Myonecrosis caused by Clostridium septicum in a horse from Southern Brazil / Mionecrose causada por Clostridium septicum em um cavalo do sul do Brasil

Clostridial myonecrosis is a highly fatal infectious disease of the muscle that is caused by pathogenic clostridia. When it occurs in equine muscle, the infection can cause a variety of clinical signs. The present report described a case of acute myonecrosis caused by Clostridium septicum in a horse, which had a favorable clinical evolution. A 6-year-old mare was admitted to a veterinary hospital due to increased volume and lameness in the left pelvic limb after an intramuscular injection of an anti-inflammatory drug. The diagnosis of myonecrosis caused by C. septicum was based on clinical and laboratory findings. The animal showed significant improvement after 5 months of treatment and was discharged from the hospital. There is very limited information regarding myonecrosis caused by C. septicum in horses in Brazil; our study showed that early diagnosis and prompt appropriate treatment can ensure significant improvement and recovery of affected animals.

Mionecroses clostridiais são doenças altamente fatais, causadas por uma ou mais espécies de Clostridium sp. patogênicos. Entretanto, no músculo equino, a infecção pode produzir uma variedade de lesões e sinais clínicos. O presente relato descreve um caso de mionecrose aguda em um cavalo causada por Clostridium septicum, que apresentou uma evolução clínica favorável. Uma égua de seis anos foi internada em um hospital veterinário devido a um aumento de volume e claudicação no membro pélvico esquerdo, observado após injeção intramuscular de anti-inflamatório. O diagnóstico de mionecrose por C. septicum foi baseado nos achados clínicos e laboratoriais. Houve uma melhora significativa do animal após cinco meses de tratamento, permitindo que o mesmo fosse liberado do hospital veterinário. Considerando que as informações sobre C. septicum causadores de mionecroses em cavalos no Brasil são muito limitadas, este relato demonstra que o diagnóstico precoce e a implementação adequada do tratamento podem garantir uma melhora significativa e a recuperação dos animais afetados.

Clostridial diseases diagnosed in herbivores in Southern Brazil

Background: The genus Clostridium includes a group of Gram-positive, anaerobic bacteria which producing endosporesand produce toxins when encounter conditions favorable to their development. These toxins can be produced and absorbedin the intestinal lumen, as occurs in cases of enterotoxemia (Clostridium perfringens), or are produced in areas of tissuenecrosis after bacterial infections, as seen in tetanus (C. tetani), blackleg (C. chauvoei) and bacillary hemoglobinuria(C. haemolyticum), or in infections by C. chauvoei, C. novyi and C. septicum frequently associated with cases of malignant edema. The aim of this research was relates the epidemiological and clinicopathological aspects of the clostridiosisobserved in the region of infl uence of the Setor de Patologia Veterinária of Universidade Federal do Rio Grande do Sul.Materials, Methods & Results: The necropsy records were reviewed from January 1996 to December 2011 to identifythe cases of clostridiosis that were diagnosed. In the period, 4.689 necropsies were performed by the (SPV-UFRGS). Atotal of 135 cases (2.88%) were associated with clostridiosis. The most prevalent clostridiosis included tetanus (48.15%)in horses, cattle, sheep and goats; botulism (17.04%) in cattle and enterotoxemia (22.96%) in goats. Additional diseaseswere blackleg (5.93%) in cattle, necrotic myositis/malignant edema in horses and sheep and bacillary hemoglobinuria incattle, both with 4 cases each (2.96%).Discussion: Tetanus, enterotoxemia, and botulism were the most prevalent clostrodiosis diagnosed at SPV-UFRGS andtogether accounted for approximately 90% of cases in the period 1996-2011. As for blackleg, bacillary hemoglobinuria,and necrotic myositis/malignant edema, together they represented slightly less than 10% of the clostridioses in the period.The most signifi cant clostridiosis in the period studied was tetanus, affecting cattle, sheep, and horses. There was a largenumber...(AU)

