis infection. The method also allows identification of serologically negative rams shedding the organism in the environment.

The article discusses a study regarding the pathology of the reproductive tract of red deer stags with active Brucella ovis infection and in stags in which B. ovis infection had resolved. The researchers examined grossly and by histopathology the epididymides and accessory sex glands of 23 slaughtered red deer stags of varying history. Results showed that only a small percentage of red deer stags with B. ovis infection develop lesions of epididymitis that can be detected by scrotal palpation.

By complement fixation test 230 blood samples from goats and he goats were examined for presence of antibodies against Brucella ovis. 134 blood samples were from goats (23 of them were from slipping goats) and 96- from he goats. The animals come from private farms in the Plovdiv and Pazardzhik regions (Southern Bulgaria). 10.87% of all tested blood samples contain antibodies against Brucella ovis. Differences in the percentage of the positive reagents of different goat categories were found. The highest percentage was among slipping goats (39.13% towards to 11.71% among the healthy goats and 3.13% among he goats). It can be concluded that goats play an important role in the epidemiology of the disease. The greatest risk is slipping goats bred together with sheep flocks. [ABSTRACT FROM AUTHOR]

Abstract: Brucella ovis is considered the most important infectious cause of reproductive disorders in sheep. The disease is characterized by epididymitis, subfertility and infertility in rams. B. ovis occasionally results in abortion in ewes, as well. The aim of this study was to evaluate kinetic changes in the reproductive organs of rams experimentally infected with B. ovis. Nine rams were experimentally inoculated intrapreputially with 2mL of a suspension containing 1.2×109 CFU (colony-forming units)/mL of B. ovis (strain ATCC25840). In addition, 50μL of a suspension containing 1.2×1010 CFU/mL of the same B. ovis strain was inoculated into each conjunctival sac, resulting in 3.6×109 CFU total per ram. Six of nine infected rams had developed clinical changes in the tail of the epididymis at 30 days post-infection (dpi), but these changes regressed in 50% of these rams. Ultrasound demonstrated an increase in the area of the tail of the epididymis (P <0.001), reduction in the area of the testes (P <0.001), and an increased length and width of the seminal vesicles (P <0.001) during the course of infection. A sperm granuloma was diagnosed on the basis of ultrasonography findings. Microscopically, there was epididymitis, testicular degeneration, and seminal vesiculitis. Inflammatory cells were detected in the semen even before the development of epididymitis. Moreover, inflammatory cells were also found in the semen of asymptomatic rams, indicating that the presence of leukocytes in the ejaculate is a valuable method for screening potential carriers of infections in the genital tract. [Copyright &y& Elsevier]

The article evaluates three serological tests using commercially available antigens for the diagnosis of Brucella ovis infection in experimentally infected rams. Serum samples were obtained from sheep of different ages belonging to a B. ovis-free flock, which was located in Belo Horizonte, Brazil. Agar gel immunodiffusion antigens were sensitive and specific for the serological diagnosis, whereas complement fixation resulted in lower sensitivity after the acute phase of infection.

Abstract: The objective was to determine the effectiveness of various antimicrobial agents added to semen extender for inactivation of B. ovis or A. seminis in ovine semen after cryopreservation. In Experiment 1, 20 ejaculates from a crossbred ram infected with B. ovis were cryopreserved in Tris-based extenders with various antimicrobial agents: (I) control without antibiotics, (II) with penicillin and streptomycin (1000 IU/mL and 1 mg/mL, respectively), (III) lincomycin (0.15 mg/mL), (IV) sulphadiazine (0.60 mg/mL), and (V) gentamicin sulphate (0.25 mg/mL). Semen was stored in 0.25 mL straws at a final concentration of 150 × 106 spermatozoa/mL. After thawing (37 °C for 30 s), sperm total motility (TM), sperm morphology, integrity of sperm membranes, and bacterial growth were assessed. In Experiment 2, six B. ovis isolates were separately inoculated into aliquots of a fresh ejaculate from a B. ovis-free ram. Mock inoculated semen was processed for cryopreservation using the five extenders described above, and bacteriologically evaluated after thawing. In Experiment 3, sensitivity of A. seminis to the same antimicrobial agents was evaluated by inoculating an ejaculate from an A. seminis and B. ovis-free ram. There were no significant differences among treatments in post-thawing sperm parameters. B. ovis was isolated from 100% (20/20), 0% (0/20), 95% (19/20), 100% (20/20), and 5% (1/20) of semen samples diluted in tris-based extender of untreated (I) and treated semen samples with antimicrobial agents II, III, IV, and V, respectively. Frequencies of isolation from samples treated with antimicrobial agent II and V were significantly lower than untreated ones (P < 0.05). There were no significant differences in the profile of antimicrobial resistance of different B. ovis isolates. A. seminis had a similar sensitivity to the antimicrobial agents. We concluded that addition of a combination of penicillin and streptomycin or gentamicin alone to ram semen cryo-extenders inactivated B. ovis and A. seminis. [Copyright &y& Elsevier]

