Preliminary molecular evaluation of the toxigenicity of Clostridium difficile strains isolated from dogs in the area of Parma (Italy)

Abstract number: P787

Ossiprandi M.C., Zerbini L.

Objectives:Clostridium difficile has been associated with canine acute and chronic large and small bowel diarrhoea, as well with acute haemorrhagic diarrhoeal syndrome. Reports have documented a variable carriage rate of C. difficile ranging from 0–40% in diarrhoeic and non-diarrhoeic dogs.

The purpose of this study was to evaluate the molecular characteristics of C. difficile strains isolated from diarrhoeic and non-diarrhoeic dogs by using PCR toxin gene profile.

Methods: Faecal samples were collected from 95 diarrhoeic and non-diarrhoeic dogs, tested for the presence of C. difficile toxins A/B with a commercially human EIA (Remel), and cultured onto pre-reduced selective medium before and/or after thermal shock.

Preliminary identification of C. difficile was based on lack aerotolerance, colony appearance, odour, and cellular morphology following Gram staining. Species identities were confirmed through a rapid latex slide agglutination test (Oxoid) and Rapid ID32A (bioMérieux). All C. difficile isolates were PCR-screened for the presence of tcdA/tcdB and cdtA/cdtB genes, as previously described by Spigaglia and Mastrantonio (2002) and Stubbs (2000), respectively. Toxigenic strains were tested for in vitro toxin production by EIA.

Results:C. difficile strains were isolated from 10 of 95 canine faecal specimens (10.5%). Eight of the samples (80%) belonged to diarrhoeic dogs: 4 dogs were subjected to antibiotic treatment and the enteritis followed the therapy, 1 with megaoesophagus was treated for enteritis and C. perfringens was also isolated, 3 were not treated.

The majority of C. difficile isolates (6/10, 60%) were toxigenic (tcdA+/tcdB+) and possessed ctdA and ctdB genes. All faecal samples tested by EIA were negative. On the contrary, all PCR-positive strains were positive for in vitro toxin production.

Conclusion: The results of this study suggest that commercially human EIA is inadequate for the diagnosis of canine C. difficile-associated diarrhoea when tested on faecal specimens, but it may be useful when used on toxigenic isolates. Moreover, based on our results, the isolation rates of C. difficile from diarrhoeic dogs (80.0%) and non-diarrhoeic dogs (20.0%) were statistically different. This is in disagreement with previous reports in which significant differences were not found in the isolation rates between the 2 groups. Probably, antibiotic administration caused the overgrowth of C. difficile in intestine of the dogs, predisposing the animals to enteritis.