Clostridial diseases diagnosed in herbivores in Southern Brazil

Background: The genus Clostridium includes a group of Gram-positive, anaerobic bacteria which producing endosporesand produce toxins when encounter conditions favorable to their development. These toxins can be produced and absorbedin the intestinal lumen, as occurs in cases of enterotoxemia (Clostridium perfringens), or are produced in areas of tissuenecrosis after bacterial infections, as seen in tetanus (C. tetani), blackleg (C. chauvoei) and bacillary hemoglobinuria(C. haemolyticum), or in infections by C. chauvoei, C. novyi and C. septicum frequently associated with cases of malignant edema. The aim of this research was relates the epidemiological and clinicopathological aspects of the clostridiosisobserved in the region of infl uence of the Setor de Patologia Veterinária of Universidade Federal do Rio Grande do Sul.Materials, Methods & Results: The necropsy records were reviewed from January 1996 to December 2011 to identifythe cases of clostridiosis that were diagnosed. In the period, 4.689 necropsies were performed by the (SPV-UFRGS). Atotal of 135 cases (2.88%) were associated with clostridiosis. The most prevalent clostridiosis included tetanus (48.15%)in horses, cattle, sheep and goats; botulism (17.04%) in cattle and enterotoxemia (22.96%) in goats. Additional diseaseswere blackleg (5.93%) in cattle, necrotic myositis/malignant edema in horses and sheep and bacillary hemoglobinuria incattle, both with 4 cases each (2.96%).Discussion: Tetanus, enterotoxemia, and botulism were the most prevalent clostrodiosis diagnosed at SPV-UFRGS andtogether accounted for approximately 90% of cases in the period 1996-2011. As for blackleg, bacillary hemoglobinuria,and necrotic myositis/malignant edema, together they represented slightly less than 10% of the clostridioses in the period.The most signifi cant clostridiosis in the period studied was tetanus, affecting cattle, sheep, and horses. There was a largenumber...

Presença de Clostridium perfringens em frangos de corte provenientes de aviários da região de Ribeirão Preto – SP / Presence of clostridium perfringens in broiler from chicken farms in Ribeirão Preto-SP

Visando estudar a presença de Clostridium perfringens em frangos de cortes provenientes de aviários da região de Ribeirão Preto-SP, amostras de conteúdo cecal de aves foram pesquisadas quanto a presença desse microrganismo. As amostras foram semeadas em meios seletivos para clostrídios e incubadas anaerobica­mente. As culturas foram identificadas e caracterizadas pelo método de Gram e séries bioquímicas. Posteriormente realizou-se teste de inoculação em camundongos para confirmar patogenicidade. De um total de 560 amostras coletadas, 374(66,78%) foram positivas para o gênero Clostridium, sendo 94 (16,78 %) Clostridium perfringens. Verificou–se que 19 (3,4%) amostras foram positivas para outras espécies de clostrídios patogênicos como Clostridium chauvoei (3 - 0,54 %), Clostridium sordelli (9 - 1,61%), Clostridium bifermentans (3 - 0,54%), Clostridium septicum (3 - 0,54%) e Clostridium tetani (1 - 0,18%).

The aim of the present study was to detect Clostridium perfringens in broiler chickens from intensive poultry farms in Ribeirão Preto-SP. We collected 516 samples of caecal content that were cultivated onto selective medium for Clostridium and incubated under anaerobic conditions. Gram smears were carried out from the positive samples and identified using biochemical tests. Mice inoculation test was performed in order to confirm pathogenicity. The results showed that 66.78% of the samples were positive for Clostridia, of which 16.78% were C. perfringens. It was confirmed that 19 (3.4%) of the samples were positive to other Clostridia pathogenic species such as Clostridium chauvoei (3 - 0.54%), Clostridium sordelli (9 - 1.61%), Clostridium bifermentans (3 - 0.54%), Clostridium septicum (3 - 0.54%) and Clostridium tetani (1 - 0.18%).

