Toxinotyping of Clostridium perfringens in Poultry from Necrotic Enteritis Cases

Clostridium perfringens is well known causative agent of necrotic enteritis in poultry and is mainly caused by Type A toxin. NetB toxin is found to be one of the newly emerging virulent toxin gene which is also responsible for necrotic enteritis. The present study was carried out to characterize and to type the different toxins associated with C. perfringens in NE cases of poultry. For the present study total 125 samples were collected from poultry birds, out of which 50 samples were of intestinal content from diseased birds, 50 cloacal swabs and 25 intestinal content from healthy birds. These samples were further processed for isolation, identification, and toxinotyping of Clostridium perfringens isolates. Onisolation of C. perfringens on blood agar total 43 isolates were found positive showing a pattern of double hemolysis on blood agar. The positive isolates of C. perfringens were further confirmed by using 16S rRNA species specific PCR. After confirmation isolates were processed for toxinotyping mainly targeting cpa, cpb and cpb2 toxins by using multiplex PCR. On toxinotyping it was found that NE in poultry birds were mainly caused by C. perfringens type A. On virulent gene detection of netB toxin, total 4 isolates were found positive for netB toxin. This study pointed out that C. perfringens type A is responsible for development of NE in poultry along with net B toxin which is a new key virulent factor. Further studies of netB toxoid and C. perfringens type A for vaccine production could minimize the clostridial problems in broiler farms

Comparative studies on the efficacy of lincomycin and bacitracin for the control of necrotic enteritis in broiler chickens

Background: The present study was conducted to evaluate the efficacy of lincomycin and/or bacitracin for control of experimentally-induced Clostridium perfringens (CP) infection in broiler chickens.Methods: A total of 100 one-day-old Cobb-mixed chicks were divided into five groups (A, B, C, D and E, each of 20 bird). At the 15th day of age, all birds (except group A) were inoculated orally with CP broth culture (109 CFU/mL). Two days later, drugs were orally administered once daily for five consecutive days as follow; Group A and B were left untreated. Group C, D, and E were treated with lincomycin (0.5 g/l), bacitracin (100 mg/l), lincomycin and bacitracin, respectively. The efficacy of used drugs was estimated based on clinical symptoms, body weight, weight gain, feed conversion rate. Hematobiochemical changes were also determined.Results: Necrotic enteritis in broiler chickens induced a significant decrease in body weight, weight gain, erythrocytic count, hemoglobin content, PCV %, serum proteins, catalase, and superoxide dismutase. Additionally, a marked decrease in serum lipids was obtained. Furthermore, a significant increase in feed conversion rate, leukocytic count, phagocytic activity, phagocytic index, serum total globulin, ? globulin and malondialdehyde coupled with a marked increase in ? and ? globulins were determined. Medication of infected broilers with lincomycin and/or bacitracin improved clinical signs and reduced mortality rate to 8, 6 and 2%, respectively, as well as restored the performance and hematobiochemical alterations.Conclusions: a combination of lincomycin and bacitracin was of considerable value for the control of necrotic enteritis in broiler chickens.

Adhesion and invasion of Clostridium perfringens type A into epithelial cells

ABSTRACT Clostridium perfringens is the causative agent for necrotic enteritis. It secretes the major virulence factors, and α- and NetB-toxins that are responsible for intestinal lesions. The TpeL toxin affects cell morphology by producing myonecrosis, but its role in the pathogenesis of necrotic enteritis is unclear. In this study, the presence of netB and tpeL genes in C. perfringens type A strains isolated from chickens with necrotic enteritis, their cytotoxic effects and role in adhesion and invasion of epithelial cells were evaluated. Six (27.3%) of the 22 C. perfringens type A strains were harboring the tpeL gene and produced morphological alterations in Vero cells after 6 h of incubation. Strains tpeL (-) induced strong cell rounding after 6 h of incubation and produced cell enlargement. None of the 22 strains harbored netB gene. All the six tpeL (+) gene strains were able to adhere to HEp-2 cells; however, only four of them (66.6%) were invasive. Thus, these results suggest that the presence of tpeL gene or TpeL toxin might be required for the adherence of bacteria to HEp-2 cells; however, it could not have any role in the invasion process.

