Streptococcus ovis sp. nov., isolated from sheep

Seven strains of an unknown Gram-positive catalase-negative chain-forming coccus-shaped organism isolated from clinical specimens from sheep were characterized by phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing studies demonstrated that the bacterium represents a new sub-line within the genus Streptococcus. The unknown bacterium was readily distinguished from recognized streptococcal species by biochemical tests and electrophoretic analysis of whole-cell proteins. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as Streptococcus ovis sp. nov. The type strain of Streptococcus ovis is CCUG 39485T (= LMG 19174T).

Brucella melitensis 16M: characterisation of the galE gene and mouse immunisation studies with a galE deficient mutant

The galE gene of Streptomyces lividans was used to probe a cosmid library harbouring Brucella melitensis 16M DNA and the nucleotide sequence of a 2.5 kb ClaI fragment which hybridised was determined. An open reading frame encoding a predicted polypeptide with significant homology to UDP-galactose-4-epimerases of Brucella arbortus strain 2308 and other bacterial species was identified. DNA sequences flanking the B. melitensis galE gene shared no identity with other gal genes and, as for B. abortus, were located adjacent to a mazG homologue. A plasmid which encoded the B. melitensis galE open reading frame complemented a galE mutation in Salmonella typhimurium LB5010, as shown by the restoration of smooth lipopolysaccharide (LPS) biosynthesis, sensitivity to phage P22 infection and restoration of UDP-galactose-4-epimerase activity. The galE gene on the B. melitensis 16M chromosome was disrupted by insertional inactivation and these mutants lacked UDP-galactose-4-epimerase activity but no discernible differences in LPS structure between parent and the mutants were observed. One B. melitensis 16M galE mutant, Bm92, was assessed for virulence in CD-1 and BALB/c mice and displayed similar kinetics of invasion and persistence in tissues compared with the parent bacterial strain. CD-1 mice immunised with B. melitensis 16M galE were protected against B. melitensis 16M challenge. Crown Copyright (C) 1999 Published by Elsevier Science B.V.

Soils of contrasting pH affect the decomposition of buried mammalian (Ovis aries) skeletal muscle tissue

Little is known about the effect of edaphic conditions on the decomposition of buried mammalian tissues. To address this, we set up a replicated incubation study with three fresh soils of contrasting pH: a Podsol (acidic), a Cambisol (neutral), and a Rendzina (alkaline), in which skeletal muscle tissue (SMT) of known mass was allowed to decompose. Our results clearly demonstrated that soil type had a considerable effect on the decomposition of SMT buried in soil. Differences in the rate of decomposition were up to three times greater in the Podsol compared with the Rendzina. The rate of microbial respiration was correlated to the rate of soft tissue loss, which suggests that the decomposition of SMT is dependent on the microbial community present in the soil. Decompositional by-products caused the pH of the immediate soil environment to change, becoming more alkaline at first, before acidifying. Our results demonstrate the need for greater consideration of soil type in future taphonomic studies.

Does repeated burial of skeletal muscle tissue (Ovis aries) in soil affect subsequent decomposition?