Presença de Clostridium perfringens em frangos de corte provenientes de aviários da região de Ribeirão Preto SP / Presence of clostridium perfringens in broiler from chicken farms in Ribeirão Preto-SP

Visando estudar a presença de Clostridium perfringens em frangos de cortes provenientes de aviários da região de Ribeirão Preto-SP, amostras de conteúdo cecal de aves foram pesquisadas quanto a presença desse microrganismo. As amostras foram semeadas em meios seletivos para clostrídios e incubadas anaerobica­mente. As culturas foram identificadas e caracterizadas pelo método de Gram e séries bioquímicas. Posteriormente realizou-se teste de inoculação em camundongos para confirmar patogenicidade. De um total de 560 amostras coletadas, 374(66,78%) foram positivas para o gênero Clostridium, sendo 94 (16,78 %) Clostridium perfringens. Verificou–se que 19 (3,4%) amostras foram positivas para outras espécies de clostrídios patogênicos como Clostridium chauvoei (3 - 0,54 %), Clostridium sordelli (9 - 1,61%), Clostridium bifermentans (3 - 0,54%), Clostridium septicum (3 - 0,54%) e Clostridium tetani (1 - 0,18%).(AU)

The aim of the present study was to detect Clostridium perfringens in broiler chickens from intensive poultry farms in Ribeirão Preto-SP. We collected 516 samples of caecal content that were cultivated onto selective medium for Clostridium and incubated under anaerobic conditions. Gram smears were carried out from the positive samples and identified using biochemical tests. Mice inoculation test was performed in order to confirm pathogenicity. The results showed that 66.78% of the samples were positive for Clostridia, of which 16.78% were C. perfringens. It was confirmed that 19 (3.4%) of the samples were positive to other Clostridia pathogenic species such as Clostridium chauvoei (3 - 0.54%), Clostridium sordelli (9 - 1.61%), Clostridium bifermentans (3 - 0.54%), Clostridium septicum (3 - 0.54%) and Clostridium tetani (1 - 0.18%).(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

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.

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

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.(AU)

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.(AU)

MIONECROSES CLOSTRIDIAIS BOVINAS

ABSTRACT 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.

RESUMO 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.

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.

Mionecrose aguda por Clostridium septicum em equinos / Acute myonecrosis by Clostridium septicum in horses

Descrevem-se dois casos de miosite necrosante causada por Clostridium septicum em equinos. Os dois equinos apresentavam aumento de volume no membro pélvico direito e que se estendia para a região abdominal ventral. Ao corte, essa área era formada por edema sanguinolento e bolhas de gás. Os músculos esqueléticos da região caudal da coxa apresentavam áreas vermelho-escuras, crepitantes, com edema sanguinolento e, ao corte, as áreas mais profundas da musculatura tinham aspecto seco. As principais alterações histopatológicas observadas foram tumefação, vacuolização, necrose hialina e necrose flocular de fibras musculares esqueléticas. Entre as fibras, havia hemorragia, edema e grande quantidade de bacilos com tamanho de 3-6μm. Na coloração de Gram, os bacilos se apresentavam roxos (gram-positivos); quando impregnados pela prata (Warthin-Starry), se mostraram enegrecidos. Nos dois casos, C. septicum foi isolado, em ambiente anaeróbio, do líquido de edema das lesões musculares.(AU)

Two cases of acute necrotizing myositis caused by Clostridium septicum in horses are described. Both horses presented swelling of the right pelvic limb extending to the ventral abdominal region. The cut surface of the affected area revealed blood-stained edema and gas bubbles. The skeletal muscles of the caudal region of the thigh of the affect limbs had dark red discolored areas of blood-stained edema and crepitation; the deep musculature was dry. The main histopathological findings were swelling, vacuolation and hyaline and floccular necrosis of skeletal myofibers; in between myofibers there were hemorrhage, edema and large amounts of bacilli. In both cases, C. septicum was isolated from the edema fluid of muscular lesions.(AU)