Epizootic amebiasis outbreak in wild black howler monkeys (Alouatta caraya) in a wildlife facility during captivity prior to translocation - Recommendations to wildlife management programs / Surto epizoótico de amebíase em Bugio preto (Alouatta caraya) provenientes de vida livre durante o cativeiro prévio a translocação - Recomendações a programas de manejo de fauna

The capture and quarantine of non-human primates could be necessary in some circumstances including those required under environmental permit. Mortality is undesirable for wildlife management programs and could be related to opportunistic pathogens, for example, deaths due to intestinal protozoa infection outbreaks as described here. Parasitological, necroscopic, microbiological, and molecular tests were used in the diagnosis of severe necrotic enteritis leading to death of three female and two male black howler monkeys (Alouatta caraya) captured and held in quarantine prior to translocation. Parasitological tests showed the presence of cysts of Entamoeba histolytica/dispar (5/5), Entamoeba coli (5/5) and Giardia duodenalis (1/5). Necroscopic assessment revealed areas of severe multifocal necrosis in the intestinal mucosa and submucosa. Histopathological examination revealed the presence of structures morphologically compatible to Entamoeba spp. in all individuals examined. Furthermore, G. duodenalis (1/5) was demonstrated by the nested PCR technique. During temporary captivity of non-human primates in management programs, proper handling protocols, including fast or immediate destination, are suggested in order to mitigate the negative effects of stress and decrease the risk of infections.(AU)

Óbitos por infecções causadas por protozoários intestinais são indesejáveis para programas de manejo de fauna, exigidos no âmbito do licenciamento ambiental e que demandam a captura e quarentena de primatas não humanos. Exames coproparasitológicos, necroscópicos, microbiológicos e moleculares foram utilizados para a confirmação do diagnóstico de uma severa enterite necrótica que levou a óbito três fêmeas e dois machos de Alouatta (A.) caraya capturados e mantidos em quarentena prévia à translocação. Exames coproparasitológicos revelaram a presença de cistos de Entamoeba (E.) histolytica/dispar (5/5), Entamoeba (E.) coli (5/5) e Giardia (G.) duodenalis (1/5). A avaliação necroscópica revelou áreas de necrose multifocal severa na mucosa e submucosa intestinal. A análise microscópica revelou a presença de estruturas morfologicamente compatíveis com trofozoítos do gênero Entamoeba spp. em todos os indivíduos examinados. Além disso, G. duodenalis (1/5) foi demonstrada pela técnica de Nested PCR. Sugere-se que quando for necessário o cativeiro temporário de primatas, deverão ser adotados protocolos de manejo adequados buscando a destinação imediata dos animais, de moto a mitigar os efeitos negativos do estresse e reduzir o risco da ocorrência de infecções.(AU)

Antibacterial activities of bark extracts from Fraxinus rhynchophylla Hance and Geranium koreanum Kom. against clinical strains of Clostridium perfringens in chickens / 대한수의학회지

Necrotic enteritis (NE) caused by Clostridium (C.) perfringens commonly occurs in domestic broiler farms since antibiotic supplementation in poultry feed has been banned. We evaluated the antibacterial activities of medicinal plant extracts against C. perfringens isolates to select alternative compounds for preventing NE. We compared antibacterial activities using two methods and evaluated susceptibilities of the isolates based on minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Two (Fraxinus rhynchophylla Hance [FRH] and Geranium koreanum Kom. [GKK]) of the 30 plant extracts had potent antibacterial activities against C. perfringens ATCC 13124 in two assays. The MIC values for FRH and GKK against 20 C. perfringens isolates were 128~256 microg/mL and 32~128 microg/mL, respectively. The geometric MIC mean values for the two extracts were 147.2 microg/mL and 68.8 microg/mL, respectively. The MBCs for the two extracts against the same strains were 1,024~2,048 microg/mL and 256~1,024 microg/mL, respectively. The geometric mean MIC and MBC for GKK were about two-fold lower than those of FRH. The modified spot-on-lawn assay may be useful for measuring primary antibacterial potential. FRH and GKK are expected to be used as feed additives to prevent or treat NE in veterinary practice.

Genotyping and antimicrobial susceptibility of Clostridium perfringens isolated from Tinamidae, Cracidae and Ramphastidae species in Brazil / Genotipagem e sensibilidade antimicrobiana de estirpes de Clostridium perfringens isolados de espécies de Tinamidae, Cracidae e Ramphastidae no Brasil

The aim of this study was to isolate, genotype and evaluate the antimicrobial susceptibility of Clostridium perfringens found in species Tinamidae, Cracidae and Ramphastidae in Brazil. C. perfringens was isolated in 13 (5%) out of 260 swab samples and five (8.3%) out of 60 stool samples. All strains were classified as C. perfringens type A, and nine (50%) were positive for the beta-2 toxin-encoding gene. No strains were positive for the necrotic enteritis toxin B-like (NetB)-encoding gene. All isolates were susceptible to penicillin, metronidazole and vancomycin, whereas four (22.2%), five (27.8%) and 13 (72.2%) strains were considered resistant to erythromycin, oxytetracycline and lincomycin, respectively.

