Occurrence of Campylobacter spp. in Italian rabbit farms

In order to investigate the occurrence of Campylobacter spp. in rabbits reared in intensive and rural farms, the caecal contents of 39 animals from 13 different farms (3 rabbits per farm) were collected from April to November 2007. The whole intestinal tract from each rabbit was obtained just after evisceration at the slaughterhouse or during necroscopy, and processed within 4 hours. Approximately 5 g of caecal contents were squeezed into 5 ml of sterile saline and shaken in order to obtain a homogenous suspension. Samples were inoculated by streaking 10 μl of each suspension directly onto four different selective fresh media: Blaser-Wang’s Agar (Oxoid), Skirrow’s Agar (Oxoid), Nutrient Agar N°2 (Oxoid) 5% sheep blood plus CAT Selective Supplement (CAT, Oxoid) and modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA, Oxoid). In addition, samples were inoculated on a non selective medium such as Nutrient Agar N°2 (Oxoid) 5% sheep blood using a modified filter technique of Steele & McDermott. All plates were incubated in a jar at 37°C±1 under a microaerobic atmosphere with hydrogen and examined daily for growth up to 12 days. From each sample, 3 colonies showing the same morphotype referable to Gram negative, curved or spiral rod bacteria, were cloned. All the selected colonies were subjected to genus-specific PCR for Campylobacter. Positive isolates were submitted to the PCRs specific for C. jejuni, C. coli, C. upsaliensis, C. helveticus and C. lari. The isolates which resulted negative to the species-specific PCRs were subjected to rpoB sequence phylogenetic analysis. A total of 36 out of 39 animals (92.3%) and all the 13 farms resulted positive for Campylobacter. All isolates were positive for Campylobacter genus PCR but negative for all the species-specific PCRs tested. Phylogenetic analysis based on the partial nucleotide rpoB sequences of 13 isolates (one strain per farm) randomly selected and the reference strains showed that all the rabbit isolates clustered together in a tight clade. This cluster was clearly separated from all the other Campylobacter species with high bootstrap values (100), indicating that these isolates may belong to a new species. This survey allowed reporting the occurrence of a probably new Campylobacter species in the caecal contents of farmed rabbits in Italy. Further studies are necessary to describe it and evaluate its possible pathogenic effect on rabbit as well as the eventual zoonotic role.