Ocorrência de clorostrídios isolados entre 1988 a 2007 / Clostrídios isolates determination between 1988 and 2007 / Ocurrencia De Clostrídios Aislados Entre 1988 A 2007

Este estudo objetivou investigar a ocorrência de clostrídios, a partir de materiais provenientes de municípios do estado do Rio Grande do Sul, entre 1988 a 2007, encaminhados para análise microbiológica. Durante o período analisado, o gênero Clostridium foi isolado em 123 protocolos, destes, 61% foram caracterizados como Clostridium perfringens, 28,45% Clostridium chauvoei, 13,8% Clostridium septicum e 0,8% Clostridium sp. Toxina botulínica foi identificada em soros e conteúdo intestinal de 10 casos analisados. Os isolados foram relacionados, principalmente, a casos de enterotoxemia, gangrena gasosa, carbúnculo sintomático e botulismo. Os resultados demonstram significativo número de isolamento de clostrídios. Ressaltamos a importância do monitoramento das clostridioses em nível nacional devido principalmente, ao forte impacto econômico que estas enfermidades podem desencadear.(AU)

The aim of this study was to determinate the occurrence of Clostridium. The samples were from cities of Rio Grande do Sul state, between 1988 and 2007. During the analyzed period, Clostridium were isolates in 123 protocols. Clostridium perfringens were isolates in 61% of cases, 28.45% Clostridium chauvoei, 13.8% Clostridium septicum and 0.8% Clostridium sp. Botulinum toxin was detected in samples from intestinal contents and serum of 10 analyzed cases. The cases with clostridiuns isolates were related, mainly, enterotoxemia, gas gangrene, blackleg and botulism. Thus, this work demonstrated a significative clostridios occurrence. Is important the nationwide monitoring of clostridial, due mainly to strong economic impact that these diseases can trigger.(AU)

El objetivo de este estudio es la determinación de la ocurrencia del aislamiento de clostrídios a partir de materiales derivados de diversos municipios del estado de Rio Grande do Sul, entre 1988 a 2007. Durante el periodo analizado, el género Clostridium se aisló en 123 protocolos, 61% de los aislados fueron Clostridium perfringens, 28,45% Clostridium chauvoei, 13,8% Clostridium septicum y 0,8% Clostridium sp. La toxina botulínica fue indentificada en suero y contenido intestinal de 10 casos analizados. Los aislamientos se refieren principalmente a casos de enterotoxemia, gangrena gaseosa, botulismo y pierna negra. Los resultados demostraron la significativa ocurrencia de clostrídios. Hacemos hincapié en la importancia de la supervisión de clostridios debido principalmente al fuerte impacto económico que estas enfermedades pueden desencadenar. (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)

Avaliação da potência de vacinas contra clostridium septicum comercializadas no brasil

ABSTRACT Clostridium septicum, a ubiquitous organism, is the main pathogen causing malignant edema. Because of its strong cytotoxic alpha toxin, infections are often lethal. Losses in livestock can be prevented through vaccination with alpha-toxoid vaccines. The antibody response to the C. septicum component of 12 commercial vaccines was evaluated by the mouse serum neutralization test using rabbit and bovine sera. The vaccines coded as T1, T2, T4, T10 and T11 induced titers of alpha antitoxin higher than the minimum test level of 2.5 IU/mL. None of the other vaccines induced the formation of antibodies. The majority of the C. septicum vaccines commercialized in Brazil were unable to induce an immune response as recommended for the control of this product.

