Diagnosis of blackleg from cattle tissue impregnated in common filter paper / Diagnóstico de carbúnculo sintomático a partir de papel filtro comum impregnado com tecido bovino

Blackleg, an acute myonecrosis caused by Clostridium chauvoei, is usually underdiagnosed since the rapid transport of adequate samples for laboratory testing is difficult. This study tested a direct polymerase chain reaction (PCR) technique using common filter paper impregnated with cattle tissue samples obtained from animals suspected with blackleg. Twenty-five samples, belonging to eleven animals from Rio Grande do Sul State, Brazil, were analyzed. The direct PCR technique identified eight positive animals corroborating with results from microbiological culture. Skeletal muscle was the most common tissue type used in this study and when the animal was positive the pathogen was always detected in this tissue. Storage time of the impregnated filter paper at room temperature did not prove to be a limiting factor for the quality of the results indicating that this procedure can be carried out in the field and samples be sent in regular mail. Our results suggested that direct PCR of common filter paper impregnated with cattle tissue is a practical and economical alternative for the diagnosis of blackleg.

Carbúnculo sintomático, uma mionecrose aguda causada por Clostridium chauvoei, costuma ser subdiagnosticada, pois o transporte rápido de amostras adequadas para exames laboratoriais é complicado. O objetivo deste estudo foi testar a técnica de reação em cadeia da polimerase (PCR) direta, utilizando papel filtro comum impregnado com amostras de tecido bovino obtidas de animais suspeitos de carbúnculo sintomático. Foram analisadas 25 amostras, pertencentes a onze animais do estado do Rio Grande do Sul, Brasil. A técnica de PCR direta identificou oito animais positivos, corroborando com os resultados da cultura microbiológica. O músculo esquelético foi o tecido mais utilizado neste estudo e quando o animal foi positivo, o patógeno sempre foi detectado neste tecido. O tempo de armazenamento do papel filtro impregnado, à temperatura ambiente, não se mostrou um fator limitante para a qualidade dos resultados, indicando que esse procedimento pode ser realizado no local e as amostras enviadas por correio normal. Nossos resultados sugerem que a PCR direta usando papel filtro comum impregnado com tecido bovino é uma alternativa prática e econômica para o diagnóstico de carbúnculo sintomático.

Diagnosis of blackleg from cattle tissue impregnated in common filter paper / Diagnóstico de carbúnculo sintomático a partir de papel filtro comum impregnado com tecido bovino

Blackleg, an acute myonecrosis caused by Clostridium chauvoei, is usually underdiagnosed since the rapid transport of adequate samples for laboratory testing is difficult. This study tested a direct polymerase chain reaction (PCR) technique using common filter paper impregnated with cattle tissue samples obtained from animals suspected with blackleg. Twenty-five samples, belonging to eleven animals from Rio Grande do Sul State, Brazil, were analyzed. The direct PCR technique identified eight positive animals corroborating with results from microbiological culture. Skeletal muscle was the most common tissue type used in this study and when the animal was positive the pathogen was always detected in this tissue. Storage time of the impregnated filter paper at room temperature did not prove to be a limiting factor for the quality of the results indicating that this procedure can be carried out in the field and samples be sent in regular mail. Our results suggested that direct PCR of common filter paper impregnated with cattle tissue is a practical and economical alternative for the diagnosis of blackleg.(AU)

Carbúnculo sintomático, uma mionecrose aguda causada por Clostridium chauvoei, costuma ser subdiagnosticada, pois o transporte rápido de amostras adequadas para exames laboratoriais é complicado. O objetivo deste estudo foi testar a técnica de reação em cadeia da polimerase (PCR) direta, utilizando papel filtro comum impregnado com amostras de tecido bovino obtidas de animais suspeitos de carbúnculo sintomático. Foram analisadas 25 amostras, pertencentes a onze animais do estado do Rio Grande do Sul, Brasil. A técnica de PCR direta identificou oito animais positivos, corroborando com os resultados da cultura microbiológica. O músculo esquelético foi o tecido mais utilizado neste estudo e quando o animal foi positivo, o patógeno sempre foi detectado neste tecido. O tempo de armazenamento do papel filtro impregnado, à temperatura ambiente, não se mostrou um fator limitante para a qualidade dos resultados, indicando que esse procedimento pode ser realizado no local e as amostras enviadas por correio normal. Nossos resultados sugerem que a PCR direta usando papel filtro comum impregnado com tecido bovino é uma alternativa prática e econômica para o diagnóstico de carbúnculo sintomático.(AU)

Molecular characterization of virulence genes cctA, nanA, and fliC in Clostridium chauvoei from Rio Grande do Sul and São Paulo State, Brazil / Caracterização molecular dos genes de virulência cctA, nanA e fliC em Clostridium chauvoei isolados no Rio Grande do Sul e São Paulo, Brasil

Clostridium chauvoei toxin A (CctA), neuraminidase (NanA), and flagellin (FliC) proteins contribute to the pathogenicity of Clostridium chauvoei, the causative agent of blackleg in cattle. The aim of this study was to analyze the genetic variability of cctA, nanA, and fliC genes in C. chauvoei isolates from the Rio Grande do Sul and São Paulo state- Brazil, during different sampling periods. The presence of these genes was verified through PCR amplification and partial gene sequencing of 17 strains. Alignment of PCR amplicons combined with bioinformatics analysis was used in an attempt to study the variability across C. chauvoei solates. The similarity among the partial sequences of cctA and nanA genes was 100%. The sequencing of fliC revealed three different paralog alleles of flagellin, and two strains were seen to be polymorphic, with amino acid alterations in the predicted protein. Overall, this study indicates that strains of C. chauvoei isolated in Brazil are highly conserved with respect to the virulence factors evaluated.(AU)

Toxina A de Clostridium chauvoei (CctA), neuraminidase (NanA) e flagelina (FliC) são proteínas que contribuem para a patogenicidade de Clostridium chauvoei, o agente causador do carbúnculo sintomático em bovinos. O objetivo deste estudo foi analisar a variabilidade genética dos genes cctA, nanA, e fliC em C. chauvoei isolados em diferentes períodos no Rio Grande do Sul e São Paulo. A presença destes genes foi verificada pela amplificação dos produtos da PCR e sequenciamento parcial dos genes de 17 cepas. Os alinhamentos da amplificação dos produtos da PCR combinados com a análise de bioinformática foram utilizados na tentativa de avaliar a variabilidade dos genes entre os isolados de C. chauvoei. A similaridade do sequenciamento parcial dos genes cctA e nanA foi 100%. O sequenciamento do fliC revelou três alelos paralogos diferentes de flagelina e duas cepas mostraram polimorfismos, causando alterações na sequência de aminoácidos. As cepas de C. chauvoei isoladas no Brasil mostraram-se altamente conservadas em relação aos fatores de virulência avaliados neste estudo.(AU)

