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)

Protective efficacy of commercial vaccines against a virulent field strain of Clostridium chauvoei / Eficácia protetiva de vacinas comerciais contra uma cepa de campo virulenta de Clostridium chauvoei

Blackleg is an acute and frequently fatal infection that mainly affects cattle and is caused by Clostridium chauvoei. Formalin-killed, whole-cell vaccines are commonly used to control blackleg. The aim of this study was to verify the protective efficacy of two commercial vaccines against the infection of guinea pigs with two strains of C. chauvoei, a virulent field strain (SBP 07/09) and the reference strain used in official tests (Manguinhos-Teixeira or MT). The strains used in the challenge were characterized by whole genome sequencing, and the minimal inhibitory concentrations of 15 antimicrobials were determined. To assess the protective efficacy, guinea pigs were vaccinated and subsequently challenged with C. chauvoei. All four vaccinated and challenged groups seroconverted after vaccination, while the control group remained seronegative, as determined by indirect ELISA. The identical performance of the two C. chauvoei strains in terms of virulence after challenge and their inability to infect vaccinated animals was correlated with their high genetic homology. Both commercial vaccines showed good protective efficacy against both the reference and field strains. Although C. chauvoei vaccination failures have been reported, the results from our study and others reported high similarity among C. chauvoei strains from all over the world, which suggests that the vaccine failures are not due to antigenic variability but inadequate vaccine management.(AU)

O carbúnculo sintomático é uma infecção aguda e frequentemente fatal que afeta principalmente os bovinos, causada pelo Clostridium chauvoei. A administração de vacinas formolizadas compostas pelas células bacterianas inteiras é comumente utilizada no controle desta doença. O objetivo deste estudo foi verificar, em cobaios, a eficácia protetiva de duas vacinas comerciais contra duas cepas de Clostridium chauvoei, uma cepa de campo virulenta (SBP 07/09) e a cepa de referência utilizada nos testes oficiais (MT, Manguinhos-Teixeira). Adicionalmente, realizar a caracterização das cepas utilizadas no desafio pelo sequenciamento completo do genoma e pela determinação da concentração inibitória mínima frente a 15 antimicrobianos. Para tanto, os cobaios foram vacinados e subsequentemente desafiados com. Os quatro grupos vacinados e desafiados soroconverteram, enquanto que o grupo controle permaneceu soronegativo pelo teste de ELISA indireto. O desempenho idêntico das duas cepas de C. chauvoei após o desafio in vivo e a incapacidade de infectar os animais vacinados está correlacionado com a homologia genética das cepas. Além disso, ambas as vacinas comerciais demonstraram uma adequada eficácia protetiva contra as cepas de campo e de referência. Embora o insucesso das vacinações contra C. chauvoei tenha sido previamente relatado, os resultados deste estudo, juntamente com a alta similaridade genética reportada previamente em cepas provenientes de diferentes continentes sugerem que as falhas vacinais não estão relacionadas à variabilidade antigênica, mas sim ao manejo vacinal inadequado.(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)

Protective efficacy of commercial vaccines against a virulent field strain of Clostridium chauvoei / Eficácia protetiva de vacinas comerciais contra uma cepa de campo virulenta de Clostridium chauvoei

Blackleg is an acute and frequently fatal infection that mainly affects cattle and is caused by Clostridium chauvoei. Formalin-killed, whole-cell vaccines are commonly used to control blackleg. The aim of this study was to verify the protective efficacy of two commercial vaccines against the infection of guinea pigs with two strains of C. chauvoei, a virulent field strain (SBP 07/09) and the reference strain used in official tests (Manguinhos-Teixeira or MT). The strains used in the challenge were characterized by whole genome sequencing, and the minimal inhibitory concentrations of 15 antimicrobials were determined. To assess the protective efficacy, guinea pigs were vaccinated and subsequently challenged with C. chauvoei. All four vaccinated and challenged groups seroconverted after vaccination, while the control group remained seronegative, as determined by indirect ELISA. The identical performance of the two C. chauvoei strains in terms of virulence after challenge and their inability to infect vaccinated animals was correlated with their high genetic homology. Both commercial vaccines showed good protective efficacy against both the reference and field strains. Although C. chauvoei vaccination failures have been reported, the results from our study and others reported high similarity among C. chauvoei strains from all over the world, which suggests that the vaccine failures are not due to antigenic variability but inadequate vaccine management.

