Clostridium perfringens is known to be a universal pathogen in humans, domestic animals and wildlife and is certainly the primary cause of clostridial enteric disease in domestic animals. C. perfringens also causes severe toxaemias in many domesticated animals . Members of the species C. perfringens can be subtyped into five toxinotypes (A, B, C, D and E) according to the production of four major toxins: α, β, ε and ι. Previously, laboratory animal tests and serological methods were used for toxinotyping of C. perfringens , but nowadays a multiplex PCR is used by which all the major toxin genes, alpha toxin gene (plc), beta toxin gene (cpb1), epsilon toxin gene (etx) and iota toxin gene (iap) can be detected . Of the four major toxin genes, only plc is located on the chromosome while the others are located on plasmids . An additional toxin and virulence factor, designated β2-toxin, has recently been found and associated with enteric diseases in domestic animals, especially piglets [4, 9, 14] and horses . The β2-toxin gene (cpb2) is also located on a plasmid.
The stability of the plasmid-borne genes in C. perfringens is crucial for the correct typing of a C. perfringens isolate. It has been discussed previously whether the plasmid-borne genes cpb1, etx and iap may be lost when strains are stored for extended periods . 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 . For typing of C. perfringens isolates, it is therefore recommended to include 5–10 individual colonies in the determination of the toxin type . Another reason for including several colonies is that more than one toxinotype of C. perfringens may be present in a clinical sample. For diagnosis of infection by anaerobic pathogenic bacteria such as C. perfringens, it is necessary to protect the clinical samples from oxygen [2, 7, 12]. A representative sampling in the field and safe transport to the laboratory is crucial for successful culturing and identification of the bacteria. For years, swabs have been used to sample and transport clinical material for bacteriological examination. Swab transport systems containing semisolid media have been developed for the transport of samples for subsequent anaerobic cultivation. These systems have been shown to protect both anaerobic and fastidious aerobic organisms . Preferably the infected material should be cultured immediately after sampling, although delay is common.
The aims of the study were to investigate the survival of C. perfringens and the stability of the plasmid-borne genes cpb1 and etx in a simulated C. perfringens containing tissue sample at 20°C and at 4°C. Furthermore the stability of plasmid-borne genes (cpb1, cpb2 and etx) was assessed after exposure of C. perfringens strains to atmospheric oxygen.