The repeated introduction of an organic resource to soil can result in its enhanced degradation. This phenomenon is of primary importance in agroecosystems, where the dynamics of repeated nutrient, pesticide, and herbicide amendment must be understood to achieve optimal yield. Although not yet investigated, the repeated introduction of cadaveric material is an important area of research in forensic science and cemetery planning. It is not currently understood what effects the repeated burial of cadaveric material has on cadaver decomposition or soil processes such as carbon mineralization. To address this gap in knowledge, we conducted a laboratory experiment using ovine (Ovis aries) skeletal muscle tissue (striated muscle used for locomotion) and three contrasting soils (brown earth, rendzina, podsol) from Great Britain. This experiment comprised two stages. In Stage I skeletal muscle tissue (150 g as 1.5 g cubes) was buried in sieved (4.6 mm) soil (10 kg dry weight) calibrated to 60% water holding capacity and allowed to decompose in the dark for 70 days at 22 °C. Control samples comprised soil without skeletal muscle tissue. In Stage II, soils were weighed (100 g dry weight at 60% WHC) into 1285 ml incubation microcosms. Half of the soils were designated for a second tissue amendment, which comprised the burial (2.5 cm) of 1.5 g cube of skeletal muscle tissue. The remaining half of the samples did not receive tissue. Thus, four treatments were used in each soil, reflecting all possible combinations of tissue burial (+) and control (−). Subsequent measures of tissue mass loss, carbon dioxide-carbon evolution, soil microbial biomass carbon, metabolic quotient and soil pH show that repeated burial of skeletal muscle tissue was associated with a significantly greater rate of decomposition in all soils. However, soil microbial biomass following repeated burial was either not significantly different (brown earth, podsol) or significantly less (rendzina) than new gravesoil. Based on these results, we conclude that enhanced decomposition of skeletal muscle tissue was most likely due to the proliferation of zymogenous soil microbes able to better use cadaveric material re-introduced to the soil.

Microbial decomposition of skeletal muscle tissue (Ovis aries) in a sandy loam soil at different temperatures

A laboratory experiment was conducted to determine the effect of temperature (2, 12, 22 °C) on the rate of aerobic decomposition of skeletal muscle tissue (Ovis aries) in a sandy loam soil incubated for a period of 42 days. Measurements of decomposition processes included skeletal muscle tissue mass loss, carbon dioxide (CO2) evolution, microbial biomass, soil pH, skeletal muscle tissue carbon (C) and nitrogen (N) content and the calculation of metabolic quotient (qCO2). Incubation temperature and skeletal muscle tissue quality had a significant effect on all of the measured process rates with 2 °C usually much lower than 12 and 22 °C. Cumulative CO2 evolution at 2, 12 and 22 °C equaled 252, 619 and 905 mg CO2, respectively. A significant correlation (P<0.001) was detected between cumulative CO2 evolution and tissue mass loss at all temperatures. Q10s for mass loss and CO2 evolution, which ranged from 1.19 to 3.95, were higher for the lower temperature range (Q10(2– 12 °C)>Q10(12–22 °C)) in the Ovis samples and lower for the low temperature range (Q10(2–12 °C)

Inactivation of formyltransferase (wbkC) gene generates a Brucella abortus rough strain that is attenuated in macrophages and in mice

Rough mutants of Brucella abortus were generated by disruption of wbkC gene which encodes the formyltransferase enzyme involved in LPS biosynthesis. In bone marrow-derived macrophages the B. abortus Delta wbkC mutants were attenuated, could not reach a replicative niche and induced higher levels of IL-12 and TNF-alpha when compared to parental smooth strains. Additionally, mutants exhibited attenuation in vivo in C57BL/6 and interferon regulatory factor-1 knockout mice. Delta wbkC mutant strains induced lower protective immunity in C56BL/6 than smooth vaccine S19 but similar to rough vaccine RB51. Finally, we demonstrated that Brucella wbkC is critical for LPS biosynthesis and full bacterial virulence. (C) 2010 Elsevier Ltd. All rights reserved.

