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PubMed1999

Typing of sheep clinical isolates and identification of enterotoxigenic Clostridium perfringens strains by classical methods and by polymerase chain reaction (PCR).

A simple procedure was developed to identify toxitypes of Clostridium perfringens of different origins. Ninety strains of C. perfringens were identified by classical bacteriological methods, typing of the strains was done by a seroneutralisation test on mice. Production of enterotoxin was tested and all strains were analysed by PCR using gene of toxin alpha, gene of toxin E, gene of toxin beta and gene of enterotoxin. Simple amplification (amplifying one gene), and duplex and triplex amplification (amplifying two and three genes simultaneously) were performed. In the conditions of the experiment, the PCR method has proved efficacious. The specificity and sensitivity are excellent and superior to those of the classical methods. The prophylaxis of enterotoxaemia in animals is achieved by vaccination, the PCR technique can thus become a first-choice tool for the identification and typing of the C. perfringens strains which initiate these diseases. In turn, this would simplify the development of vaccines adapted to the epidemiological situation.

Animals↗
PubMed1999

Molecular methods for the analysis of Clostridium perfringens relevant to food hygiene.

Clostridium perfringens continues to be a common cause of food-borne disease. It produces an enterotoxin (CPE) which is released upon lysis of the vegetative cell during sporulation in the intestinal tract. Catering premises with insufficient cooling and reheating devices often seem to be the cause of outbreaks of C. perfringens food poisoning. Typing of C. perfringens is of great importance for investigating sources of food poisoning cases and for studying the epidemiology of this microorganism. This report describes the examination of 155 C. perfringens isolates by molecular methods. Isolates were taken from 10 food poisoning outbreaks and cases (n = 34, food and fecal isolates) and from meat and fish pastes (n = 121). Isolates were characterized by plasmid profiling, ribotyping, and/or macrorestriction analysis by pulsed-field gel electrophoresis (PFGE). Results show that all three methods are suitable for classifying C. perfringens isolates below the species level. Ribopatterns and PFGE patterns can be interpreted more easily than plasmid profiling results and can be recommended for contamination studies and epidemiologic investigation of food poisonings associated with C. perfringens.

Animals↗
PubMed1999

Development and evaluation of various enzyme-linked immunosorbent assays for the detection of Clostridium perfringens beta anti-toxins.

The aim of our work was to develop an enzyme-linked immunosorbent assay for the detection of antibodies against the Clostridium perfringens beta toxin. For this purpose, five different ways of performing an enzyme-linked immunosorbent assay were investigated. Positive and negative sera of different animals and partially purified beta toxin were used. In all enzyme-linked immunosorbent assay tests, microplates were first coated with monoclonal antibodies against the C. perfringens beta toxin. Actually, the first three ways of performing enzyme-linked immunosorbent assay proved to be an inhibition or a blocking enzyme-linked immunosorbent assay. In the first of these modifications, the examined serum was added on a microplate after the toxin. In the second two tests, they were added simultaneously after they were incubated together (60 min at room temperature or overnight at 4 degrees C, respectively). An anti-toxin conjugate was used for the detection. It was also used in a competitive enzyme-linked immunosorbent assay, where it was added together with the examined serum on the microplate, to which the toxin was already bound. The fifth way of performing an enzyme-linked immunosorbent assay differed from others by the use of conjugated anti-species immunoglobulin for the detection. The biggest differences in absorbances between positive and negative sera were found in the blocking enzyme-linked immunosorbent assay, where the mixture of the toxin and the examined serum were previously incubated overnight at 4 degrees C. The smallest differences in absorbance were found when anti-species conjugates were used.

Animals↗
PubMed1999

Detection of the beta2 toxin gene of Clostridium perfringens in diarrhoeic piglets in The Netherlands and Switzerland.

The two studies presented here were done to determine the prevalence of the alpha, beta, epsilon and enterotoxin genes and the novel beta2 toxin gene of Clostridium perfringens in neonatal or pre-weaned piglets with diarrhoea or necrotic enteritis. All C. perfringens isolates were positive for the alpha and negative for the epsilon and enterotoxin gene, implying that only non-enterotoxigenic type A and C strains were detected. The most important findings were the relatively high prevalence of the beta2 toxin gene in isolates from diarrhoeic piglets in both studies, and, in one of the two studies, absence of strains with only the alpha and beta toxin gene. These data are supportive for the suggestion of a causal relationship of beta2 toxin-producing strains with digestive tract diseases in piglets.

Animals↗
PubMed1999

The control of necrotic enteritis in sucking piglets by means of a Clostridium perfringens toxoid vaccine.

