Confirmação de infecção por Brucella abortus em um rebanho bovino certificado livre em Minas Gerais: relato de caso / Brucella abortus infection in an officially brucellosis-free bovine herd in Minas Gerais state - Brazil: a case report

Relata-se a ocorrência de um surto de brucelose em um rebanho de aproximadamente 1000 animais, livre da doença há 18 anos, certificado pelo Ministério da Agricultura, Pecuária e Abastecimento desde 2006. Dois animais reagiram aos testes sorológicos de diagnóstico por ocasião dos procedimentos de recertificação em 2008. Após o sacrifício deles, Brucella abortus, biovariedade 1, amostra não vacinal, foi isolada e identificada por meio de provas bioquímicas e de biologia molecular (PCR AMOS). A origem do agente no rebanho é de difícil determinação. No entanto, a adoção de procedimentos preconizados pelo Programa Nacional de Controle e Erradicação da Brucelose permitiu evitar a disseminação da enfermidade. Ocorrências como essas, em que rebanhos livres foram infectados após anos sem a ocorrência de brucelose, nunca haviam sido relatadas no Brasil.

A Brucellosis outbreak is reported in a bovine herd free from the disease for over 18 years, officially free since 2006. One heifer and one cow tested positive in serological tests for the 2008 annual recertification in a herd of almost 1000 animals. Isolation and identification by biochemical tests and molecular biology (AMOS PCR) confirmed the infection by a Brucella abortus biovar 1 field strain. It wasn't possible to find the source of the infection. However, adoption of standard procedures prescribed by the Brazilian National Brucellosis and Tuberculosis Control and Eradication Program hindered the spread of the disease. This is the first report of a Brucella infection in an officially Brucellosis-free bovine herd in Brazil.

In vitro evaluation of Clostridium septicum alpha toxoid / Avaliação in vitro de toxoide alfa de Clostridium septicum

Aiming to investigate in vitro alternatives, a test for neutralizing antibody detection using cell culture was developed. This test was more sensitive than previous animal models, allowing for detection of substantially lower alpha toxin and anti-alpha toxin titers. Titers observed during in vivo and in vitro seroneutralization had a correlation of 99.12 percent, indicating that cell culture is a viable alternative in the evaluation of vaccine potency, screening of vaccinal seeds, and Clostridium septicum alpha toxin titration.

Padronizou-se um teste para detecção de anticorpos neutralizantes in vitro, em cultura de células. O modelo in vitro mostrou-se mais sensível que os testes com animais, permitindo a detecção de títulos de toxina e antitoxina alfa mais baixos. Os títulos observados na soroneutralização in vivo e in vitro, apresentaram correlação de 99,12 por cento, demonstrando ser a cultura de células uma alternativa viável na avaliação da potência de vacinas, triagem de sementes vacinais e titulação de toxina alfa de Clostridium septicum.

Mionecroses clostridiais bovinas / Bovine clostridial myonecrosis

As mionecroses são enfermidades altamente fatais, representadas pelo carbúnculo sintomático e edema maligno. Com o objetivo de determinar a ocorrência dos diferentes clostrídios envolvidos nas mionecroses em bovinos no Brasil, foi realizado um estudo retrospectivo a partir de 30 casos suspeitos de carbúnculo sintomático e edema maligno. Empregou-se a técnica de estreptavidina biotina peroxidase em tecidos de bovinos fixados em formol e incluídos em parafina de arquivos de patologia de diferentes estados do país. Foram detectados 21 Clostridium chauvoei (70%), cincoClostridium septicum(17%), seguidos de doisC. chauvoeiassociado aoC. septicum (7%), um C. chauvoei mais Clostridium sordellii (3%) e um C. chauvoei mais Clostridium novyi tipo A (3%). Este é o primeiro diagnóstico de C. novyi tipo A no Brasil, e o primeiro relato da ocorrência de C. chauvoei associado à lesão no miocárdio. Os achados da maior ocorrência de C. chauvoei e C. septicum corroboram com estudos anteriores no país. Estes dados sobre a ocorrência dos agentes responsáveis pelas mionecroses em diferentes estados do país são de extrema importância frente às decisões relativas ao controle dessas enfermidades.