Epizootiology of brucella infection in Australian fur seals

Novel members of the bacterial genus Brucella have recently emerged as pathogens of various marine mammal species and as potential zoonotic agents. We investigated the epizootiology of Brucella infection in Australian fur seals (Arctocephalus pusillus doriferus) by establishing demographic and temporal variations in antibody prevalence, attempting isolation of the causative agent, and determining whether this potential pathogen is involved in frequent abortions observed in this pinniped species. Two competitive enzyme-linked immunosorbent assays (cELISAs), an indirect ELISA, and a fluorescence polarization assay (FPA) were used to test sera for Brucella antibodies. The FPA and cELISA proved suitable for use in this species. Significant differences in antibody prevalence were found between age classes of seals sampled between 2007 and 2009 at one colony. Pups sampled at this site (n5134) were negative for Brucella antibodies by all serologic tests but 17 of 45 (38%) of juveniles were antibody-positive. Antibody prevalence in adult females was significantly higher than in juveniles (P50.044). Antibody prevalence for adult females between 2003 and 2009 varied significantly over time (P50.011), and for individuals sampled between 2003 and 2005, the likelihood of pregnancy was greater in individuals positive for Brucella antibodies (P50.034). Inflammatory lesions suggestive of infectious agents were found in 14 of 39 aborted Australian fur seal pups, but pathologic changes were not uniformly consistent for Brucella infection. Culture and PCR investigations on fetal tissues were negative for Brucella. Culture and PCR on selected fresh or frozen tissues from 36 juvenile and adult animals were also negative. We suspect that the prevalence of active infection with Brucella in Australian fur seals is low relative to antibody prevalence. © Wildlife Disease Association 2011.

Tipificação molecular de amostras de Brucella spp. utilização a técnica de BOX-PCR

O gênero Brucella é formado por coco-bacilos Gram negativos patogênicos ao homem e animais, sendo classificado como patógeno de grupo de risco III. A identificação dessas bactérias apresenta várias limitações como: exigência de inoculação em vários meios, tempo de incubação longo e necessidade de soros imunes e bacteriófagos. Devido à sua alta patogenicidade e ao longo tempo de exposição dos laboratoristas à bactéria, a brucelose é uma das infecções mais freqüentemente adquiridas em laboratório. Além da contaminação em laboratório, a transmissão ao homem pode ocorrer através de animais infectados e ingestão de produtos derivados, como o leite cru. A procura de métodos rápidos de identificação das espécies e biovares pode ser útil para diminuir os riscos do manuseio desta bactéria e na tomada de medidas de controle epidemiológico. O principal objetivo deste trabalho foi facilitar a classificação de cepas de referência de Brucella spp. e isoladas no Brasil utilizando a técnica de rep-PCR com oligonucleotídeos do elemento BOX, uma seqüência repetida presente no genoma de várias bactérias. Foram analisados 38 isolados representando diferentes espécies e biovares de Brucella sp. e 13 isolados de gêneros relacionados como controle da especificidade da reação. Foi realizada uma confirmação prévia dos isolados de brucela por testes bioquímicos e PCR gênero-específica. A técnica de BOX-PCR agrupou todas as espécies e biovares de Brucella em um único grupo com nível de similaridade entre 100 e 74%. Diferenças entre os isolados, quanto a presença ou ausência de bandas, puderam ser observadas. Entretanto, essas divergências não caracterizam uma espécie ou biovar. Bandas comuns a todos os isolados de Brucella sp. podem caracterizar o gênero.

Detecção de Escherichia coli, Listeria monocytogenes e Brucella sp. em queijos produzidos artesanalmente na Região Litorânea do Rio Grande do Sul

O queijo é o produto de maior importância entre os derivados do leite, pelo seu grande valor nutritivo e pelas suas características organolépticas. Na região litorânea do Rio Grande do Sul, Brasil, existe uma concentração elevada de comércio artesanal de queijo. Para a estimativa da qualidade microbiológica destes produtos, foram coletadas 80 amostras referentes a 29 bancas de estabelecimentos comerciais localizados nas rodovias RS 30 (Osório-Tramandai), RS 40 (Viamão-Pinhal) e RS 389 (Osório-Torres). Em 26% das amostras, a contagem de coliformes fecais estava acima da estabelecida pela legislação e em 32% das amostras foi possível o isolamento de E.coli., sugerindo considerável contaminação e a necessidade de orientar estes produtores para normas básicas de higiene. Foi confirmada a presença de Listeria sp. em 16% das amostras, sendo 4% L. monocytogenes. Não foram isoladas cepas de Brucella sp. As características do produto analisado como pH, atividade da água, estocagem, presença de altas contagens de microrganismos competidores, provavelmente, contribuíram significativamente para controle de Listeria sp. e Brucella sp. O presente estudo demonstra que este tipo de alimento pode tornar-se um problema de saúde pública, principalmente para os grupos de risco.

