Neurologic and cutaneous infection by Clostridium piliforme in a kitten with systemic Tyzzer disease.

Tyzzer disease (TD) is a highly fatal condition of animals caused by Clostridium piliforme and characterized pathologically by enteritis, hepatitis, myocarditis, and occasionally encephalitis. Cutaneous lesions have been reported only rarely in animals with TD, and infection of the nervous system has not been described in cats, to our knowledge. We describe here neurologic and cutaneous infection by C. piliforme in a shelter kitten with systemic manifestations of TD and coinfection with feline panleukopenia virus. Systemic lesions included necrotizing typhlocolitis, hepatitis, myocarditis, and myeloencephalitis. The cutaneous lesions consisted of intraepidermal pustular dermatitis and folliculitis, with necrosis of keratinocytes and ulceration. Clostridial bacilli were identified within the cytoplasm of keratinocytes by fluorescence in situ hybridization, and a PCR assay was positive for C. piliforme. C. piliforme can infect keratinocytes leading to cutaneous lesions in cats with the location suggesting direct contact with contaminated feces as a route of infection.

Incidence of healthcare-associated Clostridioides difficile infection in a quaternary referral university hospital in Brazil.

Clostridioides difficile infection (CDI) is an important cause of diarrhea in hospitals worldwide. The incidence of CDI in Latin America has not yet been standardized. To fill this gap, the present study performed a daily active surveillance, for three months, between April to July of 2021, at a quaternary referral university hospital in Brazil. The incidence density was 9.2 cases per 10,000 patient-days. Cases were associated mostly with ribotypes 014 and 106 (44% and 22%, respectively). Ribotype 027 was not identified. The findings strongly reinforce the need for broad epidemiological studies on the incidence of CDI in Brazilian hospitals to increase the understanding, prevention, and treatment of this infection.

Outbreak of canine distemper and coinfections in a maned wolf (Chrysocyon brachyurus) and in three giant anteaters (Myrmecophaga tridactyla).

Canine distemper outbreak and coinfections in three giant anteaters and in a maned wolf has been described. Three giant anteaters developed respiratory and digestive clinical signs after the introduction of a maned wolf to a Wildlife Rehabilitation Center. The maned wolf and two anteaters died, and one anteater was euthanized. Post mortem and histopathologic exams revealed lesions associated with numerous intraepithelial inclusion bodies, mainly in the respiratory and digestive systems. Infection by distemper virus was confirmed in all animals by RT-PCR and gene sequencing, which revealed the Europe 1/ South America 1 strain, closely related to the strain from Canis familiaris. In addition to distemper, the animals had other comorbidities, such as toxoplasmosis and salmonellosis in the maned wolf and cutaneous candidiasis in an anteater. Considering the chronology of clinical manifestation in both species and the viral characterization, it is possible that the maned wolf was the source of infection to the anteaters. This study demonstrates the importance of implementing biosecurity measures in enclosures that house animals of different species, highlighting the importance of quarantine before introduction of new animals into the same environment.

Effects of vaccination against brucellosis and clostridia on the intake, performance, feeding behavior, blood parameters, and immune responses of dairy heifers calves.

The aim of this study was to identify possible effects of different vaccination strategies (concomitantly or not) against brucellosis and clostridia on intake, performance, feeding behavior, blood parameters, and immune responses of dairy heifers calves. Fifty heifers calves were enrolled [38 Gyr (Zebu, Bos taurus indicus) and 12 5/8 Holstein × Gyr]. At 120 d of age, animals were randomly distributed among 3 groups: B (n = 18), vaccinated against brucellosis; C (n = 14), vaccinated against clostridia and CB (n = 18), vaccinated concomitantly for both. Rectal and thermographic temperatures were evaluated on days -1, 0, 1, 2, 3, 5, 7,10, 14, and 28 relatives to the vaccination day. Feed and water intake, body weight (BW), and feeding behavior were monitored daily by an electronic feeding system. Blood was sampled on days 0, 3, 7, 14, and 28, relative to the vaccination day for determination of glucose and ß -hydroxybutyrate (BHBA) concentrations. Blood sampled on day 0 (prevaccination) and on days 28 and 42 were used to evaluate the immune response against Brucella abortus and clostridia. There was an increase in rectal temperature between the first and the third day postvaccination in the 3 groups. The thermography revealed an increase of local temperature for 7 d on groups B and CB. Group C had increased local temperature for a longer period, lasting for up to 14 d. Dry mater intake was reduced for groups B and CB, but no alteration was observed for group C. No alterations regarding initial BW, final BW, average daily weight gain, and feed efficiency were observed. No differences were observed for the 3 vaccination groups for blood parameters throughout the evaluation period. The concomitant vaccination against brucellosis and clostridia led to lower neutralizing antibody titers against epsilon toxin of Clostridium perfringens and botulinum toxin type C of C. botulinum (C > CB > B). When cellular proliferation assay and serological tests to B. abortus were evaluated, no differences were observed between groups B and CB. The present results indicate that the concomitant vaccination against brucellosis and clostridia has no relevant impact on the intake, performance, and feeding behavior of dairy calves. However, the concomitant vaccination of vaccines against these 2 pathogens impacts animal immunity against clostridial infections.