Clostridial myonecroses are highly fatal diseases, characterized by black leg and malignant oedema. In order to determine the occurrence of the different agents involved in Brazilian clostridial myonecrosis, a retrospective study was conducted concerning 30 suspected cases of blackleg and gas gangrene using a streptavidin-biotin peroxidase technique in formalin-fixed, paraffin-embedded tissues of cattle from the archives of pathology of different states of Brazil. The clostridia predominantly detected wereClostridium chauvoei(70%), followed by Clostridium septicum (5 cases or 17%), C. chauvoei plus C. septicum (2 cases or 7%), C. chauvoei plus Clostridium sordellii (1 case or 3%), C. chauvoei plus Clostridium novyi type A (1 case or 3%). This is the first diagnosis of C. novyi type A in Brazil. The findings of the higher occurrence of C. chauvoei and C. septicum concur with previous works in Brazil. This article also includes the first report in Brazil of the occurrence of C. chauvoei associated with a lesion in the myocardium. Thus, this study supplies important data about the occurrence of the agents responsible for clostridial myonecrosis in different states of Brazil, these data being important for decisions relative to control of these diseases.

Molecular cloning and expression of epsilon toxin from Clostridium perfringens type D and tests of animal immunization.

Epsilon toxin produced by Clostridium perfringens types B and D causes enterotoxemia in sheep, goats and calves. Enterotoxemia can cause acute or superacute disease, with sudden death of the affected animal. It provokes huge economic losses when large numbers of livestock are affected. Therapeutic intervention is challenging, because the disease progresses very rapidly. However, it can be prevented by immunization with specific immunogenic vaccines. We cloned the etx gene, encoding epsilon toxin, into vector pET-11a; recombinant epsilon toxin (rec-epsilon) was expressed in inclusion bodies and was used for animal immunization. Serum protection was evaluated and cross-serum neutralization tests were used to characterize the recombinant toxin. To analyze the potency of the toxin (as an antigen), rabbits were immunized with 50, 100 or 200 microg recombinant toxin, using aluminum hydroxide gel as an adjuvant. Titers of 10, 30 and 40 IU/mL were obtained, respectively. These titers were higher than the minimum level required by the European Pharmacopoeia (5 IU/mL) and by the USA Code of Federal Regulation (2 IU/mL). This rec-epsilon is a good candidate for vaccine production against enterotoxemia caused by epsilon toxin of C. perfringens type D.

Clinicopathologic features of experimental Clostridium perfringens type D enterotoxemia in cattle.

This study was designed to experimentally reproduce enterotoxemia by Clostridium perfringens type D in cattle and to characterize the clinicopathologic findings of this disease. Fourteen 9-month-old calves were inoculated intraduodenally according to the following schedule: group 1 (n = 4), C. perfringens type D whole culture; group 2 (n = 3), C. perfringens type D washed cells; group 3 (n = 5), C. perfringens type D filtered and concentrated supernatant; group 4 (n = 2), sterile, nontoxic culture medium. In addition, all animals received a 20% starch solution in the abomasum. Ten animals from groups 1 (4/4), 2 (3/3), and 3 (3/5) showed severe respiratory and neurologic signs. Gross findings were observed in these 10 animals and consisted of acute pulmonary edema, excessive protein-rich pericardial fluid, watery contents in the small intestine, and multifocal petechial hemorrhages on the jejunal mucosa. The brain of one animal of group 2 that survived for 8 days showed multifocal, bilateral, and symmetric encephalomalacia in the corpus striatum. The most striking histologic changes consisted of perivascular high protein edema in the brain, and alveolar and interstitial proteinaceous pulmonary edema. The animal that survived for 8 days and that had gross lesions in the corpus striatum showed histologically severe, focal necrosis of this area, cerebellar peduncles, and thalamus. Koch's postulates have been met and these results show that experimental enterotoxemia by C. perfringens type D in cattle has similar clinical and pathologic characteristics to the natural and experimental disease in sheep.