Serosurvey for antibodies against Brucella abortus and Leptospira interrogans in pampas deer from Brazil

A survey for antibodies against Brucella abortus, and Leptospira interrogans was conducted on 17 pampas deer (Ozotocerus bezoarticus) from Pantanal Matogrossense (State of Mato Grosso do Sul, Brazil) and on 24 pampas deer from Parque Nacional de Emas (State of Goias, Brazil). Antibodies against B. abortus were detected by plate ag glutination, rose Bengal, and complement fixation tests, antibodies against Leptospira interrogans were detected by the microscopic agglutination test. All sera were negative for B. abortus antibodies and all deer sera from Parque Nacional de Emas were negative for L. interrogans antibodies. Four (24%) of 17 sera from Pantanal Matogrossense were positive for L. interrogans serovar (n = 2) hardjo, wolffi (n = 1) and mini (n = 1). While these diseases do not appear to be of major importance to the health status of Pampas deer, it appears that deer are reservoir for leptospirosis in one of the study areas.

Brucellosis due to Brucella suis in a swine herd associated with a human clinical case in the State of São Paulo, Brazil

Brucella suis has been recognized as the major etiological agent of human brucellosis in areas free from Brucella melitensis infection. However, with changes in swine management, the occurrence of swine brucellosis has decreased as has the human incidence of B. suis infection. A swine brucellosis outbreak within a herd from Jaboticabal (So Paulo, Brazil) was detected in July 2006. The herd comprised approximately 300 sows and 1,500 finishing animals. Many sows within this herd experienced abortions, while others exhibited vaginal discharge; three sows suffered posterior paralysis. Among 271 sows, 254 (93.7%) tested positive for brucellosis by complement fixation, and among 62 randomly bled finishing animals, 17 (27.4%) also tested positive. The B. suis biovar 1 was cultured from 14 aborted fetuses and six sows. Brucella was identified using routine methods. Fourteen farm workers were tested using agglutination tests, with three workers showing evidence of Brucella antibody titers. A 39-year-old woman, who worked with maternal pigs and had direct contact with aborted fetuses, presented an agglutinating titer of 480 IU/mL and displayed clinical signs of infection. Our findings suggest that despite a reduction of swine brucellosis throughout Brazil, B. suis infection still occurs, thereby posing a zoonotic risk.

Evolução de títulos sorológicos, nas provas de soroaglutinação em placa, antígeno acidificado tamponado e fixação de complemento, em bezerras Nelore vacinadas aos 18 meses de idade com Brucella abortus amostra B 19

Com o objetivo de estudar a persistência da resposta sorológica, através das provas de soroaglutinação em placa, rosa Bengala e fixação de complemento, 108 bezerras, com idade ao redor de 18 meses, foram vacinadas com uma dose padrão da vacina preparada com Brucella abortus amostra B 19. Foram obtidas amostras de soro sangüíneo antes da vacinação e após 45 dias, 6, 12 e 18 meses. Antes da vacinação, todas as bezerras apresentaram resultados negativos nos três testes. Após 45 dias, todas apresentaram título a partir de 1/100 na prova de soroaglutinação em placa e todas apresentaram resultado positivo no teste rosa Bengala, ao passo que dois animais apresentaram título 1/2 e os demais apresentaram título a partir de 1/4 na reação de fixação de complemento. Após um ano de vacinação, a grande maioria das bezerras já não apresentava título sorológico significativo. Considerando o risco a que estão sujeitos animais que vivem em áreas endêmicas e em propriedades onde a doença ocorre, e considerando a acentuada redução de título sorológico observada na grande maioria dos animais, pode-se concluir que, no caso bezerras de raças zebuínas em áreas endêmicas e que não tenham sido vacinadas na idade regulamentar, é mais vantajoso vaciná-las com a amostra B 19 aos 18 meses de idade do que deixá-las expostas a um elevado risco de infecção por Brucella.

