The development and use of multiplex PCR protocols for the detection of Clostridium perfringens toxin encoding genes cpa, cpb, etx, ia, cpe, netB, and tpeL

Abstract

Clostridium perfringens (CP) is the etiological agent of necrotic enteritis (NE) and gangrenous dermatitis (GD) in poultry as well as CP enterotoxin (CPE)-mediated food poisoning in humans. All these diseases can cause significant problems for the poultry industry. Both NE and GD cause extensive losses due to mortality, reduced performance, and carcass condemnation at processing and CPE food poisoning is one of the top foodborne illnesses in the US. Major virulence factors of CP include the production of numerous toxins and isolates are typed A-E depending upon the varied production of four major mouse-lethal toxins, α, β, ε, and ι. All three diseases are associated with type A CP, although poultry isolates are not known to produce CPE. In the past, α-toxin produced prolifically by type A organisms was thought to be the main virulence factor of NE; however, recent studies have shown that is not the case with all strains. A novel toxin, NetB, was shown to be an important virulence factor for some isolates. Evidence also suggests that isolates with both a netB gene and a gene encoding a second novel toxin, TpeL, tend to be more virulent than those with only netB. The netB gene has been found in a majority of NE isolates from countries where studies have been conducted, although there is little data describing the prevalence of netB and tpeL in the United States. The objective of this study was to develop a multiplex PCR to detect genes encoding the major lethal toxins as well as cpe, netB, and tpeL and use this to screen 238 isolates of CP from poultry disease outbreaks and poultry litter. Although optimization of a single multiplex was not successful, screening of isolates using a multiplex reaction detecting the major lethal toxins and a second multiplex detecting netB and cpe as well as a single loci PCR of tpeL completed the toxin genotyping analysis. Results showed that all isolates but 3 from poultry litter (which were determined to be C. bifermentans, Bacteroides caccae, and a Wolinella spp.) were positive for cpa and only one was positive for cpb. The netB gene was present in only two (4%) of the 49 isolates taken from NE outbreaks and were not present in any isolate from GD or poultry litter. The tpeL gene was present in one necrotic enteritis isolate and one poultry litter isolate. These results suggest that netB is not a necessary virulence factor for the development of necrotic enteritis for many strains of C. perfringens. However, all isolates analyzed in this study originated in Alabama, limiting the conclusions to populations from that region. More research is necessary to determine the prevalence of this gene on a global scale, which would indicate its importance as a virulence factor to the C. perfringens population as a whole.