Abstract: 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 1CFU/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. [ABSTRACT FROM AUTHOR]

Abstract: The establishment of infection by Brucella ovis and Brucella canis in J774.A1 macrophages was found to be dependent upon cholesterol and ganglioside GM1, two components of lipid rafts. This process also required a class A scavenger receptor of macrophages, and was not inhibited by smooth and rough lipopolysaccharides from Brucella spp. In response to infection, both bacteria induced a weak degree of macrophage activation. These results demonstrate that B. ovis and B. canis use cell surface receptors common to smooth Brucella spp. for macrophage infection, thus limiting macrophage activation and favouring intracellular multiplication and/or the survival of both bacteria. [Copyright &y& Elsevier]

Brucellosis caused by Brucella ovis is one of the most important infectious diseases of sheep. The aim of this study was to determine the presence of genes both inside and outside the specific B. ovis pathogenicity island 1 (BOPI-1) in a large collection of field isolates of B. ovis and other Brucella spp. from Argentina. The BOV_A0500 gene from B. ovis BOPI-1 was identified in all 104 B. ovis isolates studied. The BOPI-1 complete sequence was found to be conserved in 10 B. ovis strains from the collection, for which whole genome sequencing was performed. The BOV_0198 gene, which is outside BOPI-1 and considered exclusive to B. ovis , showed 90–100% identity with genomic regions of B. ovis , B. melitensis , B. abortus , B. canis , B. suis , B. microti , B. ceti and B. pinnipedialis . The results demonstrate that BOPI-1 is the only exclusive genetic region of B. ovis and marine Brucella spp. and that it is highly conserved in B. ovis field isolates from Argentina. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Infection of sheep with Brucella ovis results in ovine brucellosis, a disease characterized by infertility in rams, abortion in ewes and increased perinatal mortality in lambs. During the course of the infection both the ovine immune response and host cell gene expression are modified. The objective of this research was to conduct a preliminary characterization of differential gene expression in rams experimentally infected with B. ovis by microarray hybridization and real-time RT-PCR. Of the 600 ruminant inflammatory and immune response genes that were analyzed in the microarray, 20 and 14 genes displayed an expression fold change >1.75 with a P-value <0.05 at 15 and 60 days post-challenge (dpc), respectively. Of these genes, 16 were upregulated and 4 were downregulated in infected rams at 15dpc. At 60dpc, 11 and 3 genes were up- and down-regulated in infected rams, respectively. Only four genes, desmoglein, epithelial sodium channel, alpha subunit (ENaC-alpha), interleukin 18 binding protein (IL18BP) and macrophage migration inhibition factor (MIF) were found upregulated in infected rams at both 15 and 60dpc. The analysis of differentially expressed genes demonstrated activation of inflammatory and innate immune pathways in infected animals. B. ovis infection also resulted in upregulation of genes involved in phagocytosis and downregulation of protective host defense mechanisms, both of which may contribute to the chronicity of B. ovis infection. The gene expression profiles differed between rams with severe and moderate B. ovis infection. This is the first analysis of differential gene expression in rough brucellae and particularly in B. ovis-infected rams. The characterization of the genes and their expression profiles in response to B. ovis infection further contributes to our understanding of the molecular mechanisms of infection and the pathogenesis of brucellosis. [Copyright &y& Elsevier]

Abstract: Brucella melitensis Rev.1 is the most effective vaccine against B. ovis infection in sheep but induces antibodies interfering with B. melitensis diagnosis. Brucella BP26 and Omp31 proteins are differential diagnostic antigens. Single or double bp26 and omp31 Rev.1 deletion mutants have been proven effective against B. melitensis in sheep. Here, the CGV26 (deleted in bp26 gene) and CGV2631 (deleted in both bp26 and omp31 genes) mutants have been tested for efficacy against B. ovis in rams. Either inoculated subcutaneously or conjunctivally, both mutants conferred significant protection against B. ovis. The protection induced by CGV26 was similar to that of Rev.1 but significantly higher than that conferred by CGV2631. In conclusion, the CGV26 mutant, in association with the adequate diagnostic strategy, could be a useful alternative to Rev.1 for sheep vaccination against B. ovis infections in those countries performing simultaneously B. melitensis and B. ovis eradication campaigns. [Copyright &y& Elsevier]