RESUMO Clostridium septicum, um organismo ubiquitário, é o principal patógeno responsável pelo quadro de edema maligno. Devido à ação citotóxica da toxina alfa as infecções geralmente são fatais. Para o controle da doença é utilizada a vacinação com toxóide alfa. Neste trabalho, avaliouse a eficiência de 12 vacinas comerciais contra clostridioses, que continham em sua composição C. septicum, através da soroneutralização em camundongos com soros de coelhos e bovinos vacinados. As vacinas codificadas como T1, T2, T4, T10 e T11 apresentaram títulos de antitoxina alfa superiores ao nível mínimo exigido pelo teste de 2,5 UI/mL. As demais vacinas não induziram a formação de anticorpos. Em sua maioria, as vacinas comercializadas no Brasil, contra C. septicum, foram ineficientes em estimular resposta imune compatível com os níveis de teste recomendados para controle deste produto.

Outbreak of gas gangrene in a flock of sheeps and goats / Surto de gangrena gasosa em rebanho de ovinos e caprinos

This report describes an occasion of gas gangrene in a flock of 1200 animals between sheeps and goats in the district of Campo Formoso, Bahia, Brazil. In the period of 24 hours to 5 days after having been vaccined against clostridiosis, 40 sheep and 20 goats came to death. Clostridium novyi and Clostridium septicum were detected by the technique of direct immunofluorescence (DIF) in impression of the muscle tissue of the inoculated area.

Este relato descreve um surto de gangrena gasosa em um rebanho de 1200 animais entre ovinos e caprinos em Campo Formoso, Bahia, Brasil. No período de 24 horas a 5 dias após terem sido vacinados contra clostridioses, 40 ovinos e 30 caprinos vieram a óbito. Clostridium novyi e Clostridium septicum foram detectados pela técnica de imunofluorescência direta (IFD) em impressão do tecido do músculo da área de inoculação.

Edema maligno em suíno

Malignant edema is a fatal infection of skeletal muscles and subcutaneous tissue of ruminants and other animal species, caused mainly by Clostridium septicum, but can occur in association with Clostridium chauvoei, Clostridium sordellii, Clostridium novyi type A and Clostridium perfringens tipo A. The definitive diagnosis of malignant edema must consider clinical data and necropsy lesions, microscopic features, isolation of the agent, direct immunofluorescence, immunohistochemistry and PCR. There are few reports in the literature concerning malignant edema in swine, and, the C. septicum, has been associated with most of the cases. This paper reports a case of myonecrosis and subcutaneous edema in a piglet caused by Clostridium septicum.

Edema maligno em suíno é uma infecção fatal da musculatura esquelética e tecido subcutâneo de ruminantes e outras espécies animais, causada principalmente por Clostridium septicum, embora possa ocorrer em associação com Clostridium chauvoei, Clostridium sordellii, Clostridium novyi tipo A e Clostridium perfringens tipo A. O diagnóstico efetivo do edema maligno deve levar em consideração, além dos dados clínicos e lesões de necropsia, os achados microscópicos, isolamento do agente, imunofluorescência direta, imunoistoquímica e PCR. Embora haja poucos relatos de edema maligno em suínos na literatura, o C. septicum tem sido associado à maioria dos casos. Este trabalho relata um caso de mionecrose e edema subcutâneo em um leitão causado por C. septicum.

Edema maligno em suíno

Malignant edema is a fatal infection of skeletal muscles and subcutaneous tissue of ruminants and other animal species, caused mainly by Clostridium septicum, but can occur in association with Clostridium chauvoei, Clostridium sordellii, Clostridium novyi type A and Clostridium perfringens tipo A. The definitive diagnosis of malignant edema must consider clinical data and necropsy lesions, microscopic features, isolation of the agent, direct immunofluorescence, immunohistochemistry and PCR. There are few reports in the literature concerning malignant edema in swine, and, the C. septicum, has been associated with most of the cases. This paper reports a case of myonecrosis and subcutaneous edema in a piglet caused by Clostridium septicum.