O carbúnculo sintomático é uma infecção aguda e frequentemente fatal que afeta principalmente os bovinos, causada pelo Clostridium chauvoei. A administração de vacinas formolizadas compostas pelas células bacterianas inteiras é comumente utilizada no controle desta doença. O objetivo deste estudo foi verificar, em cobaios, a eficácia protetiva de duas vacinas comerciais contra duas cepas de Clostridium chauvoei, uma cepa de campo virulenta (SBP 07/09) e a cepa de referência utilizada nos testes oficiais (MT, Manguinhos-Teixeira). Adicionalmente, realizar a caracterização das cepas utilizadas no desafio pelo sequenciamento completo do genoma e pela determinação da concentração inibitória mínima frente a 15 antimicrobianos. Para tanto, os cobaios foram vacinados e subsequentemente desafiados com. Os quatro grupos vacinados e desafiados soroconverteram, enquanto que o grupo controle permaneceu soronegativo pelo teste de ELISA indireto. O desempenho idêntico das duas cepas de C. chauvoei após o desafio in vivo e a incapacidade de infectar os animais vacinados está correlacionado com a homologia genética das cepas. Além disso, ambas as vacinas comerciais demonstraram uma adequada eficácia protetiva contra as cepas de campo e de referência. Embora o insucesso das vacinações contra C. chauvoei tenha sido previamente relatado, os resultados deste estudo, juntamente com a alta similaridade genética reportada previamente em cepas provenientes de diferentes continentes sugerem que as falhas vacinais não estão relacionadas à variabilidade antigênica, mas sim ao manejo vacinal inadequado.

Acute myonecrosis in horse caused by Clostridium novyi type A

The objective of this study was to describe the first report involving a case of equine acute myonecrosis caused by C. novyi type A with an emphasis on clinical signs, the pathological and bacteriological analysis, and molecular identification of the microorganisms as the key of the definitive diagnosis.(AU)

Atividade antimicrobiana do oleorresina de copaíba (Copaifera reticulata) frente a Staphylococcus coagulase positiva isolados de casos de otite em cães / Antimicrobial activity of copaiba oil (Copaifera reticulata) against coagulase positive Staphylococcus of canine otitis

The aim of this study was to investigate the antimicrobial activity of the oleoresin Copaifera reticulata Ducke against Staphylococcus coagulase positive (SCP) isolated from otitis externa in dogs. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the oleoresin were determined by broth microdilution method. In addition, we verified the antimicrobial susceptibility profile of the isolates of SCP by agar diffusion method. Eight classes of antimicrobial were used to calculate the multidrug resistance. The chemical composition of the oleoresin was performed by gas chromatography coupled to the mass spectrometry (GC/MS), and β-caryophyllene, β-bisabolene, and (E)-α-bergamotene were the main compounds found. The copaiba oleoresin showed a MIC90 of 0.164mg/mL and a CBM90 of 1.3mg/mL. The multidrug resistance was found in 27% of the strains tested. The results suggest that copaiba oleoresin has bacteriostatic and bactericidal activity even in multidrug-resistant coagulase-positive strains.(AU)

O objetivo do presente trabalho foi investigar o potencial antimicrobiano do oleorresina de Copaifera reticulata Ducke em isolados de Staphylococcus coagulase positiva (SCP) provenientes de casos de otite externa em cães. O método de microdiluição em caldo foi utilizado para determinação da concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) de oleorresina de copaíba. Em adição, foi determinado o perfil de suscetibilidade aos antimicrobianos dos isolados de SCP pelo método de difusão em ágar. Oito classes de antimicrobianos foram usadas para o cálculo de multirresistência antimicrobiana. A determinação da composição química do oleorresina de copaíba foi realizada por cromatografia em fase gasosa acoplada à espectrometria de massas (GC/MS), sendo que β-cariofileno, β-bisaboleno e (E)-α-bergamoteno foram os compostos majoritários. O oleorresina de copaíba demonstrou CIM90 de 0,164mg/mL e CBM90 de 1,31mg/mL. A multirresistência foi verificada em 27% das cepas testadas. Os resultados sugerem que o oleorresina de copaíba exerceu atividade bacteriostática e bactericida mesmo em cepas multirresistentes de Staphylococcus coagulase-positiva.(AU)

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.

