Diagnosis of Clostridium perfringens Type C Enteritis in Pigs using a DNA Amplification Technique (PCR)

Abstract

Summary Clostridium perfringens type C, which produces α‐ and β‐toxin, causes severe haemorrhagic and necrotic enteritis in animals and humans. A polymerase‐chain‐reaction (PCR) assay was developed for the specific detection of the genes encoding α‐, β‐, ε‐ and enterotoxin of C. perfringens for rapid typing of C. perfringens strains, and especially for the identification of type C strains. Both the α‐ and β‐toxin genes were detected directly in porcine C. perfringens type C cultures and also in type B and type C collection strains to a sensitivity of 103 cells without purification of the DNA. The α‐toxin gene was detected in all types of C. perfringens. The ε‐toxin gene was found in type B and type D, and the enterotoxin gene in some type A strains. Nine other species of Clostridium and a variety of intestinal pathogenic bacteria showed no signal for these toxin genes in this PCR assay. The α‐ and β‐toxin genes PCR assay were used to identify C. perfringens strains isolated from intestinal contents of 36 necropsied piglets that had suddenly died or died after premonitory signs of diarrhoea. At necropsy, 20 piglets showed necrotizing enteritis (15 acute and 5 chronic cases) and were suspected to have suffered from a C. perfringens type C infection. All of them had C. perfringens which gave a positive PCR signal for α‐ and β‐toxin genes, and, hence, were identified as type C strains. From the 16 other piglets with lesions other than necrotizing enteritis, C. perfringens strains with the α‐toxin gene, but no β‐toxin gene, were isolated. The necropsy findings and the anamnesis showed a very good correlation with the PCR identification of toxin genes. It was therefore concluded that the PCR‐based toxin gene examination is a good alternative to the time‐consuming, less specific, and more expensive mouse neutralization test in the routine diagnostic laboratory.

Molecular Epidemiology of Clostridium perfringens Related to Food-Borne Outbreaks of Disease in Finland from 1984 to 1999

“…The sequences of the oligonucleotide primers for the cpe enterotoxin gene were selected from the sequence published by Van Damme-Jongsten et al in 1989, taking into account the differences noted in the publication of Czeczulin et al in 1993 (7).…”

“…Two microliters of lysed cells was added into PCR mixture as described earlier (7). PCR was performed in an Eppendorf (Hamburg, Germany) Mastercycler Gradient or Hybaid (Ashford, Middlesex, United Kingdom) PCR Sprint Temperature Cycling apparatus.…”

“…Many genotypic methods, including plasmid analysis (14,24,35), ribotyping (15,34), PCR (7,12,20), pulsed-field gel electrophoresis (PFGE) (10,25,30), and amplified-fragment length polymorphism (27), have already been used, and their discriminatory powers in the study of C. perfringens isolates have been tested. PFGE has been used to study the location of the cpe gene and the isolates associated with non-food-borne human gastrointestinal diseases (10,11,19,37).…”

Clostridial Enteric Infections in Pigs

“…Use of polymerase chain reaction (PCR) methods to detect genes for the major toxins has largely replaced toxin detection assays in typing of isolates, and can provide useful supportive findings in diagnosis. 7,28,42 For CPB detection in intestinal contents, mouse protection tests have been largely replaced by enzyme immunoassays. However, CPB is highly protease sensitive and breaks down readily in intestinal contents.…”

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“…It is assumed that C. perfringens plasmid-borne genes are unstable, a matter that has to be taken into consideration in microbiological laboratory diagnosis of clostridial infections (Buogo et al 1995). For typing of C. perfringens isolates, it is therefore recommended to include 5-10 individual colonies in the determination of the toxin type (Buogo et al 1995). Another reason for including several colonies is that more than one toxinotype of C. perfringens may be present in a clinical sample.…”