Antimicrobial susceptibility of Clostridium perfringens strains isolated from broiler chickens / Sensibilidade antimicrobiana de estirpes de Clostridium perfringens isoladas de aves de corte

Clostridium perfringens is a normal inhabitant of the intestinal tract of chickens as well as a potential pathogen that causes necrotic enteritis and colangio hepatitis. The minimum inhibitory concentration (MIC) of seven different compounds used for therapy, growth promotion or prevention of coccidiosis was determined by agar dilution method for 55 C. perfringens strains isolated from the intestines of broiler chickens. All strains showed high susceptibility to penicillin, avilamycin, monensin and narasin. Only 7.3% of the strains showed an intermediated sensitivity to lincomycin, and 49 (89.1%) were considered susceptible. For tetracycline and bacitracin, 41.8% and 47.3% of strains, respectively, were considered resistant.

Clostridium perfringens é um habitante normal da microbiota intestinal de frangos, sendo um agente potencialmente patogênico, causador de enterite necrótica e colangio-hepatite.A concentração inibitória mínima (CIM) de sete drogas utilizadas na terapêutica, como agentes promotores de crescimento ou na prevenção de coccidiose foi determinada pelo método de diluição em ágar para 55 estirpes de C. perfringens isoladas do intestino de frangos de corte. Todas as estirpes revelaram alta sensibilidade à penicilina, avilamicina, narasin e monensina, apenas 7,3% demonstraram CIM intermediário para lincomicinae 89.1% foram consideradas sensíveis. Para tetraciclina e bacitracina, 41,8% e 47.3% das amostras, respectivamente, foram consideradas resistentes.

Antimicrobial susceptibility of Clostridium perfringens strains isolated from broiler chickens.

Clostridium perfringens is a normal inhabitant of the intestinal tract of chickens as well as a potential pathogen that causes necrotic enteritis and colangio hepatitis. The minimum inhibitory concentration (MIC) of seven different compounds used for therapy, growth promotion or prevention of coccidiosis was determined by agar dilution method for 55 C. perfringens strains isolated from the intestines of broiler chickens. All strains showed high susceptibility to penicillin, avilamycin, monensin and narasin. Only 7.3% of the strains showed an intermediated sensitivity to lincomycin, and 49 (89.1%) were considered susceptible. For tetracycline and bacitracin, 41.8% and 47.3% of strains, respectively, were considered resistant.

Stability and toxicity of Clostridium perfringens type D epsilon prototoxin treated by iodine / Estabilidade e toxicidade da protoxina epsilon de Clostridium perfringens tipo D

Purified epsilon prototoxin of Clostridium perfringens type D was produced, purified, and detoxified by the stoiechiometric method of non-radioactive iodine incorporation. Different degrees of iodination were perfomed and the toxicity of the derivatives were analysed by in vivo studies. Toxicity decreased inversely to the iodine incorporation. Eletrophoretic analysis showed different levels of stability of samples kept under different temperatures 4ºC, - 20ºC, and -80 ºC. The iodinated prototoxins were stocked for a period of four months.

A prototoxina epsílon de Clostridium perfringens tipo D foi produzida, purificada e destoxificada por estoiquiometria. Diferentes quantidades de iodo foram incorporadas à estrutura protéica da prototoxina e a toxicidade dos derivados foi analisada em estudos in vivo. Verificou-se que a toxicidade diminuiu à medida que os átomos de iodo foram incorporados à prototoxina. A análise eletroforética demonstrou a estabilidade das amostras quando armazenadas a 4ºC, -20ºC e -80ºC. A prototoxina iodada foi estocada por um período de quatro meses.

