A.B. INGHAM1
J.I. ROOD2
R.J. MOORE1
1CSIRO Livestock Industries, Geelong, Vic, Australia;
2Dept of Microbiology, Monash University, Australia
Clostridium perfringens causes several diseases in humans and animals, and produces an alpha toxin that is thought to be a key virulence determinant of Necrotic Enteritis (NE) in chickens. Previously, the alpha toxin from mammalian-derived strains of C. perfringens has shown to be highly conserved in sequence and biochemical properties. Characterisation of the first alpha toxin gene isolated from an avian strain of C. perfringens from a diseased swan (SWCP), showed that the deduced toxin had only 80% amino acid sequence identity with other C. perfringens alpha toxins. We report here that 25 isolates of C. perfringens strains collected from chickens diagnosed with NE from a number of different outbreaks and locations have a highly conserved alpha toxin sequence that is significantly different from the SWCP isolate. We have shown that the predicted alpha toxin from these isolates have at least 98% amino acid sequence identity to alpha toxin from mammalian isolates. Therefore, it is concluded that divergent alpha toxin sequences are not common in avian isolates.
I. Introduction
Clostridium perfringens is a widely distributed pathogen (Hatheway, 1990) commonly isolated from the environment and the gastro-intestinal tract of birds and mammals (Hein and Timms, 1972; Willis, 1984).
C. perfringens isolates are classified into five types (A to E) according to the production of four major toxins (alpha, beta, epsilon, and iota toxins) (MacLennan, 1962; McDonel, 1980). The alpha toxin has been implicated in several diseases (Rood, 1998), including Necrotic Enteritis (NE) in chickens (Baba et al., 1992; Long and Truscott, 1976). The alpha toxin structural gene (plc or cpa) has been isolated and characterised from several strains of C. perfringens (Ginter et al., 1996) and the encoded proteins found to be highly conserved in all but one recently identified strain (Justin et al., 2002). This strain (SWCP) was isolated from a diseased swan. Justin et al. (2002) found that the SWCP alpha toxin had only 80% amino acid sequence identity to the other C. perfringens alpha toxins and questioned if this difference in sequence was typical of all avian isolates.
In this study we examined the encoded alpha toxin sequences from a range of isolates of C. perfringens derived from chickens to determine if the divergent SWCP alpha toxin sequence is common in avian isolates.
II. Methods
The C. perfringens strains used in this study were isolated from chickens displaying clinical signs of NE (Al-Sheikhly and Truscott, 1977). Genomic DNA was prepared as template for PCR by boiling crude cells in water for 3 mins. PCR conditions and reaction concentrations were as described by Meer and Songer (1997). PCR primers were designed from the sequence of C. perfringens strain 13 (Shumizu et al., 2002), and two PCR products, together encompassing the complete plc gene, were amplified and sequenced from each of the 25 strains to determine the amino acid sequence of the encoded alpha toxin. Each alpha toxin gene was sequenced twice, using independently generated templates, to confirm that the changes were not due to sequencing or PCR errors.
III. Results
In each isolate the full-length sequence was predicted to be 398 amino acids. The toxins were all highly conserved in amino acid sequence (Figure 1), and only five different alpha toxin sequence types (I–V) were identified from the 25 isolates sampled from several different NE outbreaks from different locations. All the alpha toxin sequence types from the chicken isolates closely resembled the toxin from the human isolate strain 13 (Shimizu et al., 2002), with greater than 98% identity, but differed considerably from the swan isolate (SWCP), with only 82-84% identity. The SWCP isolate has between 67-70 amino acid differences from the chicken isolates, and of all the changes in the SWCP sequence, only two amino acid changes are found in the field isolates reported here.