Abstract: The role of the outer membrane proteins of the Omp25/Omp31 family in invasiveness and intracellular survival of virulent B. ovis in phagocytes was analyzed. The absence of Omp25d or Omp22 in B. ovis abolished its invasive capacity in HeLa cells and reduced it in J774.A1 cells. Additionally, in J774.A1 cells, the Δomp25d mutant was unable to multiply, whereas the Δomp22 mutant was cleared at 24h post-infection. These findings demonstrate that Omp25d and Omp22 are essential for the invasion and survival of B. ovis inside host cells, and justify the strong attenuation in virulence of the Δomp25d and Δomp22 mutants. [Copyright &y& Elsevier]

Research was conducted during 2000 - 2004 on a number of 60.986 blood samples from rammers, 530 pairs of testicles for bacteriological examination and 76 testicle samples processed for histopathology. Serologic and morphopathologic investigations were performed in the Veterinary State Laboratory Cluj (LSVS) and the bacteriology department in the Institute of Diagnosis and Animal Health (IDSA) Bucharest. Following the examination by serological RFC resulted a number of 4200 positive tests from 60.986 samples tested. Thus, the percentage of sickness caused by infectious epididimitis in rammers is 6.88%. Investigations by bacteriological examination, conducted at IDSA Bucharest on a number of 530 samples testicles (representing evidence and aries), revealed the presence of infectious epididimitis of rammers at 77.36%, 22.64% of the samples were negative. Having established a hierarchy of isolated germs involved in the aetiology of epididimitis we observed that 62.02% of cases are caused by Brucella ovis, 6.60% of cases by B. ovis in association with Arcanobacterium pyogenes and 7,74% by Arcanobacterium pyogenes only. Histological examination was conducted on a number of 76 testicles samples, of which 46 samples (60.52%) were with pathologic changes that can be attributed to infectious orchiepididimitis of rammers. Correlation is positive between histological examination and bacteriological examination, from a proportion of 100% testing positive for pathology, we managed to isolate Brucella ovis from 83.33%. Between necropsic exam results and serological examination by RFC, the correlation is negative, only 4.17% of the proportion of 100% diagnosed serological, presents pathological lesions of epididimitis. This type of relationship is valid also between serological examination by RFC and palpatory clinical examination. Considering RFC as a standard method to diagnose infectious epididimitis and comparing results with those of bacteriological examination carried out on the same samples that were diagnosed positive with RFC, we found that between these two methods there is a positive correlation. If RFC detected positive samples in the proportion of 100%, bacteriological examination detected the evidence of infected germs in a proportion of 77.36%, of which the Brucella ovis is 69.62%. Following bacteriological tests carried out on the same samples diagnosed as positive by serological examination (RFC), we find that the reaction of complement fixation although very sensitive, does not meet all the requirements, especially in regard of specificity, detecting also other bacterial antigens similar to B . ovis. For these reasons, in order to avoid false positive reactions from rammers with high economic and biologic value, testing is required to be made by at least two different methods (RFC and ELISA or RFC and bacteriological examination of semen). [ABSTRACT FROM AUTHOR]

Because some batch-to-batch variation in the preparation of rough lipopolysaccharide (RLPS) from Brucella ovis has been experienced, several protocols were tested to establish the most reliable method for detection of antibody in indirect enzyme immunoassay. An early version of the assay gave a performance index (PI=sum of optimum percent sensitivity and percent specificity, determined by receiver operator characteristic analysis) of 198.6. This assay used RLPS from B. ovis as the antigen and a monoclonal antibody specific for bovine IgG1 heavy chain-enzyme conjugate for detection. This was not repeatable using other batches of antigen. Newer versions of the assay generally had decreased sensitivity values, giving PIs of 193. Use of a recombinant protein A/G-enzyme conjugate did not improve the PI (PI=190), giving reduced specificity and higher sensitivity. The final version used B. abortus RB51 RLPS as the antigen and protein A/G-enzyme conjugate for detection, giving a PI of 197. Because of the batch uniformity of the B. abortus RB51 RLPS and the versatility of the protein A/G-enzyme conjugate, the latter version appears to be the most useful for diagnostic serology. [ABSTRACT FROM AUTHOR]