Blackleg in cattle: current understanding and future research needs / Carbúnculo sintomático: compreensão atual e futuras necessidades de pesquisa

Blackleg is an endogenous acute infection that principally affects cattle, whose etiologic agent is the anaerobic bacterium Clostridium chauvoei. In recent years, the major virulence factors of C. chauvoei have been discovered and described. However, the pathogenesis of blackleg in cattle, and in particular, the movement of the pathogen from the point of entry to the affected tissues is not yet fully elucidated. Disease control is based on appropriate management and vaccination. This review summarizes the latest research findings that contribute toward the understanding of the disease in cattle, provide a foundation to preventive strategies, and identify future research needs.

O carbúnculo sintomático é uma infecção endógena, aguda, que acomete principalmente bovinos, cujo agente etiológico é a bactéria anaeróbica Clostridium chauvoei. Recentemente, os principais fatores de virulência do C. chauvoei foram descobertos e descritos. Contudo, a patogênese do carbúnculo sintomático em bovinos, especialmente a circulação do patógeno desde o ponto de entrada até os tecidos acometidos ainda não está completamente elucidada. O controle desta enfermidade é baseado em medidas adequadas de manejo e vacinação. Esta revisão reúne as mais recentes descobertas que contribuíram para o entendimento da doença em bovinos, fornece embasamento para medidas preventivas e aponta futuras necessidades de pesquisa.

Blackleg in cattle: current understanding and future research needs / Carbúnculo sintomático: compreensão atual e futuras necessidades de pesquisa

Blackleg is an endogenous acute infection that principally affects cattle, whose etiologic agent is the anaerobic bacterium Clostridium chauvoei. In recent years, the major virulence factors of C. chauvoei have been discovered and described. However, the pathogenesis of blackleg in cattle, and in particular, the movement of the pathogen from the point of entry to the affected tissues is not yet fully elucidated. Disease control is based on appropriate management and vaccination. This review summarizes the latest research findings that contribute toward the understanding of the disease in cattle, provide a foundation to preventive strategies, and identify future research needs.(AU)

O carbúnculo sintomático é uma infecção endógena, aguda, que acomete principalmente bovinos, cujo agente etiológico é a bactéria anaeróbica Clostridium chauvoei. Recentemente, os principais fatores de virulência do C. chauvoei foram descobertos e descritos. Contudo, a patogênese do carbúnculo sintomático em bovinos, especialmente a circulação do patógeno desde o ponto de entrada até os tecidos acometidos ainda não está completamente elucidada. O controle desta enfermidade é baseado em medidas adequadas de manejo e vacinação. Esta revisão reúne as mais recentes descobertas que contribuíram para o entendimento da doença em bovinos, fornece embasamento para medidas preventivas e aponta futuras necessidades de pesquisa.(AU)

Blackleg in cattle in the state Mato Grosso do Sul, Brazil: 59 cases / Carbúnculo sintomático em bovinos em Mato Grosso do Sul: 59 casos

This study aimed to review cases of blackleg (Clostridium chauvoei infection) diagnosed in cattle from Midwestern Brazil from 1994 to 2014 considering epidemiological, clinical, necropsy and histopathological findings. Also the following laboratory tests were used for the diagnosis of some cases of blackleg: microbiological culture and identification of the agent, microbiological culture and identification of the agent by the polymerase chain reaction (PCR), and identification of the agent in formalin fixed paraffin embedded tissues (FFPE). Criteria for presumptive diagnosis of blackleg included necrohemorrhagic emphysematous myositis consisting of inflammatory infiltrate, coagulative necrosis of myofiber, interstitial edema, hemorrhage, and gas bubbles between myofibers. Fifty nine cases from 51 outbreaks of blackleg were found, which corresponded to 1.1% of 5,375 cattle deaths investigated. In five of those outbreaks, samples of affected muscles cultures for the identification of pathogenic clostridia were made. Another three samples of similar material were cultured for clostridia with subsequent identification of the isolate by PCR. Twelve samples of FFPE affected muscle fragments were submitted to PCR for identification of the etiological agent. Except for January, cases were observed in each month of the year, with higher numbers in July-October. Most affected cattle were in the age of 7-12 years, but calves younger than 6 month-old and older than 24 months were also observed. Vaccination histories were scarce. In 32 outbreaks some vaccination history was available, but only in two of those vaccination has been carried out properly. In 56 six cases the skeletal muscles were involved. Muscles of the hind limbs were the most affected. In ten cases muscles of the tongue, myocardium and diaphragm were also affected. In three of the cases the visceral form was observed. Deaths occurred after a clinical course of 6-24 hours, but in most cases cattle were found death. Sudden death was the outcome in visceral cases (cardiac) blackleg. Clostridium chauvoei was confirmed to be the cause by culturing in 5 cases, and by PCR and histopatology in 8 cases. Bacterial culture followed by PCR did not demonstrate C. chauvoei. Calculation of the economic impact indicates that blackleg is a frequent disease in the state of Mato Grosso do Sul (MS) that inflicts significant economic loss. The amount of these losses would be reduced through proper vaccination programs against the prevalent strains of C. chauvoei in the region.(AU)