IV. Discussion
Sequence type I has only one amino acid difference from the strain 13 sequence and was the most common alpha toxin found in the sampled group. The threonine to alanine substitution is within the putative signal peptide sequence (Titball et al., 1989) and would not be present in the mature protein and therefore cannot affect the properties of the mature toxin (Ginter et al., 1996). Alanine is also found in this position in the alpha toxin signal sequence from strain NCTC8237 (Leslie et al., 1989). Sequence type II has two amino acid differences compared to the strain 13 sequence, and includes the threonine to alanine change at position 13, and an isoleucine to valine substitution at position 373. Sequence type IV contains two amino acid changes – the threonine to alanine change (position 13), and a leucine to methionine alteration (position 54). The latter amino acid substitution is also seen in the alpha toxin from C. perfringens strain 8-6 (Saint-Joanis et al., 1989) and the phospholipase C from Clostridium novyi (Tsutsui et al., 1995). The type V alpha toxin sequence contains three amino acid substitutions compared to strain 13 – the common threonine to alanine substitution at position 13, an aspartic acid to alanine change at position 202 (also found in the SWCP sequence), and an alanine to threonine substitution at position 205. The most distinct alpha toxin sequence type seen in this study was type III. It contains six amino acid changes compared to the strain 13 alpha toxin, including the isoleucine to valine substitution at position 373, and a methionine residue that replaces a lysine residue at position 54. Overall, the amino acid differences detected in this study were minimal compared to the sequences differences observed between SWCP and strain 13. The differences that were found in the alpha toxin sequences of the chicken isolates were all the result of single base substitutions, and did not significantly alter the predicted physical properties of the encoded proteins (MW 45.5kDa, pI 5.58, overall negative charge). Plating each of the strains onto egg-yolk agar (Awad et al., 1995) produced a zone of precipitation around the colonies, indicating that each strain was able to produce functional alpha toxin, although the levels of toxin activity varied. Toxin activity of some strains with identical alpha toxin sequence was markedly different, indicating that the variable toxin levels must be due to differences in strain growth or expression rather than differences in specific activity.
None of the predicted amino acid differences occurred in the active site (Guillouard et al., 1996; Nagahama et al., 1997; Nagahama et al., 1995) or in the calcium binding pocket in the C-terminal domain (Justin et al., 2002), regions of the protein thought to play a key role in membrane-protein interactions. The valine to isoleucine change (position 373) is located in a region that is predicted to be a flexible surface-exposed loop linking two helices (Ginter et al., 1996). However, this change is a conservative substitution that is unlikely to affect the tertiary structure of the protein. In regions of the protein that are reported to be important for structural integrity, such as between residues 87-95 and 100-118 (Naylor et al., 1998), there are no amino acid changes in the chicken isolates.
Williamson and Titball (1993) showed that the protective antibody response to alpha toxin is directed against the C-terminal domain of the protein (amino acids 247-370 in the mature toxin). Unlike SWCP, none of the chicken isolates have any amino acid changes in this C-terminal domain of the toxin. Therefore, it is predicted that animals vaccinated with the C-terminal domain would elicit an immune response against the alpha toxin from these chicken-derived strains of C. perfringens.
In conclusion, the C. perfringens strains from chickens suffering Necrotic Enteritis have highly conserved alpha toxin sequences that closely resemble those of the alpha toxin found in mammalian isolates of C. perfringens, but are significantly different from that of the SWCP isolate obtained from a diseased swan, the only avian derived strain previously characterised.
These results are encouraging for the development of diagnostic tests and vaccines for the control and treatment of C. perfringens infections of commercial chickens as they signify that vaccines and tests used for other C. perfringens infections may be able to be used in this host species.
Acknowledgements
We thank Dr Pat Blackall (Queensland Department of Primary Industries) who supplied several strains of C. perfringens, Dr Ambrosio Rubite and Dr Peter Scott for facilitating access to poultry farms for sample collection, Dr Mark Ford (CSIRO Livestock Industries) for animal handling and disease diagnosis, and the kind support of the Australian Rural Industries Research and Development Corporation (RIRDC) through which this work was funded.
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From Proceedings of the "17th Australian Poultry Science Symposium", New South Wales, Australia.