Necrotic enteritis in sucking piglets constitutes a serious problem in piglet rearing units because of the high morbidity and mortality associated with the disease. The primary causal agent is Clostridium perfringens type C. The beta-toxin plays a decisive role in the pathogenesis of this disease. A toxoid vaccine for use in sows has been developed and studied in field trials. The European Pharmacopoeia Monograph on vaccines for use in animals lays down a method of the efficacy testing based on the immunization of rabbits, the collection of pooled sera and the subsequent assay of anti-toxin antibodies in mice using an appropriate test toxin. The vaccine is regarded as effective if it induces a minimum of 10 IU of beta-anti-toxin per ml of rabbit serum. We have established a range of 17.14-98.23 IU beta-anti-toxin per ml rabbit serum induced by a sample of C. perfringens toxoid vaccine. The vaccine has been used under field conditions in different rearing units at the same time, mostly in the form of emergency vaccinations following the outbreak of disease. The outcome of vaccination was evaluated by recording the total numbers of piglets born alive and the piglet losses. Use of the vaccine, coupled with other measures, resulted in an approximately 30% reduction in the number of losses.

Animals↗
PubMed1999

Liver lesions seen at slaughter as an indicator of necrotic enteritis in broiler flocks.

Historical data of necrotic enteritis incidence and carcass condemnations owing to liver lesions in south-eastern Norway during 1978-1998 revealed covariance in the occurrence of the two variables. In histological examination during the first half of 1998, 33 of 45 sampled carcasses condemned owing to liver lesions showed histopathological changes consistent with earlier described Clostridium perfringens-associated hepatitis. The results suggest that the occurrence of necrotic enteritis in broiler flocks may be monitored by using meat inspection data on liver lesions.

Animals↗
PubMed1999

Direct detection of Clostridium perfringens enterotoxin in patients' stools during an outbreak of food poisoning.

An outbreak of diarrhoea in a hotel affected 25 time keepers attending the 1997 Mediterranean Games. Epidemiological investigation implicated a 'pasta al ragù' consumed at the hotel's restaurant and Clostridium perfringens food poisoning was identified by direct detection of C. perfringens enterotoxin in patients' stools. This report confirms that a careful evaluation of epidemiological features, together with the availability of direct and rapid laboratory methods, may lead to a prompt identification of C. perfringens food poisoning.

Adolescent↗
PubMed1994

Clostridium perfringens in stool, intrapartum antibiotics and gastrointestinal signs in a neonatal intensive care unit.

In 1989, we observed in our neonatal intensive care unit (NICU), an increased number of infants with gastrointestinal signs, including five cases of necrotizing enterocolitis. Clostridium perfringens was found in 26% of newborns (n = 168) and was associated significantly with the occurrence of flatulence, distended abdomen, foul-smelling stools, diarrhea and blood in stool (all p < 0.001). C. difficile was found in 17% of the newborns (n = 72). Cesarean section, low gestational age and low birth weight were significantly associated with C. perfringens in stools (all p < 0.001). Treatment with antibiotics was not associated with occurrence of C. perfringens. However, in infants with C. perfringens, intrapartum antibiotics were associated with increased appearance of abdominal distension (p < 0.05). Thus the antibiotics, which disturb primary colonization, may also favor the pathogenic role of opportunistic gut bacteria, such as C. perfringens.

Anti-Bacterial Agents↗
PubMed1985

The occurrence of antibiotic resistance in Clostridium perfringens from pigs.

Porcine faecal specimens were collected from piggeries in south western Australia. Clostridium perfringens strains were isolated, identified, and examined by disc susceptibility tests for their resistance to several antibiotics. The resultant data were correlated with the known exposure of the animals to antimicrobial agents in the feed or water. The results showed that the percentage of C. perfringens isolates resistant to tetracycline or the macrolide-lincosamide antibiotics was significantly higher from weaners fed one of a number of combinations of antimicrobial agents than was the percentage of resistant strains isolated from the one piggery that did not use antimicrobials. No differences in the levels of chloramphenicol or penicillin resistance were observed. Genetic analyses showed that 3 of the resistant strains carried conjugative R-plasmids which carried the tetracycline resistance determinants.

Animals↗
PubMed1996

Clostridium perfringens food-borne outbreak: an epidemiological investigation.

On 3 April 1994, the Western Sector Public Health Unit was notified of an outbreak of gastroenteritis at a Christian youth camp. Attending the camp were 820 people; 241 (42 per cent) of 574 camp participants who completed a questionnaire reported illness. Of these, 230 met the case definition. Main symptoms reported were stomach cramps (78 per cent), diarrhoea (67 per cent) and nausea (46 per cent). Bacterial analysis of leftover chicken grew 2.3 x 10(7) and 3.3 x 10(7) colonies/g of Clostridium perfringens, and we identified Type A enterotoxin of C. perfringens in four of seven stool samples collected from ill people. Camp participants who consumed chicken at lunch on the second day of the camp were nearly four times as likely to be ill than those who did not (relative risk 3.81, 95 per cent confidence interval 3.07 to 4.72). There were deficiencies in hygiene and food preparation. We highlight the importance of time and temperature controls in food preparation and storage to prevent contamination and subsequent poisoning by C. perfringens or other food pathogens. This investigation demonstrates the importance of a multidisciplinary team when investigating disease outbreaks.

Adolescent↗