Este estudo foi realizado com o objetivo de descrever casos de carbúnculo sintomático (infecção por Clostridium chauvoei) diagnosticados em bovinos do Centro-Oeste brasileiro de 1994-2014, avaliando a epidemiologia, os sinais clínicos, os achados de necropsia e a histopatologia; objetivou-se também avaliar os seguintes testes laboratoriais para o diagnóstico de carbúnculo sintomático: cultura microbiológica e identificação do agente, cultura microbiológica e identificação do agente por reação em cadeia de polimerase (PCR) e identificação do agente em material fixado em formol e incluído em parafina (FFIP). Os critérios para o diagnóstico presuntivo de carbúnculo sintomático incluíram miosite necro-hemorrágica enfisematosa, caracterizada por infiltrado inflamatório, necrose de coagulação de miofribras, edema intersticial, hemorragia e bolhas de gás em meio às miofribras. Cinquenta e nove casos oriundos de 51 surtos foram encontrados, o que corresponde a 1,1% das 5.375 mortes de bovinos investigadas. Em cinco desses casos, amostras do músculo afetado foram cultivadas para clostrídios patogênicos. Amostras semelhantes de outros três animais foram cultivadas para clostrídios e os isolamentos identificados subsequentes por PCR. Doze fragmentos de músculo afetado FFIP foram submetidos a PCR para identificação do agente etiológico. Com exceção de janeiro, os casos de carbúnculo sintomático foram observados em todos os meses do ano com uma maior incidência em junho-outubro. A faixa etária da maioria dos bovinos afetados era de 7-12 anos de idade, mas bovinos mais jovens que 6 meses e mais velhos que 24 meses foram também afetados. Os históricos de vacinação eram escassos nesses surtos. Em 32 surtos havia alguma informação sobre a vacinação, mas em apenas dois casos a vacinação tinha sido realizada adequadamente. Cinquenta e seis casos de carbúnculo sintomático deste estudo eram casos clássicos afetando os músculos esqueléticos. Os músculos mais afetados foram os dos membros pélvicos. Em dez casos os músculos da língua, miocárdio e diafragma estavam também afetados. Apenas três dos casos apresentaram a forma visceral (cardíaca). O curso clínico foi de 6-24 horas, mas na maioria dos casos os bovinos foram encontrados mortos. Em casos da forma visceral ocorria morte súbita. Clostridium chauvoei foi confirmado como o agente causal por cultura em cinco casos e por PCR em amostra FFIP em 8 casos. Cultura bacteriana seguida de PCR do isolado não demonstrou C. chauvoei. Carbúnculo sintomático é uma doença frequente em bovinos no Mato Grosso do Sul podendo provocar importantes prejuízos para os produtores rurais. Esses prejuízos podem ser reduzidos através de um programa de vacinação adequado usando-se vacinas eficazes contra cepas de C. chauvoei prevalentes na região.(AU)

Blackleg in cattle in the state Mato Grosso do Sul, Brazil: 59 cases / Carbúnculo sintomático em bovinos em Mato Grosso do Sul: 59 casos

This study aimed to review cases of blackleg (Clostridium chauvoei infection) diagnosed in cattle from Midwestern Brazil from 1994 to 2014 considering epidemiological, clinical, necropsy and histopathological findings. Also the following laboratory tests were used for the diagnosis of some cases of blackleg: microbiological culture and identification of the agent, microbiological culture and identification of the agent by the polymerase chain reaction (PCR), and identification of the agent in formalin fixed paraffin embedded tissues (FFPE). Criteria for presumptive diagnosis of blackleg included necrohemorrhagic emphysematous myositis consisting of inflammatory infiltrate, coagulative necrosis of myofiber, interstitial edema, hemorrhage, and gas bubbles between myofibers. Fifty nine cases from 51 outbreaks of blackleg were found, which corresponded to 1.1% of 5,375 cattle deaths investigated. In five of those outbreaks, samples of affected muscles cultures for the identification of pathogenic clostridia were made. Another three samples of similar material were cultured for clostridia with subsequent identification of the isolate by PCR. Twelve samples of FFPE affected muscle fragments were submitted to PCR for identification of the etiological agent. Except for January, cases were observed in each month of the year, with higher numbers in July-October. Most affected cattle were in the age of 7-12 years, but calves younger than 6 month-old and older than 24 months were also observed. Vaccination histories were scarce. In 32 outbreaks some vaccination history was available, but only in two of those vaccination has been carried out properly. In 56 six cases the skeletal muscles were involved. Muscles of the hind limbs were the most affected. In ten cases muscles of the tongue, myocardium and diaphragm were also affected. In three of the cases the visceral form was observed. Deaths occurred after a clinical course of 6-24 hours, but in most cases cattle were...(AU)

Este estudo foi realizado com o objetivo de descrever casos de carbúnculo sintomático (infecção por Clostridium chauvoei) diagnosticados em bovinos do Centro-Oeste brasileiro de 1994-2014, avaliando a epidemiologia, os sinais clínicos, os achados de necropsia e a histopatologia; objetivou-se também avaliar os seguintes testes laboratoriais para o diagnóstico de carbúnculo sintomático: cultura microbiológica e identificação do agente, cultura microbiológica e identificação do agente por reação em cadeia de polimerase (PCR) e identificação do agente em material fixado em formol e incluído em parafina (FFIP). Os critérios para o diagnóstico presuntivo de carbúnculo sintomático incluíram miosite necro-hemorrágica enfisematosa, caracterizada por infiltrado inflamatório, necrose de coagulação de miofribras, edema intersticial, hemorragia e bolhas de gás em meio às miofribras. Cinquenta e nove casos oriundos de 51 surtos foram encontrados, o que corresponde a 1,1% das 5.375 mortes de bovinos investigadas. Em cinco desses casos, amostras do músculo afetado foram cultivadas para clostrídios patogênicos. Amostras semelhantes de outros três animais foram cultivadas para clostrídios e os isolamentos identificados subsequentes por PCR. Doze fragmentos de músculo afetado FFIP foram submetidos a PCR para identificação do agente etiológico. Com exceção de janeiro, os casos de carbúnculo sintomático foram observados em todos os meses do ano com uma maior incidência em junho-outubro. A faixa etária da maioria dos bovinos afetados era de 7-12 anos de idade, mas bovinos mais jovens que 6 meses e mais velhos que 24 meses foram também afetados. Os históricos de vacinação eram escassos nesses surtos. Em 32 surtos havia alguma informação sobre a vacinação, mas em apenas dois casos a vacinação tinha sido realizada adequadamente. Cinquenta e seis casos de carbúnculo sintomático deste estudo eram casos clássicos afetando os músculos esqueléticos. Os músculos mais...(AU)