Abstract: The diagnostic techniques most widely used for detecting brucellosis caused by Brucella ovis are serological tests such as complement fixation (CFT), agar gel immunodiffusion (AGID), and ELISAs. However, to our knowledge, milk tests, with the advantage that samples may be taken in a non invasive manner, have not been investigated as diagnostic tools. We studied 144 samples of milk and sera from lactating ewes, comparing bacteriological studies, serological and milk tests using Brucella canis and B. ovis antigens. A group of 75 ewes in an uninfected flock were serologically negative to rapid slide agglutination test (RSAT), indirect ELISA (IELISA)-B. canis, AGID and IELISA-B. ovis. The milk of these ewes had an IELISA-B. canis mean (%P) value of 16.18 (S.D. 5.63), while the IELISA-B. ovis had a mean (%P) value of 12.52 (S.D. 4.94). A cut-off value of (%P) 27.44 (+2 S.D.) or (%P) 33 (+3 S.D.) was determined by milk-ELISA-B. canis and (%P) 22.4 (+2 S.D.) and (%P) 27.34 (+3 S.D.) by milk-IELISA-B. ovis. These cut-off values were adjusted by receiver–operator characteristics (ROC) analysis using 23 positive samples from infected ewes, which indicated a milk-IELISA-B. canis cut-off value of (%P) 33 and milk-IELISA-B. ovis of (%P) 26 with 100% sensitivity and specificity. Based on our results, we propose that, following a study of a larger number of samples, the milk-IELISA-B. canis could be considered a suitable test for the diagnosis of B. ovis brucellosis in lactating ewes. [Copyright &y& Elsevier]

Abstract: Due to the important drawbacks of the Brucella melitensis Rev 1 vaccine, a safer vaccine based on an outer membrane complex from Brucella ovis encapsulated in poly-ɛ-caprolactone (PEC) microparticles (MP) was developed and tested in rams. Homogeneous batches of microparticles were prepared by a new double emulsion solvent evaporation method called “Total Recirculation One-Machine System” (TROMS). Such microparticles presented a mean diameter of 2μm and displayed an antigen loading of about 13μg HS per mg of microparticles. Subcutaneous vaccination of rams with 800μg HS (hot saline antigenic extract of B. ovis) in PEC microparticles induced an adequate serological response against B. ovis antigens and conferred similar protection against challenge with B. ovis to that induced by the living attenuated B. melitensis Rev 1 reference vaccine. By contrast, lower doses (80μg) of HS-PEC evoked reduced serological responses against B. ovis antigens and did not induce significant protection. The revaccination with 800μg of HS-PEC increased the intensity and duration of the serological response against B. ovis antigens but did not improve the protection conferred by the single vaccination. Sample sera taken from any of the animals immunized with Rev 1 were seropositive in both Rose Bengal and the Complement Fixation tests (RBT, CFT) used for the diagnosis of smooth Brucella infections. By contrast, no positive reactors in both tests were recorded in the animals vaccinated with HS-PEC, being this a target objective of this study. HS-PEC microparticles can be used as a safe vaccine against brucellosis in rams, but further studies using higher doses of antigens are necessary to exploit their full potential for the prophylaxis of brucellosis in sheep. [Copyright &y& Elsevier]

Abstract: Brucella ovis is recognized worldwide as an important pathogen of sheep, and has also been identified in farmed deer in New Zealand. Previously, only one strain type of B. ovis has been identified. The objective of this paper was to perform pulsed-field gel electrophoresis (PFGE) on field isolates of B. ovis to determine whether strain variations exist, whether sheep and deer are affected by the same strains, and to compare the performance of the rare-cutting restriction enzymes XbaI and SwaI. Ten B. ovis isolates from sheep and two from deer in New Zealand, as well as the type strain, were subjected to PFGE analysis using both XbaI and SwaI. PFGE of XbaI restriction fragments produced two banding patterns consisting of 27–28 bands, which were found to be 98% similar by cluster analysis, and were named X1 and X1a. PFGE of SwaI restriction fragments resulted in three banding patterns consisting of 13–15 bands each. Ten of the isolates had identical banding patterns and were named S1. One isolate differed by one band, representing a subtype named S1a. Two isolates differed by six bands, representing a different strain type of B. ovis and this was named S2. Cluster analysis showed S2 to be 78% similar to the S1/S1a cluster. Both strain types were isolated from both sheep and deer. Thus, two distinct strain types of B. ovis were identified in New Zealand, which is the first report of more than one strain type being identified worldwide. Neither strain was species-specific for sheep or deer. The restriction endonuclease SwaI was found to be more discriminatory than the enzyme XbaI, which has been used in previous studies. [Copyright &y& Elsevier]