Gas gangrene in mammals: a review.

Gas gangrene is a necrotizing infection of subcutaneous tissue and muscle that affects mainly ruminants and horses, but also other domestic and wild mammals. Clostridium chauvoei, C. septicum, C. novyi type A, C. perfringens type A, and C. sordellii are the etiologic agents of this disease, acting singly or in combination. Although a presumptive diagnosis of gas gangrene can be established based on clinical history, clinical signs, and gross and microscopic changes, identification of the clostridia involved is required for confirmatory diagnosis. Gross and microscopic lesions are, however, highly suggestive of the disease. Although the disease has a worldwide distribution and can cause significant economic losses, the literature is limited mostly to case reports. Thus, we have reviewed the current knowledge of gas gangrene in mammals.

Type C botulism outbreak in feedlot cattle fed contaminated corn silage.

A large outbreak of botulism in feedlot steers fed corn silage contaminated with Clostridium botulinum neurotoxin type C (BoNT/C) is reported occurring in Midwestern Brazil in August 2017. The onset of the outbreak occurred 15 days after 1700 steers started to be fed the contaminated corn silage. Affected steers were alert and afebrile with varying degrees of flaccid paralysis in various muscle groups. A total of 1100 steers were affected, 1090 of which died within four days. Ten steers recovered after treatment with antitoxin. No gross or microscopic lesions were found in affected steers. The diagnosis was based on epidemiological data, characteristic clinical signs, and positive mouse bioassay results. This outbreak is interesting due to the high number of fatally affected cattle and the on-site diagnostic approach. This case report demonstrates the difficulties in diagnosing and treating botulism in cattle.

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

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

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

Investigation of Clostridium botulinum group III's mobilome content.

Clostridium botulinum group III is mainly responsible for botulism in animals. It could lead to high animal mortality rates and, therefore, represents a major environmental and economic concern. Strains of this group harbor the botulinum toxin locus on an unstable bacteriophage. Since the release of the first complete C. botulinum group III genome sequence (strain BKT015925), strains have been found to contain others mobile elements encoding for toxin components. In this study, seven assays targeting toxin genes present on the genetic mobile elements of C. botulinum group III were developed with the objective to better characterize C. botulinum group III strains. The investigation of 110 C. botulinum group III strains and 519 naturally contaminated samples collected during botulism outbreaks in Europe showed alpha-toxin and C2-I/C2-II markers to be systematically associated with type C/D bont-positive samples, which may indicate an important role of these elements in the pathogenicity mechanisms. On the contrary, bont type D/C strains and the related positive samples appeared to contain almost none of the markers tested. Interestingly, 31 bont-negative samples collected on farms after a botulism outbreak revealed to be positive for some of the genetic mobile elements tested. This suggests loss of the bont phage, either in farm environment after the outbreak or during laboratory handling.

Antitoxin therapy of natural avian botulism outbreaks occurred in Brazil.

Botulism commonly affects water birds and it has recently been observed to be emerging in poultry production. In the present work, outbreaks of botulism in wild native species, such as the black-fronted Piping-guan (Aburria jacutinga), wild duck (Cairina moschata) and its crosses with mallard (Anas platyrhynchos), and domestic chickens (Gallus gallus domesticus) are described. Following treatments with a commercial botulism antitoxin CD, 28 (96.5%) out of 29 animals fully recovered after 24-72 h. The antitoxin therapy was shown to be a useful option for the treatment of affected birds, including those that were severely affected.

Draft Genome Sequences of Five Brazilian Clostridium botulinum Group III Type D/C Strains.

Animal botulism is mainly associated with Clostridium botulinum group III-producing neurotoxin types C, C/D, D, and D/C. In this report, we present the draft genome sequences of the first five strains of Clostridium botulinum type D/C isolated in Brazil and used for vaccination purposes.