Blackleg in cattle in the state Mato Grosso do Sul, Brazil: 59 cases

ABSTRACT: This study aimed to review cases of blackleg (Clostridium chauvoei infection) diagnosed in cattle from Midwestern Brazil from 1994 to 2014 considering epidemiological, clinical, necropsy and histopathological findings. Also the following laboratory tests were used for the diagnosis of some cases of blackleg: microbiological culture and identification of the agent, microbiological culture and identification of the agent by the polymerase chain reaction (PCR), and identification of the agent in formalin fixed paraffin embedded tissues (FFPE). Criteria for presumptive diagnosis of blackleg included necrohemorrhagic emphysematous myositis consisting of inflammatory infiltrate, coagulative necrosis of myofiber, interstitial edema, hemorrhage, and gas bubbles between myofibers. Fifty nine cases from 51 outbreaks of blackleg were found, which corresponded to 1.1% of 5,375 cattle deaths investigated. In five of those outbreaks, samples of affected muscles cultures for the identification of pathogenic clostridia were made. Another three samples of similar material were cultured for clostridia with subsequent identification of the isolate by PCR. Twelve samples of FFPE affected muscle fragments were submitted to PCR for identification of the etiological agent. Except for January, cases were observed in each month of the year, with higher numbers in July-October. Most affected cattle were in the age of 7-12 years, but calves younger than 6 month-old and older than 24 months were also observed. Vaccination histories were scarce. In 32 outbreaks some vaccination history was available, but only in two of those vaccination has been carried out properly. In 56 six cases the skeletal muscles were involved. Muscles of the hind limbs were the most affected. In ten cases muscles of the tongue, myocardium and diaphragm were also affected. In three of the cases the visceral form was observed. Deaths occurred after a clinical course of 6-24 hours, but in most cases cattle were found death. Sudden death was the outcome in visceral cases (cardiac) blackleg. Clostridium chauvoei was confirmed to be the cause by culturing in 5 cases, and by PCR and histopatology in 8 cases. Bacterial culture followed by PCR did not demonstrate C. chauvoei. Calculation of the economic impact indicates that blackleg is a frequent disease in the state of Mato Grosso do Sul (MS) that inflicts significant economic loss. The amount of these losses would be reduced through proper vaccination programs against the prevalent strains of C. chauvoei in the region.

RESUMO: Este estudo foi realizado com o objetivo de descrever casos de carbúnculo sintomático (infecção por Clostridium chauvoei) diagnosticados em bovinos do Centro-Oeste brasileiro de 1994-2014, avaliando a epidemiologia, os sinais clínicos, os achados de necropsia e a histopatologia; objetivou-se também avaliar os seguintes testes laboratoriais para o diagnóstico de carbúnculo sintomático: cultura microbiológica e identificação do agente, cultura microbiológica e identificação do agente por reação em cadeia de polimerase (PCR) e identificação do agente em material fixado em formol e incluído em parafina (FFIP). Os critérios para o diagnóstico presuntivo de carbúnculo sintomático incluíram miosite necro-hemorrágica enfisematosa, caracterizada por infiltrado inflamatório, necrose de coagulação de miofribras, edema intersticial, hemorragia e bolhas de gás em meio às miofribras. Cinquenta e nove casos oriundos de 51 surtos foram encontrados, o que corresponde a 1,1% das 5.375 mortes de bovinos investigadas. Em cinco desses casos, amostras do músculo afetado foram cultivadas para clostrídios patogênicos. Amostras semelhantes de outros três animais foram cultivadas para clostrídios e os isolamentos identificados subsequentes por PCR. Doze fragmentos de músculo afetado FFIP foram submetidos a PCR para identificação do agente etiológico. Com exceção de janeiro, os casos de carbúnculo sintomático foram observados em todos os meses do ano com uma maior incidência em junho-outubro. A faixa etária da maioria dos bovinos afetados era de 7-12 anos de idade, mas bovinos mais jovens que 6 meses e mais velhos que 24 meses foram também afetados. Os históricos de vacinação eram escassos nesses surtos. Em 32 surtos havia alguma informação sobre a vacinação, mas em apenas dois casos a vacinação tinha sido realizada adequadamente. Cinquenta e seis casos de carbúnculo sintomático deste estudo eram casos clássicos afetando os músculos esqueléticos. Os músculos mais afetados foram os dos membros pélvicos. Em dez casos os músculos da língua, miocárdio e diafragma estavam também afetados. Apenas três dos casos apresentaram a forma visceral (cardíaca). O curso clínico foi de 6-24 horas, mas na maioria dos casos os bovinos foram encontrados mortos. Em casos da forma visceral ocorria morte súbita. Clostridium chauvoei foi confirmado como o agente causal por cultura em cinco casos e por PCR em amostra FFIP em 8 casos. Cultura bacteriana seguida de PCR do isolado não demonstrou C. chauvoei. Carbúnculo sintomático é uma doença frequente em bovinos no Mato Grosso do Sul podendo provocar importantes prejuízos para os produtores rurais. Esses prejuízos podem ser reduzidos através de um programa de vacinação adequado usando-se vacinas eficazes contra cepas de C. chauvoei prevalentes na região.

Blackleg in a free-range brown brocket deer (Mazama gouazoubira) / Carbúnculo sintomático em veado virá de vida livre (Mazama gouazoubira)

A case of blackleg in a brown brocket deer (Mazama gouazoubira) associated with trauma from being hit by a car in southern Rio Grande do Sul is reported. The clinical signs included fever, dehydration and lethargy that worsened progressively until 36 hours after the accident, when the animal died. In the fore right limb, there was a comminuted closed fracture of the radius and ulna but no skin wounds were observed. Grossly, the musculature of the pelvic limbs presented hemorrhage, edema and emphysema. Microscopically, the muscles of both rear legs had necrosis, edema, hemorrhage and mild inflammatory infiltration of neutrophils. Clostridium chauvoei was cultured from affected skeletal muscles, and it was also detected by immunohistochemistry, confirming a diagnosis of blackleg. The overlapping habitat of cattle and brown brocket deer is proposed as a predisposing factor in this case and alerts to spillover cases maybe happening in this region. In addition, blackleg should be included as differential diagnoses of deer with post-traumatic myositis.(AU)