O objetivo do presente estudo foi isolar, genotipar e avaliar a sensibilidade antimicrobiana de estirpes de Clostridium perfringens de espécies de Tinamidae, Cracidae e Ramphastidae no Brasil. C. perfringens foi isolado de 13 (5%) dos 260 suabes e de cinco (8,3%) das 60 amostras de fezes. Todos os isolados foram classificados como C. perfringens, tipo A, e nove (50%) foram positivos para o gene cpb2, responsável pela produção da toxina beta-2. Nenhuma estirpe foi positiva para o gene que codifica a produção da toxina NetB. Todos os isolados avaliados foram sensíveis à penincilina, metronidazol e vancomicina, enquanto que quatro (22,2%), cinco (27,8%) e 13 (72,2%) foram considerados resistentes à eritromicina, oxitetraciclina e lincomicina, respectivamente.

Clostridium perfringens e a enterite necrótica em frangos: principais fatores de virulência, genéticos e moleculares

Clostridium perfringens é o causador da enterite necrótica que afeta a produção de frangos de corte no mundo todo. Essa bactéria produz diversas toxinas e causa lesões no intestino, tendo como consequências a elevada mortalidade e perdas econômicas devido à baixa produtividade. Nesta revisão são apresentados os principais fatores de virulência, a susceptibilidade aos antimicrobianos e a diversidade genética de C. perfringens isolados de frangos com enterite necrótica.

Clostridium perfringens cause necrotic enteritis affecting the poultry production worldwide. This bacterium produces various toxins and causes lesions in the intestine producing high mortality and economic loss due to the low productivity.In this review, the major virulence factors, antimicrobial susceptibility and genetic diversity of C. perfringens from chickens with necrotic enteritis are showed.

Molecular detection and characterization of cpb2 gene in Clostridium perfringens isolates from healthy and diseased chickens

Clostridium perfringens is an important pathogen in both human and veterinary medicine. Necrotic enteritis (NE) is the most clinically dramatic bacterial enteric disease of poultry induced by C. perfringens. The pathogenicity of this bacterium is associated with the production of extracellular toxins produced by some of its strains, such as beta2 toxin. The exact role of beta2 toxin in NE pathogenesis is still controversial. In the present study, C. perfringens isolates from healthy and diseased poultry flocks from different parts of Iran were analyzed by PCR assay to determine the presence of all variants of the beta2 toxin gene (cpb2). The products of two positive cpb2 PCR reactions were sequenced, compared to each other and to the cpb2 sequences published in GenBank (by multiple alignment and phylogenetic analysis). The current work represents the first study of cpb2 in poultry C. perfringens isolates in Asia, and reports the highest percentage of cpb2-positive isolates in both apparently healthy chickens (97.7%) and those afflicted with NE (94.4 %). The sequenced isolates were classified as atypical. This study did not show a direct correlation between NE occurrence and cpb2 presence.(AU)

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

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

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

Genotipicação de Clostridium perfringens isolados de frangos de corte através da PCR múltipla / Genotyping Clostridium perfringens broiler chickens isolates by multiplex PCR products analyses

Clostridium perfringens (Cp) é uma bactéria aneróbica gram positiva que, além de provocar gangrena gasosa e enterotoxemia em humanos e animais, constitui-se na principal causa de enterite necrótica em aves de criações intensificadas. A identificação dos isolados foi realizada pela reação de lecitinase em ágar TSC-gema de ovo, colônias com dupla hemólise em ágar sangue desfibrinado de eqüino, coloração de Gram e provas bioquímicas. Das amostras analisadas, 171Cp foram isolados em jejuno e íleo de frangos de corte provenientes de um frigorífico da região de Pará de Minas-MG. Cp foi isolado em 62 (49,6 por cento) de 125 amostras de conteúdo lumenal de jejunos e em 109 (87,2 por cento) de igual número de íleos dos frangos de corte. Utilizando-se a técnica da PCR múltipla para genotipicacão das estirpes de Cp, de acordo com os genes para as toxinas principais e letais (cpa, cpb, etx e iA), da toxina cpb2 (cpb2) e enterotoxina (cpe), as estirpes de Cp isoladas foram classificadas em cinco tipos toxigênicos (A-E). Das 62 estirpes de Cp isoladas do jejuno, foram obtidos 42/62 (67,7 por cento) tipo A, 1/62 (1,6 por cento) tipo A com produto de amplificação para o gene da toxina beta2, 0/62 (0 por cento) tipo B, 17/62 (27,4 por cento) tipo C, 1/62 (1,6 por cento) tipo D. Das 109 amostras de Cp isolados do íleo das aves foram obtidos 62/109 (56,9 por cento) tipo A, 3/109 (2,7 por cento) tipo A com produto de amplificação para o gene da toxina beta2, 1/62 (0,9 por cento) tipo B, 38/109 (34,9 por cento) tipo C, 1/109 (0,9 por cento) tipo D. Cp A (60,8 por cento) e Cp C (32,2 por cento) foram os tipos toxigênicos predominantes em conteúdo intestinal de frango de corte. Cinco (2,9 por cento) das 171 amostras de Cp isolados não foram tipificadas. Não foram detectados os genes codificadores das toxinas iota (iA) e enterotoxina (cpe) em nenhuma das 171 estirpes de Cp caracterizados.