PCR multiplex para identificação de isolados de Clostridium chauvoei e Clostridium septicum / Multiplex PCR for identification of Clostridium chauvoei and Clostridium septicum

Padronizou-se uma técnica de reação em cadeia da polimerase múltipla (PCR multiplex) para detecção de Clostridium chauvoei e Clostridium septicum em culturas puras. Foram utilizados pares de iniciadores para segmentos específicos dos genes que codificam a flagelina de C. chauvoei e a toxina alfa de C. septicum. Para avaliaçã o da PCR multiplex, foram testados 16 isolados clínicos de C. chauvoei e 15 isolados de C. septicum provenientes de ruminantes, quatro sementes vacinais de cada um desses agentes. Amostras de referência de ambos os microrganismos foram usadas como controle. Para avaliar a especificidade, DNAs genômicos dos seguintes microrganismos foram usados: C. sordellii, C. novyi tipo A, C. novyi tipo B, C. perfringens tipo A, C. haemolyticum, C. botulinum tipo D, Pseudomonas aeruginosa, Staphylococcus aureus, Enterobacter aerogenes, Escherichia coli e Salmonella typhimurium. Todos os isolados e sementes vacinais de C. chauvoei e C. septicum foram detectados pela técnica. Não foram observadas reações cruzadas com as outras espécies de clostrídios, outras espécies bacterianas ou entre C. Chauvoei e C. septicum. As menores concentrações de DNA de C. chauvoei e C. septicum detectadas foram 45pg/µl e 30pg/µl, respectivamente. A PCR multiplex pode ser utilizada para a identificação específica de C. chauvoei e C. septicum em culturas puras.

Multiplex PCR was optimized to detect Clostridium chauvoei and Clostridium septicum in pure cultures. In each reaction, a pair of primers for a specific segment of the flagellin gene of C. chauvoei and a pair of primers for a specific segment of alpha toxin gene of C. septicum were employed. Reference strains of both microorganisms were used as control. The multiplex PCR was evaluated by testing 16 clinical isolates of C. chauvoei from ruminants, 15 clinical isolates of C. septicum from ruminants and, four vaccine strains of each one of these agents. Reference strains of both microorganisms were used as control. To evaluate the specificity, genomic DNA of the following microorganisms was used: C. sordellii, C. novyi type A, C. novyi type B, C. perfringens type A, C. haemolyticum, C. botulinum type D, Pseudomonas aeruginosa, Staphylococcus aureus, Enterobacter aerogenes, Escherichia coli, and Salmonella typhimurium. All the isolates and vaccine strains of C. chauvoei and C. septicum were positive by PCR assay and cross reactions were not observed with the other species of clostridia, the other bacterial species or amongst both investigated agents. The smallest concentrations of DNA detected from C. chauvoei and C. septicum were 45pg/µl and 30pg/µl, respectively. The multiplex PCR was useful for the specific identification of C. chauvoei and C. septicum in pure cultures.

Detection of several clostridia by a direct fluorescent antibody test in formalin-fixed, paraffin-embedded tissues

Descreve-se a aplicabilidade de uma técnica de imunofluorescência direta, para o diagnóstico de mionecroses causadas por clostrídios, a partir de tecidos fixados em formol e incluídos em parafina. Essa técnica pode auxiliar no diagnóstico do carbúnculo sintomático e da gangrena gasosa, contribuindo para determinar a real prevalência dessas doenças no país

Infection of internal umbilical remnant in foals by Clostridium sordellii.

Omphalitis and the resulting septicemia contribute to perinatal mortality in several animal species. In foals, the most important causes of omphalitis are Escherichia coli and Streptococcus zooepidemicus. However to date, no information has been published about the role of Clostridium sordellii in these infections. In this paper, we describe 8 cases of perinatal mortality in foals associated with internal umbilical remnant infection by C. sordellii. The foals studied were between 12 and 21 days old at the time of death, and various breeds were represented in the group. Five of the foals were male and 3 were female. The diagnosis was established on the basis of the detection of C. sordellii by 3 methods (culture, fluorescent antibody test, and immunohistochemistry) and on gross and histopathologic findings. All foals had acute peritonitis, and the internal umbilical remnant was thickened by edema, hemorrhage, and fibrosis. A moderate amount of serosanguinous fluid with fibrin strands was present in the pericardial sac and pleural cavity. Histopathologically, the urachus and umbilical arterial walls were thickened by edema and exhibited hemorrhage, fibrin, and leukocytic infiltration. Gram-positive bacterial rods were observed in subepithelial areas of the urachus, the adventicia of umbilical arteries, and interstitium of the internal umbilical remnant. On the basis of these findings, we suggest that C. sordellii should be considered in the differential diagnosis for infections of the internal umbilical remnant in foals.