Descreve-se um caso de carbúnculo sintomático em um veado-virá (Mazama gouazoubira), macho, jovem, resgatado após atropelamento em uma rodovia na região sul do Rio Grande do Sul. O cervídeo apresentou febre, desidratação e letargia, progredindo para a morte em 36 horas. No membro torácico direito foi observado fratura cominutiva fechada de rádio e ulna sem a presença de feridas perfurantes. Na necropsia foi observada hemorragia, edema e enfisema na musculatura dos membros pélvicos. Microscopicamente, os músculos dos membros pélvicos apresentaram necrose, edema, hemorragia e discreto infiltrado inflamatório neutrofílico. Houve o isolamento de Clostridium chauvoei e marcação positiva na técnica de IHQ com anticorpo monoclonal anti-C. chauvoei, confirmando o diagnóstico de carbúnculo sintomático. A sobreposição de habitat entre bovinos domésticos e cervídeos pode ser um fator de risco para esta doença e chama a atenção para casos de "spillover" que podem estar ocorrendo na região. Adicionalmente, sugere-se que o carbúnculo sintomático seja incluído nos diagnósticos diferenciais de cervídeos que apresentam miosite pós-traumática.(AU)

Blackleg in a free-range brown brocket deer (Mazama gouazoubira) / Carbúnculo sintomático em veado virá de vida livre (Mazama gouazoubira)

A case of blackleg in a brown brocket deer (Mazama gouazoubira) associated with trauma from being hit by a car in southern Rio Grande do Sul is reported. The clinical signs included fever, dehydration and lethargy that worsened progressively until 36 hours after the accident, when the animal died. In the fore right limb, there was a comminuted closed fracture of the radius and ulna but no skin wounds were observed. Grossly, the musculature of the pelvic limbs presented hemorrhage, edema and emphysema. Microscopically, the muscles of both rear legs had necrosis, edema, hemorrhage and mild inflammatory infiltration of neutrophils. Clostridium chauvoei was cultured from affected skeletal muscles, and it was also detected by immunohistochemistry, confirming a diagnosis of blackleg. The overlapping habitat of cattle and brown brocket deer is proposed as a predisposing factor in this case and alerts to spillover cases maybe happening in this region. In addition, blackleg should be included as differential diagnoses of deer with post-traumatic myositis.(AU)

Descreve-se um caso de carbúnculo sintomático em um veado-virá (Mazama gouazoubira), macho, jovem, resgatado após atropelamento em uma rodovia na região sul do Rio Grande do Sul. O cervídeo apresentou febre, desidratação e letargia, progredindo para a morte em 36 horas. No membro torácico direito foi observado fratura cominutiva fechada de rádio e ulna sem a presença de feridas perfurantes. Na necropsia foi observada hemorragia, edema e enfisema na musculatura dos membros pélvicos. Microscopicamente, os músculos dos membros pélvicos apresentaram necrose, edema, hemorragia e discreto infiltrado inflamatório neutrofílico. Houve o isolamento de Clostridium chauvoei e marcação positiva na técnica de IHQ com anticorpo monoclonal anti-C. chauvoei, confirmando o diagnóstico de carbúnculo sintomático. A sobreposição de habitat entre bovinos domésticos e cervídeos pode ser um fator de risco para esta doença e chama a atenção para casos de "spillover" que podem estar ocorrendo na região. Adicionalmente, sugere-se que o carbúnculo sintomático seja incluído nos diagnósticos diferenciais de cervídeos que apresentam miosite pós-traumática.(AU)

Histopathological, immunohistochemical and biomolecular diagnosis of myocarditis due to Clostridium chauvoei in a bovine / Diagnóstico anatomopatológico, imuno-histoquímico e biomolecular de um caso de miocardite por Clostridium chauvoei em um bovino

The aim of this study was to report a case of clostridial myocarditis in a bovine in Brazil with emphasis on the pathological findings, isolation and molecular identification associated with the in situ localization of C. chauvoei. The animal, a male Brangus bull with nine months of age, was found dead without prior clinical signs. Multifocal and coalescent areas of necrosis were observed in the myocardium. Rod cells in the cardiac muscle fibers were positive immunostaining for C. chauvoei, while this bacterium was also isolated and identified by polymerase chain reaction (PCR).(AU)

O objetivo deste estudo foi relatar um caso de miocardite clostridial em um bovino no Brasil, com ênfase nos achados patológicos, isolamento e identificação molecular do agente e visualização in situ de C. chauvoei. O animal, um macho da raça Brangus com nove meses de idade, foi encontrado morto sem sinais clínicos prévios. Foram observadas áreas de necrose multifocal e coalescente no miocárdio, com a presença de bastonetes imunomarcados para C. chauvoei nas fibras musculares cardíacas. Uma estirpe de C. chauvoei foi isolada do tecido e sua identidade foi confirmada por reação em cadeia da polimeras (PCR).(AU)

Edema maligno em equino causado por Clostridium chauvoei / Malignant Edema Caused by Clostridium chauvoei in a Horse

Background: Malignant edema occurs when a wound is contaminated by one or a combination of the following bacteria: Clostridium septicum, C. sordellii, C. chauvoei, C. novyi type A, and C. perfringens type A. Although malignant edema has been recognized as a disease of economic importance in domestic ruminants, it has rarely been reported in horses in Brazil. Immunohistochemistry is a method that combines histological, immunological and biochemical techniques which, with the use of specific antibodies, detects the agent in situ. This paper reports a case of malignant edema by Clostridium chauvoei in a horse in Bahia, Brazil. Case: A 4.5-year-old male horse was submitted for necropsy. The clinical history was obtained from the owner. Samples from the abdominal cavity, thoracic cavity, central nervous system and integumentary system were collected. The samples were fixed in 10% formalin, processed routinely for histology, embedded in paraffin, sectioned at five microns, and stained with hematoxylin and eosin. Additionally, histological sections were subjected to immunohistochemistry anti-Clostridium perfringens type A, C. septicum, C. chauvoei, C. novyi type A, and C. sordellii. Positive and negative controls were included. The animal presented difficulty in walking, swelling of the pelvic limbs two days after intramuscular anti-inflammatory treatment, and died seven hours after the onset of clinical signs. Macroscopically, large amounts of hemorrhagic edema and tissue emphysema in the subcutaneous tissue and skeletal muscles were seen. Microscopically, there was moderate bleeding, particularly in subcutaneous areas and between myofiber bundles, with few and discrete degeneration foci. Immunohistochemistry technique was positive for Clostridium chauvoei. Discussion: The diagnosis of malignant edema was confirmed considering clinical and pathological alterations as well as the positive immunohistochemistry result for Clostridium chauvoei. Cases of malignant edema are occasionally observed in horses, and Clostridium perfringens and Clostridium septicum are the major etiological agents involved. Two cases have been reported in Brazil, both caused by Clostridium septicum. As in the present report, the animals developed the disease after administration of intramuscular anti-inflammatory medication. The clinical course of the disease in horses is considered severe, with the first clinical signs occurring between 24 and 48 h post infection. Clinical signs, particularly the marked difficulty in walking, edema, and crepitus of the affected areas, may be considered characteristic. The pathological findings were similar to those described by other authors, which are attributed to toxins produced by the bacteria; however, the lesions described in this study were similar to those reported in less severe infections by Clostridium septicum. Bacterial culture and polymerase chain reaction (PCR) are standards for the identification of Clostridium species; however, immunohistochemistry is considered a very sensitive and specific diagnostic technique. To control the disease, animals may be treated with high doses of penicillin or broad-spectrum antibiotics, which are efficient when used during the early stages of the disease. Prophylactic measures include good hygiene while handling syringes and surgical instruments, as well as annual vaccination of the animals.