Clostridium perfringens (Cp) is an anaerobic gram-positive bacterium which causes gaseous gangrene and enterotoxaemias in humans and domestic animals, besides being the primary cause of necrotic enteritis in poultry. Cp isolates were preliminary identified according to the lecithinase test on agar TSC-egg yolk, colony with double haemolysis in desfibrinated horse blood agar, Gram staining and biochemical tests. Cp isolates (171) were obtained from the intestinal content of broiler chickens sampled in a slaughterhouse in Pará de Minas city, MG, Brazil. Cp was isolated in 62/125 (49.6 percent) strains from jejunum content and in 109/125 (87.2 percent) of ileum. Cp strains were classified into five toxigenic types (A-E), using multiplex PCR assay for genotyping of the principal and lethal toxins in the detection of genes coding for toxins alfa, beta, epsilon e iota, nomely genes cpa, cpb, etx e iA genes, beta2 toxin (cpb2) and enterotoxin (cpe). From a total of 62 Cp jejunum isolates obtained 42/62 (67.7 percent) were type A, 1/62 (1.6 percent) type A with the amplification of products for beta2 toxin gene (A/B2), 0/62 (0 percent) type B, 17/62 (27.4 percent) type C and 1/62 (1.6 percent) type D. A total of 109 ileum Cp isolates were obtained being 62/109 (56.9 percent) type A, 3/109 (2.7 percent) type A/B2 toxin gene, 1/62 (0.9 percent) type B, 38/109 (34.9 percent) type C, 1/109 (0.9 percent) type D. Cp A (60.8 percent) and Cp C (32.2 percent) toxigenic types were the most prevalent types in the analyzed intestinal contents of broiler chickens Cp A 104/171 (60.8 percent) and 55/171 (32.2 percent) toxigenic types which were the most prevalent types analyzed into two partes of the intestinal content of broiler chickens. Five (2.9 percent) out of 171 Cp isolates were not typified. The iota toxin (iA) and enterotoxin gene (cpe) codifying genes were not identified.

Enterite necrótica aviária: [revisão] / Avian necrotic enteritis: [review]

A Enterite Necrótica Aviária (ENA) é uma enterotoxemia aguda que aparece subitamente e provoca morte rápida, afetando principalmente animais jovens. Embora seu impacto negativo na produção, devido ao aumento da conversão alimentar e da condenação de carcaças seja já conhecido, questões relacionadas à etiologia, à patogenia e ao controle desta importante enfermidade necessitam de maiores esclarecimentos. Nos últimos anos, o controle da ENA baseou-se na aplicação de antibióticos na ração animal, prática banida pelo mercado consumidor, que exigiu o desenvolvimento de novas estratégias de controle. Esta revisão aborda informações sobre a etiologia, a epizootiologia, a patogenia, o diagnóstico e o controle da doença, em especial a utilização de probióticos e vacinas como alternativas de controle da ENA.

Avian Necrotic Enteritis is an acute enterotoxaemia that appears suddenly producing rapid deaths, affecting mainly young animals. Although its negative impact in poultry production is already known, factors related to etiology, pathogenesis and control of this important disease need better clarifications. For a long time its control was based on the use of antibiotics in poultry feed, whose the use was banned by several consumer markets, requiring the development of new control strategies. Informations on the etiology, epizootiology, pathogenesis, diagnosis and control are reviewed, emphasizing the role of probiotics and vaccines as control alternatives.