Enterotoxemia em bovino / Bovine enterotoxaemia

This report describes a case of bovine enterotoxaemia in Morro da Garça, Minas Gerais, Brazil. Clostridium perfringens type D was isolated in pure culture and was characterized by biochemical reactions and PCR. By the mouse neutralization test, the presence of epsilon toxin from intestinal content was detected.

Macrorhabdus ornithogaster in ostrich, rhea, canary, zebra finch, free range chicken, turkey, guinea-fowl, columbina pigeon, toucan, chuckar partridge and experimental infection in chicken, japanese quail and mice

Since 2000, Macrorhabdus ornithogaster "megabacteriosis" has been diagnosed in the avian diseases laboratory in a diversity of avian species and varied spectrum of disease. The disease in some species (chickens, turkeys, guinea fowls) was clinically characterized by emaciation, prostration, loss of appetite, cachexia and death, with a typically chronic course. A more acute disease was observed in finches (canary-Serinus and zebra-Taeniopygia) and budgerigars (Melopsittacus undulatus). The large rod shaped organism, visible from 100 times magnification, with and without staining, could be detected in sick and also in reasonably normal individuals of some species, such as chickens, turkeys, quails and pigeons. In rheas (Rhea americana), ostriches (Struthio camelus), canaries, zebra-finches, guinea-fowl (Numida meleagris) and budgerigars. The disease was severe, causing to up to 100 percent mortality. The infection could be detected in some species along with other infectious or disease problems, such as endoparasites (helminths, coccidia) and ectoparasitism (order Mallophaga or/and order Acarina). The cultivation of M. ornithogaster was successfully achieved in solid and liquid media, originated from chickens (four isolates), guinea fowl (1 isolate), chuckar partridge (1 isolate) and canary (1 isolate). A very interesting finding at microscopy was motility of M. ornithogaster, as detected both in cultures obtained on agar for pathogenic fungi and passaged into thioglycolate broth, as well as on samples observed in wet preparations from in vivo. Differences in colony aspects were noted among the isolates. Experimental infections were attempted in chicken and japanese quail, using a chicken isolate, allowing the detection of the organism in the proventriculus and liver in apparently normal birds. One chicken isolate was injected intraperitoneally in Balb/c mice and resulted in 100 percent mortality.

Desde 2000, diversos casos de infecção e doença por Macrorhabdus ornithogaster (megabacteria) foram diagnosticados no Setor de Doenças das Aves (Escola de Veterinária da UFMG). A doença clínica foi caracterizada por emagrecimento, prostração, perda do apetite, caquexia e morte, em curso crônico, embora com forma mais aguda em canários e periquitos. O microrganismo grande, em forma de bastão, visível a partir de 100 aumentos sem e com coloração, pode também ser detectado em aves de aspecto clínico normal, principalmente galinhas, perus, codornas e pombos. Em emas (Rhea), avestruzes (Struthio camelus), canários, mandarins, galinhas da Angola (Numida meleagris) e periquitos Australianos (Melopsittacus undulatus), a severidade da doença foi sempre maior, ocasionando até 100 por cento de mortalidade em alguns plantéis. Na maioria das espécies a doença foi detectada em aves com endo e/ou ectoparasitismo. O cultivo de M. ornithogaster foi obtido em meio sólido (ágar para fungos patogênicos) e subcultivado em meio líquido (thioglicolato), do proventriculo de galinha, galinha da Angola, perdiz de chuckar e canário. O resultado mais surpreendente na microscopia de M. ornithogaster foi a presença de motilidade, detectada tanto de cultivos in vitro quanto de preparações úmidas de in vivo. Diferenças nos aspectos das colônias foram notadas entre os isolados. Infecções experimentais em galinha (SPF) e codorna japonesa permitiram a detecção do organismo nos proventrículos das aves de aspecto normal. Nas codornas, à necropsia notaram-se hemorragias hepáticas. A infecção experimental em camundongos via intraperitoneal resultou em 100 por cento de mortalidade, também com lesões hepáticas. Aspectos do cultivo, a importância da doença, as espécies de aves susceptíveis e seu papel na epidemiologia são discutidos.