Malignant edema caused by Clostridium chauvoei in a Horse / Edema maligno em equino causado por Clostridium chauvoei

Background: Malignant edema occurs when a wound is contaminated by one or a combination of the following bacteria: Clostridium septicum, C. sordellii, C. chauvoei, C. novyi type A, and C. perfringens type A. Although malignant edema has been recognized as a disease of economic importance in domestic ruminants, it has rarely been reported in horses in Brazil. Immunohistochemistry is a method that combines histological, immunological and biochemical techniques which, with the use of specifi c antibodies, detects the agent in situ. This paper reports a case of malignant edema by Clostridium chauvoei in a horse in Bahia, Brazil.Case: A 4.5-year-old male horse was submitted for necropsy. The clinical history was obtained from the owner. Samples from the abdominal cavity, thoracic cavity, central nervous system and integumentary system were collected. The samples were fi xed in 10% formalin, processed routinely for histology, embedded in paraffi n, sectioned at fi ve microns, and stained with hematoxylin and eosin. Additionally, histological sections were subjected to immunohistochemistry anti-Clostridium perfringens type A, C. septicum, C. chauvoei, C. novyi type A, and C. sordellii. Positive and negative controls were included. The animal presented diffi culty in walking, swelling of the pelvic limbs two days after intramuscular anti-infl ammatory treatment, and died seven hours af

Background: Malignant edema occurs when a wound is contaminated by one or a combination of the following bacteria: Clostridium septicum, C. sordellii, C. chauvoei, C. novyi type A, and C. perfringens type A. Although malignant edema has been recognized as a disease of economic importance in domestic ruminants, it has rarely been reported in horses in Brazil. Immunohistochemistry is a method that combines histological, immunological and biochemical techniques which, with the use of specifi c antibodies, detects the agent in situ. This paper reports a case of malignant edema by Clostridium chauvoei in a horse in Bahia, Brazil.Case: A 4.5-year-old male horse was submitted for necropsy. The clinical history was obtained from the owner. Samples from the abdominal cavity, thoracic cavity, central nervous system and integumentary system were collected. The samples were fi xed in 10% formalin, processed routinely for histology, embedded in paraffi n, sectioned at fi ve microns, and stained with hematoxylin and eosin. Additionally, histological sections were subjected to immunohistochemistry anti-Clostridium perfringens type A, C. septicum, C. chauvoei, C. novyi type A, and C. sordellii. Positive and negative controls were included. The animal presented diffi culty in walking, swelling of the pelvic limbs two days after intramuscular anti-infl ammatory treatment, and died seven hours af

Molecular identification of Clostridium chauvoei from common filter paper / Molecular identification of Clostridium chauvoei from common filter paper

Background: Blackleg is an acute and often fatal infection in bovine caused by the bacterium Clostridium chauvoei. The absence of conclusive diagnosis of blackleg usually occurs due to absence of practical and economical methods to send samples to microbiology laboratory. The goal of this work was to verify the possibility of using ordinary fi lter paper as a practical and economically feasible method for collecting, storing and shipping material to the laboratory to be used in a rapid and direct PCR approach to detect Clostridium chauvoei DNA.Materials, Methods & Results: The PCR technique for the diagnosis of blackleg from common fi lter paper was tested for specifi city, sensitivity and feasibility. To test the specifi city, the papers were impregnated with a suspension of the following microorganisms: C. chauvoei, C. perfringens, C. septicum, Bacillus anthracis, Staphylococcus aureus, and Escherichia coli. To test the sensitivity different concentration of C. chauvoei (ATCC 10092) were pipetted on common fi lter paper. To both test, DNA extraction of impregnated ordinary fi lter paper and their respective controls followed the method previously described and tested under different storage times (0 h, 24 h, 72 h and a week later). To test the feasibility, 12 bovine livers were collected and tissues samples were impregnate on common fi lter paper with suspension of C. cha

Background: Blackleg is an acute and often fatal infection in bovine caused by the bacterium Clostridium chauvoei. The absence of conclusive diagnosis of blackleg usually occurs due to absence of practical and economical methods to send samples to microbiology laboratory. The goal of this work was to verify the possibility of using ordinary fi lter paper as a practical and economically feasible method for collecting, storing and shipping material to the laboratory to be used in a rapid and direct PCR approach to detect Clostridium chauvoei DNA.Materials, Methods & Results: The PCR technique for the diagnosis of blackleg from common fi lter paper was tested for specifi city, sensitivity and feasibility. To test the specifi city, the papers were impregnated with a suspension of the following microorganisms: C. chauvoei, C. perfringens, C. septicum, Bacillus anthracis, Staphylococcus aureus, and Escherichia coli. To test the sensitivity different concentration of C. chauvoei (ATCC 10092) were pipetted on common fi lter paper. To both test, DNA extraction of impregnated ordinary fi lter paper and their respective controls followed the method previously described and tested under different storage times (0 h, 24 h, 72 h and a week later). To test the feasibility, 12 bovine livers were collected and tissues samples were impregnate on common fi lter paper with suspension of C. cha

