Rodrigo Otávio Silveira Silva

Potency against enterotoxemia of a recombinant Clostridium perfringens type D epsilon toxoid in ruminants.

Enterotoxemia, a disease that affects domestic ruminants, is caused mainly by the epsilon toxin from Clostridium perfringens type D. Its eradication is virtually impossible, control and prophylaxis are based on systematic vaccination of herds with epsilon toxoids that are efficient in inducing protective antibody production. The use of recombinant toxins is one of the most promising of these strategies. This work evaluates the potency of a Cl. perfringens type D epsilon toxoid expressed by Escherichia coli administered to goats, sheep, and cattle. The etx gene was cloned into the pET-11a plasmid of E. coli strain BL21 to produce the recombinant toxin. Rabbits (n=8), goats, sheep, and cattle (n=5 for each species) were immunized with 0.2mg of the insoluble recombinant protein fraction to evaluate vaccine potency of the epsilon toxoid studied. Antibody titers were 40, 14.3, 26, and 13.1 IU/mL in the rabbit, goat, sheep, and cattle serum pools, respectively. The epsilon toxoid produced and tested in this work is adequate for immunization of ruminants against enterotoxemia.

Detection of A/B toxin and isolation of Clostridium difficile and Clostridium perfringens from foals

REASONS FOR PERFORMING THE STUDY Toxin detection and screening could contribute to knowledge of the transmission patterns, risk factors and epidemiology of Clostridium difficile and Clostridium perfringens. OBJECTIVE To isolate C. difficile and C. perfringens and to detect A/B toxins in faecal samples from diarrhoeic and nondiarrhoeic foals. STUDY DESIGN Cross-sectional observational study. METHODS A total of 153 samples from foals were collected: 139 samples from farms and 14 samples from diarrhoeic foals admitted to a veterinary hospital. The A/B toxins were detected by cytotoxicity assay. All suspected colonies of C. perfringens were subjected to polymerase chain reaction for detection of the major toxin genes (α, β, ε and ι) and for detection of β2-, NetB- and enterotoxin-encoding genes. Furthermore, C. difficile and C. perfringens isolates were evaluated for in vitro antimicrobial susceptibility. RESULTS Seven of 153 (4.6%) samples, all from diarrhoeic foals, were positive for C. difficile A/B toxin. Of these, 5 of 14 (35.7%) were from hospitalised foals, and only 2 of 63 (3.2%) diarrhoeic foal samples were from farms (P = 0.002). Clostridium perfringens was isolated from 31 (20.3%) foals, of which 21 of 76 (27.6%) were diarrhoeic and 10 of 76 (13.2%) were nondiarrhoeic, demonstrating a difference between these 2 groups (P = 0.045). Only 4 strains were positive for the β2-encoding gene (cpb2). All C. difficile and C. perfringens isolates were susceptible to metronidazole and vancomycin. CONCLUSIONS The present report highlights the need for laboratory diagnostics to differentiate C. difficile-associated infection in foals from other causes of diarrhoea to facilitate adequate antimicrobial therapy. POTENTIAL RELEVANCE More studies are needed to clarify the role of C. perfringens as a primary agent of diarrhoea in foals.

Antimicrobial susceptibility of Clostridium perfringens strains isolated from broiler chickens

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

Characterization of polymorphisms and isoforms of the Clostridium perfringens phospholipase C gene (plc) reveals high genetic diversity

Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is encoded by the plc gene and has been implicated in several diseases; however, only a few studies have described polymorphisms in this gene. The aim of this study was to analyze polymorphisms in the Cp-PLC nucleotide and amino acid sequences obtained from isolates from different regions and to compare them to Clostridium phospholipase C sequences deposited in the NCBI database. Environmental samples (sediment, poultry feed, sawdust) and stool samples (from poultry, bovine, swine, horse, caprine, bird, dog, rabbit, toucan) were collected from healthy and sick animals. A total of 73 isolates were analyzed with the majority of samples belonging to the toxin type A subtype and possessing the gene encoding for the beta-2 toxin. Comparison of plc gene sequences from respective isolates revealed a high genetic diversity in the nucleotide sequences of mature Cp-PLC. Sequence comparisons identified 30 amino acid substitutions and 34 isoforms including some isoforms with substitutions in amino acids critical to toxin function. Comparison of sequences obtained in this study to Cp-PLC sequences obtained from the NCBI database resulted in the identification of 11 common haplotypes and 22 new isoforms. Phylogenetic analysis of phospholipase C sequences obtained from other Clostridium species identified relationships previously described. This report describes a broad characterization of the genetic diversity in the C. perfringens plc gene resulting in the identification of various isoforms. A better understanding of sequences encoding phospholipase C isoforms may reveal changes associated with protein function and C. perfringens virulence.

Detection of toxins A/B and isolation of Clostridium difficile and Clostridium perfringens from dogs in Minas Gerais, Brazil.

