Breast blisters and cellulitis are of serious concern to the turkey industry. The lesions are accompanied by high mortality and carcass condemnation in the processing plants, resulting in heavy losses for turkey producers. Cellulitis in turkeys was named by the US Animal Health Association's Transmissible Diseases of Poultry Committee as one of the top three industry concerns in 2008.

Clostridium perfringens is frequently isolated from breast blister lesions. The preliminary studies at the University of Minnesota have demonstrated that turkeys immunised with a vaccine made from clinical isolates of virulent C. perfringens type A were protected against clostridial cellulitis. It is believed that there are genetic differences in C. perfringens strains that may be associated with their phenotypic expression of virulence or their toxin production.

The objectives of this study included:

• Determine the genetic variation among C. perfringens isolates from cases of cellulitis in turkeys using Multi locus sequence typing (MLST).
• Characterise the toxin profile of C. perfringens and identify immunogenic components recognized by the turkey immune system to clostridial organisms using Difference Gel Electrophoresis (DiGE).

Nagaraja and colleagues characterised seven housekeeping genes and one toxin gene of C. perfringens isolates. The housekeeping genes examined were ddlA, dut, glpK, gmk, recA, sod, tpi and the toxin gene was plc. Among the eight loci examined plc locus had a maximum number of alleles corroborating the fact that plc is highly diverse in nature genetically. The dN/dS ratio indicates that most of these polymorphisms found in the eight genes examined resulted in synonymous substitutions. We compared the level of sequence divergence by MLST from a dendrogram. Our studies indicated two major clusters among the C. perfringens isolates examined. The MLST results indicate that there is considerable genetic diversity among C. perfringens isolates from cellulitis cases unlike previous studies. C. perfringens from cellulitis cases showed no ancesteral relationship with any of the reference strains like SM101, ATCC13124 and Strain13 whose genome sequences are published.

The SDS-PAGE analysis demonstrated more than six C. perfringens reactive proteins of interest. The major secretory toxins we identified from C. perfringens type A isolates cultured from breast blisters and cellulitis cases by MALDI-TOF mass spectrometry were phospholipase, collagenase, hyaluronidase, Dnase, enolase, muramidase, pyruvate kinase and some hypothetical proteins. Hypothetical proteins were observed only in profiles of cellulitis producing C. perfringens isolates for which roles need to be elucidated.

The results suggest involvement of different toxins of C. perfringens that may play a role in pathogenesis and possibly in protective immune response against cellulitis in turkeys, according to Nagaraja and colleagues. Cellulitis inducing C. perfringens we examined differed in their secretory protein profile from non-cellulitis inducers but not in their genomic profiles.

The results of this study enable a better understanding of the genotypic and phenotypic characteristics of C. perfringens type A involved in cellulitis in turkeys, which may contribute to the selection of isolates as vaccine candidates.

April 2010