Molecular identification of Clostridium chauvoei from common filter paper

Background: Blackleg is an acute and often fatal infection in bovine caused by the bacterium Clostridium chauvoei. The absence of conclusive diagnosis of blackleg usually occurs due to absence of practical and economical methods to send samples to microbiology laboratory. The goal of this work was to verify the possibility of using ordinary filter paper as a practical and economically feasible method for collecting, storing and shipping material to the laboratory to be used in a rapid and direct PCR approach to detect Clostridium chauvoei DNA. Materials, Methods & Results: The PCR technique for the diagnosis of blackleg from common filter paper was tested for specificity, sensitivity and feasibility. To test the specificity, the papers were impregnated with a suspension of the following microorganisms: C. chauvoei, C. perfringens, C. septicum, Bacillus anthracis, Staphylococcus aureus, and Escherichia coli. To test the sensitivity different concentration of C. chauvoei (ATCC 10092) were pipetted on common filter paper. To both test, DNA extraction of impregnated ordinary filter paper and their respective controls followed the method previously described and tested under different storage times (0 h, 24 h, 72 h and a week later). To test the feasibility, 12 bovine livers were collected and tissues samples were impregnate on common filter paper with suspension of C. chauvoei. The filter paper was stored for 48 h, 72 h and one week. Subsequently, a rapid and direct PCR approach to detect C. chauvoei was performed. All procedures were performed in triplicate and was performed by PCR using the same primers employed to amplify the flic gene encoding flagellin (FliC). There was no cross reaction with any tested microorganism, confirming the specificity of the flic gene previously studied. It was possible to visualize the amplification until the corresponding to 100 CFU. Specific PCR amplification products were visualized in 100% of the trials at 48 h, 70% at 72 h, and 90% within one week of storage at room temperature using direct PCR. Discussion: This report describes a rapid, highly sensitive method for the detection of C. chauvoei DNA from liver tissue bovine samples stored on filter papers. It was observed a high sensitivity and a specificity of 100%. The selection of hepatic tissue was based on previous studies that identified C. chauvoei in this tissue by PCR assays. Besides, blackleg in visceral form can be detected in hepatic tissue but does not in muscle. According to others researchers, the direct PCR procedure exhibits several advantages, such as costs and time reduction through omission of DNA extraction as well as avoid any cross contamination with other agents. However, current substances in the blood and tissues may inhibit the PCR amplification. For this reason, a methanol fixation and preheating the samples before the direct PCR assay was performed, mainly because the amplicon is relatively large (535 bp). Some authors consider the use of direct PCR from filter paper simple and inexpensive which offer a handy tool for epidemiologic studies and to clinicians, particularly in many tropical countries where collection and storage of clinical specimens for this purpose are logistically complicated. Furthermore, this procedure can simplify the material shipment for laboratory diagnosis, since it can also be transported in standard envelops by regular mail. The current results propose the use of the direct PCR from common filter paper as practical and economical alternative to diagnosis of blackleg.

Carbúnculo sintomático em bovinos no Mato Grosso dos Sul: 59 casos

Esse estudo foi realizado com o objetivo de descrever casos de carbúnculo sintomático (infecção por Clostridium chauvoei) diagnosticado em bovinos do Mato Grosso do Sul de 1994-2014, para avaliar a epidemiologia, os sinais clínicos, os achados de necropsia e histopatologia e os seguintes testes laboratoriais para o diagnóstico de carbúnculo sintomático: cultura microbiológica e identificação do agente, cultura microbiológica e identificação do agente por reação em cadeia de polimerase (PCR) e identificação do agente em material fixado em formol e incluído em parafina (FFIP). Os critérios para o diagnóstico presuntivo de carbúnculo sintomático incluiu miosite necro-hemorrágica enfisematosa, associada a infiltrado inflamatório, necrose de coagulação de miofribras, edema intersticial, hemorragia e bolas de gás em meio às miofribras. Cinquenta e nove casos oriundos de 51 surtos foram encontrados, o que corresponde a 1,10% das 5.375 mortes de bovinos investigadas. Em cinco desses casos, amostras de músculo afetado foram cultivadas para clostrídios patogênicos. Outras três amostras de material semelhante foram cultivadas para clostrídios e identificação subsequente por PCR. Doze fragmentos de músculo afetado FFIP foram submetidos a PCR para identificação do agente etiológico. Com exceção de janeiro, os casos de carbúnculo sintomático foram observados em todos os meses do ano com uma maior incidência em junho-outubro. A faixa etária da maioria dos bovinos afetados era 7-12 anos de idade, mas bovinos mais jovens que 6 meses e mais velhos que 24 meses foram também afetados. Os históricos de vacinação eram escassos nesses surtos. Em 32 surtos havia alguma informação sobre a vacinação, mas em apenas dois casos a vacinação tinha sido realizada adequadamente. Cinquenta e seis casos de carbúnculo sintomático deste estudo eram casos clássicos afetando os músculos esqueléticos. Os músculos mais afetados foram os dos membros pélvicos. Em dez casos os músculos da língua, miocárdio e diafragma estavam também afetados. Apenas três dos casos apresentaram a forma visceral (cardíaca). Foi estimado que as mortes ocorreram com curso de 6-24 horas e, na maioria dos casos, os bovinos foram encontrados mortos. Em casos da forma visceral ocorreu morte súbita. C. chauvoei foi confirmado em cinco casos como etiologia pela cultura e identificação do agente e por PCR em amostras de músculo FFIP em oito casos.

This study was performed aiming to describe cases of blackleg (Clostridium chauvoei infection) diagnosed in cattle from Mato Grosso do Sul from 1994-2014 considering epidemiological, clinical, necropsy and histopathological findings and to evaluate the use of the following laboratory tests for the diagnosis of blackleg: microbiological culture and identification, of the agent, microbiological culture and identification of the agent by the polymerase chain reaction (PCR) and identification of the agent in formalin fixed paraffin embedded tissues (FFPE). Criteria for presumptive diagnosis of blackleg included necrohemorrhagic emphysematous myositis associated inflammatory infiltrate, coagulative necrosis of myofiber, interstitial edema, hemorrhage, and gas bubbles interspersed among myofibers. Fifty nine cases from 51 outbreaks of blackleg were found, which corresponded of 1,10% of 5,375 cattle deaths investigated. In five of those samples of affected muscles cultures for the identification of pathogenic clostridia were made. Another three samples of similar material were cultured for clostridia and subsequent identification by PCR. In twelve samples of FFPE affected muscle fragments were submitted to PCR for the identification of the etiological agent. Except for January, cases were observed in every month of the year, with higher numbers in July-October. Most affected cattle were in the age range of 7-12 year old but calves younger than 6 month-old and older than 24month-old were also observed. Vaccination histories were scarce in those outbreaks. In 32 outbreaks some vaccination history was available but only in two of those, vaccination was carried out properly. Fifty six cases of the disease were of classical blackleg affecting the skeletal muscle. Muscle of the pelvic members were the most affected. In ten cases muscles of the tongue, myocardium and diaphragm were also affected. Only three of the cases had the visceral (cardiac) form. It was estimated that deaths occurred within 6-24 hours and in most cases cattle was found death. In cases of visceral form there was sudden death. C. chauvoei was confirmed as the etiology by culture and identification in five cases and by PCR in FFPE samples in 8 cases.