Estabilidade do antígeno de célula total de Brucella abortus para uso no diagnóstico sorológico da brucelose bovina pela reação de fixação de complemento

A reação de fixação de complemento é um dos testes usados no diagnóstico confirmatório da brucelose bovina, e para sua realização emprega-se o mesmo antígeno usado na prova de soroaglutinação lenta, porém não foi possível encontrar na literatura estudos sobre a estabilidade desse antígeno para uso na prova de fixação de complemento, de modo a estabelecer um prazo de validade para o mesmo. Por isso, esta investigação teve por objetivo avaliar a estabilidade do antígeno de célula total de Brucella conservado sob refrigeração, para uso na reação de fixação de complemento. Analisaram-se 14 partidas de antígeno, preparado com Brucella abortus amostra 1119/3 e padronizado para uso na prova de soroaglutinação lenta, com tempo de fabricação variando de 9 meses a 23 anos e 11 meses. Testaram-se 167 soros bovinos com títulos variáveis de anticorpos contra Brucella, adotando-se a técnica com incubação a 37ºC nas duas fases da reação e 5 unidades hemolíticas 50% de complemento. Considerou-se como positivo o soro com pelo menos 25% de fixação de complemento na diluição 1:4. Compararam-se os resultados obtidos com as 13 partidas de antígeno com aqueles obtidos com a partida com 9 meses de fabricação, usando o teste de chi2 de McNemar e o coeficiente kappa. A grande maioria dos soros apresentou resultados muito próximos quando testados com as diversas partidas de antígeno, e não se observou relação entre tempo de fabricação do antígeno e diferenças nos resultados obtidos.

Prevalence and Molecular Characterization of Mycoplasma ovis in Selected Free-Ranging Brazilian Deer Populations

Mycoplasma ovis is a hemoplasma that may cause anemia and mortality in small ruminants. Our aim was to determine whether M. ovis infects populations of free-ranging deer in Brazil. Bully coat samples from 64 Blastocerus dichotomus from Porto Primavera, 18 Ozotocerus bezoarticus from Pantanal, and 21 O. bezoarticus from Emas National Park were tested. Using a M. ovis PCR protocol to amplify extracted DNA, 46/64 (72%) of deer froth Porto Primavera, 10/18 (56%) from Pantanal, and 4/21 (19%) from Emas National Park were positive, giving an overall positive rate of 58% for hemoplasma in these wild deer. Sequencing and phylogenetic analysis of the 168 rRNA gene revealed 3 genetically distinct hemoplasmas including M. ovis, 'Candidatus Mycoplasma erythrocervae', and a hemoplasma most closely related to M. ovis. Phylogenetic analysis of the 23S rRNA gene from selected sequences confirmed these relationships.

Diagnóstico sorológico da brucelose bovina em animais adultos vacinados com dose reduzida da cepa 19 de Brucella abortus

Com o presente trabalho avaliou-se o uso de dose reduzida da vacina produzida com a amostra 19 de Brucella abortus, em rebanho adulto negativo para a enfermidade, por meio de técnicas de diagnóstico sorológico preconizadas pelo Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose Animal e por um ensaio indireto de imunoadsorção enzimática (ELISA ID). A prova de fixação de complemento detectou 46,77% de positivos, o antígeno acidificado tamponado 67,74%, o 2-mercaptoetanol com soroaglutinação lenta 87,09% e o ELISA ID 100%. A dose reduzida interferiu no diagnóstico sorológico. Nenhuma das técnicas apresentou especificidade adequada para uso em rebanho nestas condições, até 3 meses após a vacinação.