First study of Brucella ovis antibodies in purebred sheep flocks in the State of Parana, Brazil

ABSTRACT Brucella ovis, a non-zoonotic species, is the etiological agent of ovine brucellosis, an infectious disease of clinical or subclinical occurrence in sheep flocks. Until then, there is no serological study of anti-Brucella ovis antibodies in purebred sheep herds. This study aimed to determine the presence of anti-Brucella ovis antibodies in purebred sheep flocks with breeding purposes from Parana State. Blood samples from 728 animals, of which 563 were females and 165 males, between 8 and 56 months of age from the six major sheep producing mesoregions of Parana, were submitted to detection of anti-Brucella ovis antibodies by the Agar Gel Immunodiffusion technique using an antigen from the bacteria Brucella ovis (Reo 198). The results indicate the presence of this disease in purebred sheep from Parana State in a low occurrence of 0.27% (2/728). The only two positive animals were rams, Santa Inês breed, from the same flock in the East Center region of Parana, without clinical disease. In conclusion, Brucella ovis is present in purebred sheep in Parana State, Brazil, and this low occurrence may have occurred due to rigorous breeding systems that may contribute to reduce the transmission of this disease.

Diagnostic Findings in a Confirmed Outbreak of Brucella ovis Infection in a Traditional Sheep Farm in Sicily (South-Italy)

Aim of this study is to report a laboratory investigation performed following the isolation of Brucella ovis, causing ovine epididymitis, in a traditional sheep farm in Sicily (South Italy). This disease represents a newly emerging risk for Italian livestock and is listed among diseases of EU priority (EU Reg 2016/429). Blood samples from 56 rams and 143 ewes were analyzed by both Enzyme-Linked Immunosorbent Assay (ELISA) and Complement Fixation Test (CFT). Genital swabs from all rams and 15 lactating ewes were collected to perform real-time PCR. Eighteen serologically positive rams were slaughtered and postmortem-inspected. Samples of testicle, epididymis, lymph nodes, and urine were also collected in order to perform microbiological, molecular, and histopathological analysis. Twelve slaughtered rams showed anatomo-pathological lesions. Real-time PCR for B. ovis BOV_A0504 gene was positive for 13 testicles and epididymis and 11 urine while B. ovis was isolated from epididymis and testicles of 7 slaughtered rams. This is the first exhaustive laboratory report of a microbiological, molecular, and serological pattern of the disease in sheep in Italy. Despite the impact on health and animal welfare, the epidemiology of B. ovis infection is still unknown, particularly in our country where the disease is considered endemic.

Rickettsia burneti and Brucella melitensis co-infection: a case report and literature review

Rickettsia is the pathogen of Q fever, Brucella ovis is the pathogen of brucellosis, and both of them are Gram-negative bacteria which are parasitic in cells. The mixed infection of rickettsia and Brucella ovis is rarely reported in clinic. Early diagnosis and treatment are of great significance to the treatment and prognosis of brucellosis and Q fever. Here, we report a case of co-infection Rickettsia burneti and Brucella melitensis. The patient is a 49-year-old sheepherder, who was hospitalized with left forearm trauma. Three days after admission, the patient developed fever of 39.0°C, accompanied by sweating, fatigue, poor appetite and headache. Indirect immunofluorescence (IFA) was used to detect Rickettsia burneti IgM. After 72 hours of blood culture incubation, bacterial growth was detected in aerobic bottles, Gram-negative bacilli were found in culture medium smear, the colony was identified as Brucella melitensis by mass spectrometry. Patients were treated with doxycycline (100 mg bid, po) and rifampicin (600 mg qd, po) for 4 weeks. After treatment, the symptoms disappeared quickly, and there was no sign of recurrence or chronic infection. Q fever and Brucella may exist in high-risk practitioners, so we should routinely detect these two pathogens to prevent missed diagnosis.

Development of a real-time PCR assay for the detection of Brucella ovis DNA in clinical samples

The etiological agent of infectious ovine epididymitis is Brucella ovis and for its direct indication in clinical samples several PCR protocols are proposed. This study describes a design and selection of the oligonucleotides for real-time PCR targeting conservative BOV_A0504 gene. The specificity of a real-time PCR was validated using 25 B. ovis field isolates and 14 microorganisms of closely related species. The detection limit of B. ovis in bacterial culture was determined as 3.5×101 CFU/mL with Ct value of 37.8. There are no detectable fluorescence signals in the clinical samples from intact animals, whereas bacteriologically confirmed material such as urine and testicle tissue samples were positive. It confirms that the assay is highly specific for detection of B. ovis DNA. Thus, the proposed real-time PCR assay enables fast detection and quantification of B. ovis in clinical material, which can be used as additional test for estimation of the health status of a sheep herd

Sororreatividade para Brucella abortus e Brucella ovis em pequenos ruminantes no Sertão de Itaparica, PE

Objetivou-se realizar um levantamento soro-epidemiológico da brucelose nos rebanhos caprinos e ovinos no sertão de Itaparica. Foram coletadas amostras de 239 caprinos e 435 ovinos, totalizando 674 amostras, procedentes de 16 propriedades. As amostras foram obtidas por meio de venopunção da jugular, sendo acondicionadas em caixa de isopor com gelo para envio ao laboratório. Para a pesquisa de anticorpos anti-Brucella abortus, utilizou-se o teste do antígeno acidificado tamponado (TAAT) e a técnica de fixação do complemento. Neste trabalho, todas as amostras de soro caprino analisadas (n=161) mostraram resultado negativo no TAAT. Com relação à espécie ovina, das 343 amostras submetidas ao TAAT, observou-se uma soropositividade de 0,87% (n=03), contudo, essas amostras foram negativas no teste de fixação do complemento. A infecção natural pela Brucella abortus não ocorre nos rebanhos de caprinos e ovinos avaliados neste trabalho. Registra-se a soropositividade para Brucella ovis na espécie ovina, porém em baixa freqüência. Dessa forma, salienta-se a importância da continuidade de estudos como esse no Estado de Pernambuco e em outros estados geograficamente próximos, devido a importância econômica que a doença apresenta.

Brucella ovis cysteine biosynthesis contributes to peroxide stress survival and fitness in the intracellular niche

Brucella ovis is an ovine intracellular pathogen with tropism for the male genital tract. To establish and maintain infection, B. ovis must survive stressful conditions inside host cells, including low pH, nutrient limitation, and reactive oxygen species. These same conditions are often encountered in axenic cultures during stationary phase. Studies of stationary phase may thus inform understanding of Brucella infection biology, yet the genes and pathways that are important in Brucella stationary phase physiology remain poorly defined. We measured fitness of a barcoded pool of B. ovis Tn-himar mutants as a function of growth phase and identified cysE as a determinant of fitness in stationary phase. CysE catalyzes the first step in cysteine biosynthesis from serine, and we provide genetic evidence that two related enzymes, CysK1 and CysK2, function redundantly to catalyze cysteine synthesis at steps downstream of CysE. Deleting either cysE (ΔcysE) or both cysK1 and cysK2 (ΔcysK1 ΔcysK2) results in premature entry into stationary phase, reduced culture yield and sensitivity to exogenous hydrogen peroxide. These phenotypes can be chemically complemented by cysteine or glutathione. ΔcysE and ΔcysK1 ΔcysK2 strains have no defect in host cell entry in vitro but have significantly diminished intracellular fitness between 2 and 24 hours post infection. Our study has uncovered unexpected redundancy at the CysK step of cysteine biosynthesis in B. ovis, and demonstrates that cysteine anabolism is a determinant of peroxide stress survival and fitness in the intracellular niche.