Moraxella bovoculi em casos de ceratoconjuntivite infecciosa bovina no Rio Grande do Sul / Moraxella bovoculi in cases of infectious bovine keratoconjunctivitis in Rio Grande do Sul, Brazil

A ceratoconjuntivite infecciosa (CI), embora raramente fatal, resulta em perdas econômicas significativas para os rebanhos bovinos e ovinos. Os principais agentes causadores dessa enfermidade são Moraxella bovis e Moraxella ovis. Em 2007 foi descrita uma nova espécie também responsável pela CI e denominada Moraxella bovoculi, que até o presente momento, não havia sido relatada no Brasil. Assim, objetivou-se com este trabalho caracterizar e distinguir 54 isolados de Moraxella spp. de amostras clínicas oriundas de 34 bovinos e 17 ovinos, encaminhadas ao Laboratório de Bacteriologia da Universidade Federal de Santa Maria no período de 1990 a 2011, visando a identificação de M. bovoculi. A distinção dos isolados foi fundamentada nas características genotípicas, pela amplificação parcial da região intergênica 16S-23S e clivagem dos produtos da amplificação com enzima RsaI. Como resultados, 25 (46%) isolados foram caracterizados como M. bovis, 17 (32%) como M. ovis e 12 (22%) como M. bovoculi. Logo, conclui-se que M. bovoculi encontra-se presente no rebanho bovino do Rio Grande do Sul e, portanto, no Brasil.(AU)

Infectious keratoconjunctivitis (IK), although rarely fatal, results in significant economic losses for cattle and sheep farmers. The main causative agents of this disorder are Moraxella bovis and Moraxella ovis. In 2007, a new species also responsible for IK was described. This newly described pathogen, called Moraxella bovoculi, was never reported in Brazil. Therefore, the aim of this study was confirmed the M. bovoculi among the samples analyzed. For this, 54 isolates of Moraxella spp. from clinical samples derived from 34 cattle and 18 sheep, sent to the laboratory of bacteriology from 1991 to 2011 was characterized. Differentiation among the species was based on genotypic characteristics, using partial amplification of 16S-23S intergenic region and cleavage products of amplification with enzyme RsaI. Results showed that 25 isolates (46%) were characterized as M. bovis, 17 (32%) as M. ovis, and 12 (22%) as M. bovoculi. This means that M. bovoculi is present among cattle herds in Rio Grande do Sul and, therefore, in Brazil.(AU)

Necrotizing enteritis associated with Clostridium perfringensType B in chinchillas (Chinchilla lanigera) / Enterite necrosante associado a infecção por Clostridium perfringenstipo B em chinchilas (Chinchilla lanigera)

Four 3-4 month-old chinchillas (Chinchilla lanigera) from a commercial flock of 395 chinchillas, were found dead with evidence of previous diarrhea and prolapsed rectum. A fifth 8 month-old chinchilla died 8 hours after being found recumbent, apathetic, diarrheic and with a prolapsed rectum. Two chinchillas were necropsied and observed gross lesions consisted of extensive hemorrhagic enteritis, mild pulmonary edema and enlarged and yellow liver; this latter finding was particularly prominent in the chinchilla presenting longer clinical course. Histologically there was necrotizing enteritis associated with abundant bacterial rods aggregates in the intestinal surface epithelium and within the lamina propria. In the lungs there were small amounts of pink proteinaceous material (edema) in the interstitium and marked vacuolar hepatocellullar degeneration (lipidosis) in the liver. Anaerobic cultures from the intestinal contents of one of the affected chinchillas yielded Clostridium perfringens. Genotyping of this C. perfringens isolate was achieved by multiplex polymerase chain reaction (mPCR) as C. perfringenstype B due to detection of alpha, beta and epsilon-toxin genes. These findings suggest C. perfringens type B as an important cause of sudden or acute death in chinchillas.(AU)

Quatro chinchilas (Chinchilla lanigera) com 3-4 meses de idade, pertencentes a um criadouro comercial com 395 chinchilas, foram encontradas mortas com evidências de diarreia prévia e prolapso de reto. Uma quinta chinchila, de oito meses de idade, foi encontrada em decúbito, apática, com diarreia e prolopaso de reto, e morreu após oito horas. Duas chinchilas foram submetidas à necropsia. As lesões macroscópicas consistiam de extensa enterite hemorrágica, moderado edema pulmonar e fígado pálido e aumentado de volume; este achado foi particularmente proeminente na chinchila que apresentou curso clínico mais longo. Histologicamente foi observado enterite necrosante associada a numerosos agregados bacterianos na superfície epitelial com invasão da lâmina própria. Nos pulmões foi observada pequena quantidade de material proteináceo róseo amorfo (edema) no interstício e marcada degeneração hepatocelular vacuolar (lipidose). Cultura anaeróbica do conteúdo intestinal de uma chinchila afetada revelou crescimento de Clostridium perfringens. A genotipificação de C. perfringensisolado, realizada por reação em cadeia de polymerase multiplex(mPCR), revelou C. perfringenstipo B pela detecção das tóxinas alfa, beta e épisilon. Estes achados sugerem que infecção por C. perfringenstipo B é uma importante causa de morte súbita ou aguda em chinchilas.(AU)

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