The objective of this study was to detect C. difficile A/B toxins and to isolate strains of C. perfringens and C. difficile from diarrheic and non-diarrheic dogs in Brazil. Stool samples were collected from 57 dogs, 35 of which were apparently healthy, and 22 of which were diarrheic. C. difficile A/B toxins were detected by ELISA, and C. perfringens and C. difficile were identified by multiplex PCR. C. difficile A/B toxins were detected in 21 samples (36.8%). Of these, 16 (76.2%) were from diarrheic dogs, and five (23.8%) were from non-diarrheic dogs. Twelve C. difficile strains (21.1%) were isolated, of which ten were A+B+ and two were A−B−. All non-toxigenic strains were isolated from non-diarrheic animals. The binary toxin gene cdtB was found in one strain, which was A+B+ and was derived from a non-diarrheic dog. C. perfringens strains were isolated from 40 samples (70.2%). Of these, 18 (45%) were from the diarrheic group, and 22 (55%) belonged to the non-diarrheic group. All isolates were classified as C. perfringens type A and there was an association between the detection of the cpe gene and the presence of diarrhea. Interestingly, ten strains (25%) were positive for the presence of the cpb2 gene. The high rate of detection of the A/B toxins in non-diarrheic dogs suggests the occurrence of subclinical disease in dogs or carriage of its toxins without disease. More studies are needed to elucidate the epidemiology of C. difficile and C. perfringens in dogs and to better our understanding of C. difficile as a zoonotic agent. This is the first study to report the binary toxin gene in C. difficile strains isolated from dogs in Brazil.

Detection of enterotoxin A and cytotoxin B, and isolation of Clostridium difficile in piglets in Minas Gerais, Brazil.

Clostridium difficile has emerged as a major cause of neonatal colitis in piglets, displacing classic bacterial pathogens. However, there is no information regarding the distribution of this microorganism in pig farms in Brazil. In the present study, the presence of toxins A/B and of C. difficile strains in stool samples from 60 diarrheic or non-diarrheic newborn piglets (one to seven days old), from 15 different farms, was studied. The presence of toxins A/B was detected by ELISA and PCR was used to identify toxin A, toxin B and binary toxin gene in each isolated strain. C. difficile A/B toxins were detected in ten samples (16.7%). Of these, seven were from diarrheic and three were from non-diarrheic piglets. C. difficile was recovered from 12 out of 60 (20%) fecal samples. Of those, three strains were non-toxigenic (A-B-) and nine were toxigenic. Of the nine toxigenic strains, four were A+B+ strains and five were A-B+ strains. The presence of binary toxin observed in the present study was much higher (50%) than in previously reported studies. All three non-toxigenic strains were isolated from otherwise healthy piglets. The results suggest the occurrence of neonatal diarrhea by C. difficile in farms in Brazil.

Clostridium difficile infection: main features and occurrence in domestic species in Brazil

Clostridium difficile is an emerging enteropathogen of humans and domestic animals. The bacterium was recently confirmed to be present in foals and dogs in Brazil, with some recent studies suggesting that C. difficile is one of the most important causes of piglet diarrhea in the country. Moreover, some reports also suggest the transmission of this microorganism between animals and humans, raising the possibility that C. difficile is a zoonotic disease. Therefore, the aim of the present review is to describe the main features of C. difficile infection in domestic animals and outline the occurrence of the disease in horses, dogs and pigs in Brazil.

Botulismo em ruminantes causado pela ingestão de cama-de-frango

Botulismo e uma intoxicacao causada pela ingestao das toxinas produzidas pelo Clostridium botulinum, que acomete mamiferos e aves. Neste trabalho e descrito um surto de botulismo em ruminantes, ocorrido em duas propriedades localizadas no municipio de Patos, no Estado da Paraiba, Brasil. Em uma das propriedades, de um total de 88 bovinos, 85 (96,6%) vieram a obito. Na segunda, morreram 145 ovinos (96,7%), 233 caprinos (57,8%) e 30 bovinos (96,8%). Os animais acometidos apresentavam paralisia progressiva, dificuldade de locomocao, sialorreia e dispneia. A morte ocorreu entre 24 e 48 horas apos o inicio dos sinais, por parada cardio-respiratoria. Nenhuma alteracao significativa foi observada no exame post-mortem. O diagnostico de botulismo foi confirmado pela demonstracao das toxinas C e D no conteudo intestinal e na cama-de-frango utilizada na alimentacao dos animais, pela tecnica de soroneutralizacao em camundongos.

Carriage of Clostridium difficile in free-living South American coati (Nasua nasua) in Brazil

The objective of this study was to isolate and characterize Clostridium difficile strains in stool samples from a wild urban mammal, a South American coati (Nasua nasua) in Brazil. Forty-six free-living N. nasua were trapped, and stool samples were collected. C. difficile was isolated from three (6.5%) sampled animals, two strains were toxigenic (A+B+CDT-, PCR ribotype 014/020 and 106) and one was non toxigenic (A-B-CDT-, PCR ribotype 053). The present work confirms that ring-tailed coati (N. nasua) could harbor C. difficile strains, including those PCR ribotypes commonly reported in C. difficile infection in humans.

A surveillance of enteropathogens in piglets from birth to seven days of age in Brazil

ABSTRACT.- Cruz Junior E.C., Salvarani F.M., Silva R.O.S., Silva M.X., Lobato F.C.F. & Guedes R.M.C. 2013. A surveillance of enteropathogens in piglets from birth to seven days of age in Brazil . Pesquisa Veterinaria Brasileira 33(8):963-969 . Escola de Veterinaria, Univer-sidade Federal de Minas Gerais, Av. Antonio Carlos 6627, Cx. Postal 567, Belo Horizonte, MG 30123-970, Brazil. E-mail: guedesufmg@gmail.comThe purpose of the study was to evaluate the real importance of anaerobic enteropa-thogens and rotavirus in contrast to more common agents as cause of diarrhea in piglets within the first week of life. Sixty 1- to 7-day-old piglets, 30 diarrheic and 30 non-diarrheic (control), from 15 different herds were selected, euthanized and necropsied. Samples of the jejunum, ileum, colon, cecum and feces were collected from the piglets and analyzed to determine the presence of the following enteropathogens: enterotoxigenic Escherichia coli (ETEC), Clostridium perfringens types A and C, Clostridium difficile, rotavirus and