Vaccination protocol and bacterial strain affect the serological response of beef calves against blackleg / Esquemas de vacinação e cepa bacteriana influenciam na resposta sorológica contra o carbúnculo sintomático em bezerros de corte

The serological response of beef calves was evaluated with different vaccination regimens against blackleg, using an official strain (MT) and a field-collected strain of Clostridium chauvoei as antigens. Sixty calves were randomly allocated to four different groups and were submitted to distinct vaccination protocols with a commercial polyvalent vaccine. Group G1 was first vaccinated at four months of age and a booster shot was given after weaning, at eight months. Group G2 was given the first dose at eight months and a booster shot 30 days later. Group G3 was vaccinated only once at eight months and the control group was not vaccinated. These alternative vaccination regimens were proposed in an effort to adequately protect cattle under open-field farming conditions. Serological evaluations were made by Elisa at 4, 8, 9 and 10 months of age. Both groups receiving booster shots had a significantly increased serological response 30 days later. However, the serum IgG levels against C. chauvoei were significantly higher in the calves that were first vaccinated at four months. At 10 months, the two booster shot groups (G1 and G2) had similar serological responses, while the calves that were treated with a single dose of vaccine at weaning (G3) had a response that was similar to that of the control group. The serological response of the calves was significantly inferior at several of the evaluation times when the field strain of the bacteria was used as a challenge antigen instead of the official MT strain. The serological response of calves that are vaccinated twice was found to be satisfactory, independent of the first injection being made at four or eight months of age. It was also concluded that it would be useful to include local bacterial strains in commercial vaccine production.(AU)

Foi avaliada a resposta sorológica de bezerros de corte submetidos a diferentes esquemas de vacinação contra o carbúnculo sintomático, empregando-se como antígenos duas cepas distintas de Clostridium chauvoei: uma oficial (MT) e a outra uma cepa de campo. Os animais (n=60) foram randomizados em quatro grupos (G1, G2, G3 e Controle) e submetidos a três protocolos distintos de vacinação com um produto comercial polivalente. O G1 foi primovacinado aos 4 meses de idade e recebeu o reforço após desmama (8 meses de idade). O G2 recebeu a primeira dose aos 8 meses de idade e reforço 30 dias após. O G3 foi vacinado somente aos 8 meses de idade e o Controle não foi vacinado. As avaliações sorológicas pelo ensaio imunoenzimático (Elisa) foram realizadas aos 4, 8, 9 e 10 meses de idade dos bezerros. Nos dois esquemas em que os animais receberam o booster (G1 e G2), houve um aumento significativo (p<0,05) da resposta sorológica quando foram avaliados 30 dias após. No entanto, os valores séricos de IgG contra C. chauvoei foram significativamente maiores nos animais primovacinados aos 4 meses de idade. Aos 10 meses, os dois grupos que receberam o reforço vacinal (G1 e G2) não diferiram entre si na resposta sorológica e os bezerros que receberam uma única dose de vacina na desmama (G3) não diferiu do Controle. A resposta sorológica dos bezerros foi significativamente inferior (p<0,05) em diversos momentos da avaliação, quando a cepa de campo foi empregada como antígeno e quando comparada à da cepa MT. Pode-se deduzir que a resposta sorológica dos bezerros vacinados aos 4 e 8 meses de idade foi satisfatória e que existiram diferenças significativas nos valores séricos de anticorpos contra C. chauvoei quando na avaliação foi empregada cepa de campo.(AU)

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.(AU)

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.(AU)

Identification of Clostridium chauvoei in clinical samples cultures from blackleg cases by means of PCR

C. chauvoei presence was detected by means of polymerase chain reaction (PCR) from supernatant of culture in cooked meat medium of liver, muscle and metatarsian bone marrow samples of seven calves with blackleg symptoms. The isolation under anaerobic conditions of one muscle sample revealed Clostridium perfringens in pure culture.

Foi detectada presença de Clostridium chauvoei pela reação de PCR a partir de cultivo em cooked meat medium de amostras de fígado, músculo e medula óssea metatarsiana de sete bezerros acometidos de carbúnculo sintomático. O isolamento de uma amostra de músculo sob condições anaeróbias revelou Clostridium perfringens em cultura pura.

Carbúnculo sintomático: ocorrência, evolução clínica e acompanhamento da recuperação de bovino acometido de manqueira

ABSTRACT Clostridium chauvoei infection typically develops in young cattle between 6 months and 2 years of age, and is rarely found affecting calves younger than 6 months of age. Mortality is nearly 100%. In the Clinic of Bovines and Small Ruminants of the FMVZ-USP, assistance was given to a one-year old beef calf with no definite race, vaccinated against Clostridium sp. The animal showed movement troubles of the right anterior member, gas presence with volume and temperature increase on the affected area. The animal was submitted to aseptical cleanliness on the affected region and treated with penicillin against C. chauvoei. Six months later the lesion was reduced and the animal showed only a moderate limp.

RESUMO Clostridium chauvoei causa uma infecção conhecida no Brasil como manqueira e geralmente desenvolve-se em bovinos entre os 6 meses e 2 anos de idade, raramente acomete animais com menos de 6 meses de vida, sendo que a mortalidade é de quase 100%. Um animal de um ano de idade e sem raça definida, vacinado contra as clostridioses entre 10 a 15 dias antes de ser atendido na Clínica de Bovinos do Centro de Pesquisa e Diagnóstico de Enfermidades de Ruminantes (CPDER), FMVZ-USP, apresentava dificuldade de locomoção do membro anterior direito, sendo que na região acometida observou-se mionecrose com crepitação ao toque, e alterações na temperatura do membro acometido. O animal foi tratado com antibioticoterapia sistêmica e limpeza do local da ferida obtendo cura das lesões após 6 meses do início do tratamento, sendo confirmado o diagnóstico de clostridiose pelo isolamento bacteriano do Clostridium chauvoei.