Production and purification of beta-toxin from Clostridium perfringens type C

Empregaram-se os métodos cromatográficos de afinidade metálica e de imunoafinidade para purificação da toxina beta em sobrenadante de cultivo de Clostridium perfringens tipo C. Observaram-se, na eletroforese das primeiras frações eluidas nos dois métodos de purificação, uma banda de peso molecular aproximado de 38kDa, característica da forma monomérica de toxina beta de Clostridium perfringens tipo C, e bandas de peso moleculares superiores, referentes às suas formas oligoméricas. Maior rendimento foi obtido com a utilização do método de imunoafinidade.

Characterization of the venom from the Brazilian Brown Spider Loxosceles similis Moenkhaus, 1898 (Araneae, Sicariidae).

Accidents caused by brown spiders (Loxosceles genus) are frequent in Brazil and are associated with dermonecrotic lesions and, eventually, systemic reactions that may be lethal. The major species implicated with human envenoming have been: L. intermedia, L. gaucho and L. laeta. In this study we characterized the venom from Loxosceles similis, a species of spider normally found inside caves. L. similis venom was characterized by two-dimensional gel electrophoresis and enzymatic activity (dermonecrosis and haemolysis). The lethal dose to mice and the capacity of commercial anti-serum to neutralize this venom were also analysed. The cross-reactivity with anti-venoms against L. intermedia, L. laeta and L. gaucho were studied. Our results showed that this venom was able to induce severe dermonecrotic lesions and showed the presence of the bacteria Clostridium septicum in association with the fangs. In addition, we have cloned the DNA coding for a dermonecrotic protein (LsD1), using the genomic DNA of L. similis. The deduced amino acid sequence showed a toxin of approximately 31.2 kDa with an estimated pI of 7.37 and sequence similar to LiD1, a protein from the dermonecrotic family of Loxosceles intermedia spider venom, a synanthropic species of medical importance.

Diarréia em leitões lactentes por Clostridium perfringens tipo A em granjas tecnificadas nos estados de Minas Gerais e São Paulo / Clostridium perfringens type A diarrhea in suckling piglets in industrial swine farms in the states of Minas Gerais and São Paulo

Diarrhea in suckling piglets caused by Clostridium perfringens type A was diagnosed in industrial (technified) swine farms of the states of Minas Gerais and São Paulo (Brazil), based on isolation and identification of bacterium by biochemical tests, detection of alpha toxin in animal bioassays, and PCR. This seems to be the first report of clostridial enterotoxaemia in piglets by C. perfringens type A in Brazil and allowed specific procedures to control the disease.

Avaliação de vacinas contra Clostridium novyi tipo B / Evaluation of vaccines against Clostridium novyi type B

Avaliou-se a eficiência de 13 vacinas comerciais contra clostridioses que continham em sua composição Clostridium novyi tipo B, pela titulação de antitoxina alfa em soro de coelhos e de bovinos vacinados e pelo teste de desafio direto em cobaias. As vacinas codificadas como T1 e T10 apresentaram, em coelhos, títulos de antitoxina alfa de 8 e 12UI/ml respectivamente, superiores ao nível mínimo de teste de 3,5UI/ml, recomendado para controle desse produto, e as vacinas T2 e T5, títulos de 2 e 3UI/ml, respectivamente. As vacinas T1, T2, T5 e T10 apresentaram níveis de antitoxina alfa detectáveis em bovinos, mas somente T1 e T10 induziram títulos compatíveis com o nível de teste. Pelo método de desafio direto em cobaias, as vacinas T1, T2, T5, T10 e T11 atenderam aos requisitos, protegendo todos os animais desafiados. Em sua maioria, as vacinas comercializadas no Brasil contra Clostridium novyi tipo B foram ineficientes em estimular títulos sorológicos compatíveis com os níveis de teste recomendado para controle desse produto.