Edema maligno em suíno é uma infecção fatal da musculatura esquelética e tecido subcutâneo de ruminantes e outras espécies animais, causada principalmente por Clostridium septicum, embora possa ocorrer em associação com Clostridium chauvoei, Clostridium sordellii, Clostridium novyi tipo A e Clostridium perfringens tipo A. O diagnóstico efetivo do edema maligno deve levar em consideração, além dos dados clínicos e lesões de necropsia, os achados microscópicos, isolamento do agente, imunofluorescência direta, imunoistoquímica e PCR. Embora haja poucos relatos de edema maligno em suínos na literatura, o C. septicum tem sido associado à maioria dos casos. Este trabalho relata um caso de mionecrose e edema subcutâneo em um leitão causado por C. septicum.

Immunohistochemical detection of Clostridia species in paraffin-embedded tissues of experimentally inoculated guinea pigs / Detecção imunohistoquímica de várias espécies de Clostridia em tecidos parafinizados de cobaias inoculadas experimentalmente

O carbúnculo sintomático é causado pelo Clostridium chauvoei, enquanto o edema maligno é causado pelo C. chauvoei, C. septicum, C. sordellii, C. perfringens tipo A, e/ou C. novyi tipo A. Corpos policlonais anti-C. chauvoei, anti-C. septicum, anti-C. sordellii and anti-C.novyi tipo A foram produzidos em coelhos e purificados em uma coluna de DEAE-celulose. Alíquotas das imunoglobulinas foram conjugadas com isotiocianato de fluoresceína e o restante foi usado na técnica de streptavidina biotina peroxidase (SBP). SBP foi padronizada para detectar C. chauvoei, C. septicum, C. sordellii e C. novyi tipo A em tecidos de cobaias fixados em formol e incluídos em parafina. A mesma foi comparada com a técnica de imunofluorescência direta (IFD). Secções e impressões do músculo da área de inoculação, coração, fígado, baço e rim, foram obtidas para ambas as técnicas. Nenhuma reação cruzada foi observada quando tecidos inoculados com outras espécies de Clostridia foram tratadas com esses quatro anticorpos primários. C. chauvoei e C. septicum foram detectados em todos os espécimes provenientes dos animais inoculados com esses microrganismos, enquanto somente secções de músculo obtidas de todos os animais inoculados com C. sordellii e C. novyi tipo A foram positivas. Os mesmos resultados observados pela técnica de SBP, foram obtidos em impressões de tecidos desses microrganismos corados pela IFD. Os resultados indicam que a técnica de SBP permite a detecção de C. chauvoei, C. septicum, C. sordellii e C. novyi tipo A em tecidos de cobaias fixados em formol e incluídos em parafina.(AU)

Blackleg is caused by Clostridium chauvoei, whereas malignant oedema is caused by C. chauvoei, C. septicum, C. sordellii, C. perfringens type A, and/or C. novyi type A. Anti-C. chauvoei, anti-C. septicum, anti-C. sordellii and anti-C. novyi type A polyclonal antibodies were produced in rabbits and purified in a column of DEAE-cellulose. Aliquots of the antisera were conjugated with fluorescein isothiocyanate and the remaining was used for the streptavidin biotin peroxidase technique (SBPT). SBPT was standardized to detect C. chauvoei, C. septicum, C. sordellii and C. novyi type A in formalin-fixed, paraffin-embedded tissues of guinea pigs. SBPT was compared to a fluorescent antibody technique (FAT). Sections and smears of muscle from inoculation area (MIA), heart, liver, spleen and kidney, were obtained for both SBPT and FAT. Cross-reactions between the different Clostridial species were not observed. C. chauvoei and C. septicum were detected in all specimens from the animals inoculated with these microorganisms, while only sections of muscle obtained from all the animals inoculated with C. sordellii and C. novyi type A were positive. The same results observed by the SBPT, were obtained on tissue smears of these microorganisms stained by the FAT. The results indicate that SBPT is suitable for detection of C. chauvoei, C. septicum, C. sordellii and C. novyi type A in formalin-fixed, paraffin-embedded tissues of guinea pigs.(AU)