ISOLATION AND GENOTYPING OF CLOSTRIDIUM PERFRINGENS FROM FREE-LIVING SOUTH AMERICAN COATI (NASUA NASUA).

The importance of Clostridium perfringens for most wild animal species remains unclear. This study aimed to isolate and genotype C. perfringens in stool samples from free-living South American coati (Nasua nasua) in Brazil. Forty-six free-living N. nasua were trapped and stool samples were collected. Two different protocols for C. perfringens isolation were tested: direct plating onto selective agar and pre-enrichment in broth followed by plating in selective agar. Clostridium perfringens type A was isolated from 15 (32.6%) animals by direct plating and 36 (78.3%) animals by broth PE, and the rate of isolation was significantly different between these two methods (P < 0.01). Twelve of the 36 (33.3%) isolated strains by the PE protocol were positive for the ß-2 toxin-encoding gene (cpb2) whereas the enterotoxin-encoding gene (cpe) and necrotic enteritis like-B toxin gene (netb) were not found. These results suggest that C. perfringens is commonly part of the microbiota of free-living coatis. Additionally, the use of a PE protocol appears to be essential for studies on C. perfringens in this species.

Complete genome sequence of Peptoclostridium difficile strain Z31.

BACKGROUND: Peptoclostridium (Clostridium) difficile is a spore-forming bacterium responsible for nosocomial infections in humans. It is recognized as an important agent of diarrhea and colitis in several animal species and a possible zoonotic agent. Despite the known importance of P. difficile infection in humans and animals, no vaccine or other effective measure to control the disease is commercially available. A possible alternative treatment for P. difficile infection is the use of a nontoxigenic strain of P. difficile as a competitive exclusion agent. However, a thorough knowledge of this strain is necessary for this purpose. We selected P. difficile Z31, a nontoxigenic strain (PCR ribotype 009), for investigation because it prevents P. difficile infection in a hamster model. RESULTS: The genome sequence of P. difficile Z31 is a circular chromosome of 4298,263 bp, with a 29.21 % GC content, encoding 4128 proteins, and containing 78 pseudogenes. This strain belongs to ST 3, clade 1, and has five phage regions in its genome. Genes responsible for resistance to tetracycline and erythromycin were detected and more importantly, Z31 also contains genes that promote spore production and stability, cell attachment, intestinal adherence, and biofilm formation. CONCLUSION: In this study, we present the first complete genome sequence of nontoxigenic P. difficile strain Z31. When the Z31 genome was compared with those of other isolates available in GenBank, including a draft genome of a nontoxigenic strain, several unique regions were evident. Z31 contains no toxin genes, but encodes several non-toxin virulence factors, which may favor host colonization.

Hemorrhagic colitis associated with Salmonella enterica serotype Infantis infection in a captive western lowland gorilla (Gorilla gorilla gorilla) in Brazil.

BACKGROUND: Enteric diseases are among the most common causes of morbidity and mortality in gorillas, and it is often caused by bacteria. METHODS: A thirteen-year-old captive female western lowland gorilla (Gorilla gorilla gorilla) developed hemorrhagic diarrhea. Despite the treatment, the animal died 7 days after the onset of clinical signs. The animal was submitted to a thorough pathological and microbiological evaluation. RESULTS: Pathologic examination revealed a severe acute hemorrhagic colitis, neutrophilic splenitis, glomerulitis, and interstitial pneumonia. Salmonella enterica serotype Infantis was isolated from a mesenteric lymph node. CONCLUSION: A diagnosis of hemorrhagic colitis associated with Salmonella enterica serotype Infantis was established.

A surveillance of enteropathogens in piglets from birth to seven days of age in Brazil / Levantamento dos enteropatógenos de leitões do nascimento a sete dias de idade no Brasil

The purpose of the study was to evaluate the real importance of anaerobic enteropathogens 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 Isospora suis. Among diarrheic piglets, 23.3% were positive for C. difficile, 70% for C. perfringens type A cpb2+, 14.3% for rotavirus and 10% for ETEC. Among non-diarrheic control piglets, 10% were positive for C. difficile, 76.7% for C. perfringens type A cpb2+, 0% for rotavirus, 3.3% for ETEC and 3.3% for I. suis. C. perfringens type C was not detected in any of the animals. Histological lesions characteristic of C. difficile, E. coli and rotavirus were observed. However, no C. perfringens type A suggestive lesions were detected. There was a positive correlation between mesocolon edema and the presence of C. difficile toxins. Although C. perfringens type A cpb2+ was the most frequently detected enteropathogen, there was no association between its presence and diarrhea or macro or microscopic changes. C. difficile and Rotavirus were the most relevant pathogens involved with neonatal diarrhea in this study, and histopathology associated with microbiological test proved to be the key to reach a final diagnosis.