Detergent-extracted recombinant Omp31 (rOmp31 extract) from Brucella melitensis produced in Escherichia coli was previously identified as a protective immunogen against B. ovis in mice. In this study, we evaluated the immunogenicity of rOmp31extract in rams. This immunogen was emulsified in an oil adjuvant and administered three times with 4 and 8 weeks intervals. Antibody response was measured in serum by whole B. ovis ELISA. Specific antibodies to purified rOmp31 (pET-Omp31) were detected by Western blotting and indirect ELISA. In addition, isotype specific antibodies were measured in tears. Serum bactericidal activity against B. ovis in the presence of complement was measured in vitro. Cellular immune response was explored by intradermal testing with purified rOmp31. Immunization with rOmp31 extract induced IgG specific antibodies in serum able to bind to whole B. ovis cells. Furthermore, strong inhibition in a competitive ELISA (with an Omp31-specific monoclonal antibody) suggested that a proportion of Omp31-specific antibodies were directed against a loop containing a protective epitope. Serum antibodies killed efficiently B. ovis in vitro in the presence of either guinea pig or ovine serum. Tears had both IgG and IgA antibodies to equivalent titers. Finally, immunized rams showed skin reactivity to Omp31. These data demonstrate that B. melitensis Omp31, a protective antigen identified in the mouse model, induces antibody and cellular immune mechanisms in sheep. [Copyright &y& Elsevier]

Rough lipopolysaccharide (RLPS) antigens were prepared from cultures of Brucella abortus RB51, B. ovis, and B. canis. The preparations were standardized by weight and tested with sera from cattle immunized with B. abortus RB51, sheep infected with B. ovis, and dogs infected with B. canis. Populations of unexposed animals of each species were also tested. The tests used were the indirect enzyme immunoassay (IELISA) using RLPS and the fluorescence polarization assay (FPA) using RLPS core fractions, labeled with fluorescein isothiocyanate. The IELISA using B. abortus RB51 RLPS antigen resulted in sensitivity and specificity values of 94.8% and 97.3%, respectively, when testing bovine sera, 98.5% and 97.8% when testing ovine sera, and 95.8% and 100% when testing dog sera. The IELISA using B. ovis RLPS antigen gave sensitivity and specificity values of 80.5% and 91.7%, respectively with bovine sera, 98.9% and 93.8% with sheep sera, and 70.8% and 79.8% with dog sera. The IELISA using B. canis RLPS antigen resulted in sensitivity and specificity values of 97.0% and 97.4%, respectively, with bovine sera, 96.2% and 96.3% with sheep sera, and 95.8% and 98.8% with dog sera. Labeling RLPS core from B. ovis and B. canis with fluorescein was not successful. B. abortus RB51 core labeled with fluorescein resulted in sensitivity and specificity values of 93.5% and 99.8%, respectively, with bovine sera and 78.1% and 99.0% with sheep sera. It was not possible to test the dog sera in the FPA. [ABSTRACT FROM AUTHOR]

An indirect enzyme-linked immunoassay (IELISA) for the detection of antibodies to Brucella ovis was evaluated. Relative to the complement fixation test (CFT) the sensitivity of the IELISA was 96.3% and the specificity was 99.6%. The sensitivity and specificity obtained in this study were comparable to ELISAs and CFTs of other studies in which B. ovis isolation was used for evaluation making it a choice to replace current serological tests such as the agar gel immunodiffusion test and the CFT. [Copyright &y& Elsevier]

The sensitivity and specificity of a PCR assay with primers derived from the insertion sequence IS6501 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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

O diagnóstico sorológico por imunodifusão radial da brucelose ovina causada por Brucella ovis é uma importante arma contra esta enfermidade, podendo também ser utilizada para a brucelose canina por Brucella canis, uma vez que há reação cruzada entre as duas bactérias. Contudo, ao utilizar-se antígenos de diferentes origens no diagnóstico, podem ocorrer diferenças quanto à reação produzida, mesmo utilizando-se a mesma cepa bacteriana e técnica de produção semelhante. Foram analisados antígenos produzidos por dois laboratórios (antígeno I, IIa e IIb) e um antígeno padrão, a fim de verificar se há diferenças entre padrões de bandas em eletroforese em gel de poliacrilamida (PAGE) destes produtos. A composição antigênica e a identificação de um antígeno padrão foram comparadas com antígenos comerciais pela técnica de PAGE e imunodifusão radial, onde foram encontradas 2 linhas de precipitação no antígeno IIa e uma linha de precipitação nos demais antígenos estudados. A concentração proteica dos diferentes antígenos foi obtida através do SENSIPROT .