O objetivo do presente estudo foi avaliar a real importância de enteropatógenos anaeróbios e rotavirus em comparação à outros agentes mais comuns como causa de diarreia em leitões até cinco dias de idade. Leitões com 0 a 7 dias de vida, 30 diarreicos e 30 não diarreicos (controles) de 15 granjas diferentes foram eutanasiados e necropsiados. Amostras de jejuno, íleo, colon e ceco foram coletadas e submetidas à detecção dos seguintes enteropatógenos: Escherichia coli enterotoxigênica (ETEC), Clostridium perfringens, Clostridium difficile, rotavirus e Isospora suis. Entre os animais diarréicos, 23.3% foram positivos para C. difficile, 70% para C. perfringens tipo A cpb2+, 14.3% para rotavirus e 10% para ETEC. Entre os leitões não-diarréicos, 10% foram positivos para C. difficile, 76.7% para C. perfringens tipo A cpb2+, 3.3% para ETEC e 3.3% for I. suis. C. perfringens tipo C não foi detectado em nenhum animal. Lesões histológicas características de C. difficile, E. coli e rotavirus foram observadas. Por outro lado, nenhuma lesão sugestiva de C. perfringens foi detectada. Foi possível observar uma correlação positiva entre edema de mesocolon e presença das toxinas A/B. Apesar de C. perfringens tipo A cpb2+ ter sido o patógeno mais encontrado, nenhuma associação com lesões foi encontrada. C. difficile e Rotavirus foram os agentes mais relevantes associados à diarreia neonatal, e ficou demonstrada a relevância de associação de histopatologia com testes de detecção microbiológica para se firmar um diagnóstico.

Vaccination with recombinant Clostridium perfringens toxoids α and ß promotes elevated antepartum and passive humoral immunity in swine.

Due to the increasingly restricted use of antimicrobials in animal production systems, the prevention and control of Clostridium perfringens type A- and C-induced diarrhea in piglets should be based on passive immunization via the prepartum vaccination of sows. Given the current obstacles in the production of conventional clostridial vaccines, the use of recombinant proteins has been considered to represent a promising alternative. In the present study, the neutralizing antibody response of immunized sows and their litters to a bivalent vaccine containing the C. perfringens recombinant toxoids alpha (rTA) and beta (rTB) produced in Escherichia coli was assessed. Rabbits (n=8) and pregnant sows (n=7) were immunized with 200µg of each recombinant antigen using Al(OH)3 as adjuvant. The alpha and beta antitoxin titer detected in the rabbits' serum pool was 9.6 and 20.4IU/mL, respectively. The mean alpha and beta antitoxin titers in the sows' sera were 6.0±0.9IU/mL and 14.5±2.2IU/mL, and the corresponding individual coefficients of variation (CV) were 16.04% and 14.91%, respectively. The mean alpha and beta antitoxin titers in the litters' serum pools were 4.2±0.4IU/mL and 10.9±1.7IU/mL, and the CV between litters was 9.23% and 9.85%, respectively. The results showed that the rTA and rTB proteins produced and tested in the present study induced an immune response and can be regarded as candidates for the development of a commercial vaccine against C. perfringens type A- and C-induced diarrhea in pigs.

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.

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.

Selection of a Clostridium perfringens type D epsilon toxin producer via dot-blot test.

Clostridium perfringens type D produces enterotoxemia, an enteric disease in ruminants, also known as pulpy kidney disease. Caused by epsilon toxin, enterotoxemia is a major exotoxin produced by this microorganism. Epsilon toxin is also the main component of vaccines against this enteric disorder. In this study, a standardized dot-blot was used to choose strains of C. perfringens type D that are producers of epsilon toxin. Clones producing epsilon toxin were chosen by limiting dilution; after three passages, lethal minimum dose titers were determined by soroneutralization test in mice. These clones produced epsilon toxin 240 times more concentrated than the original strain. The presence of the epsilon toxin gene (etx) was verified by polymerase chain reaction. All clones were positive, including those determined to be negative by dot-blot tests, suggesting that mechanisms in addition to the presence of the etx gene can influence toxin production. The dot-blot test was efficient for the selection of toxigenic colonies of C. perfringens type D and demonstrated that homogeneous populations selected from toxigenic cultures produce higher titers of epsilon toxin.