Prevalence of Clostridium sPP. in diarrhoeic and healthy dogs ...

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Ann. Fac. Medic. Vet. di Parma (Vol. XXVII, 2007) pag. 143 - pag. 156Antibiotic administration, especially in hospital environments, is the mostcommonly reported as predisposing factor for the development of CDAD in peopleand horses, although there have been reports of CDAD in the absence of antibioticadministration. In contrast, administration of antibiotics does not seem to predisposedogs to CDAD [7].Toxigenic C. difficile can be isolated from up to 94% of neonate dogs in theabsence of clinical signs of disease. Clinical signs that have been associated withcanine C. difficile infection range from asymptomatic carriage to a potentially fatalacute hemorrhagic diarrhoeal syndrome. As with C. perfringens, there does not seemto be a specific anatomic localization of clinical signs, and recent studies have shownthat dogs with suspected C. difficile-associated diarrhoea commonly have signsof small and large intestinal diarrhoea as well as diffuse disease characterized byconcurrent involvement of both the small and large intestine [7].As with C. perfringens, isolation of C. difficile from diarrhoeic specimensis of little diagnostic value, except for procuring strains for detection of toxin genesand typing. Limitations in the isolation of C. difficile in dogs are underscored by thefindings of several studies demonstrating no significant difference in isolation rates(0-40%) between non-diarrhoeic and diarrhoeic dogs, as above mentioned.Diagnosis of C. difficile-associated diarrhoea traditionally has been basedon positive fecal assays for toxin A or toxin B. The current gold standard assay isthe cell culture cytotoxicity assay, which detects toxin B activity; however, thisassay is expensive and requires up to 48 hours for conformation of a negative result.There are currently several commercially available immunoassays for detection ofC. difficile toxin A or for detection of both toxins A and B in fecal samples (for example,Premier Toxins A & B, Meridian Bioscience, Inc, Cincinnati, OH; C. difficile TOXA/B II, TechLab, Inc., Blacksburg, VA; ProSpecT Clostridium difficile Toxin A/B,Remel, USA). Because of increasing reports of toxin A-negative and toxin B-positivestrains isolated from clinical cases, the use of ELISA kits that detect both toxins isgaining preference. None of the commercial ELISA kits currently available havebeen validated in the dog, however, and there is no standard assay used by veterinarydiagnostic laboratories. This fact is concerning, given the wide range of specificities(66-100%) and sensitivities (33-95%) that have been reported for many of the kitswhen analyzed for human fecal toxin detection.Detection of toxigenic C. difficile strains after culture has been shown to be oflittle diagnostic value in the dog. First, there is no significant difference in detection oftoxigenic strains between diarrhoeic and non-diarrhoeic dogs, and, second, isolationof the microrganism is difficult, costly, and time-consuming. Nevertheless, studies inhuman beings have begun bypassing culture of the microrganism and looking for thepresence of specific toxin genes in DNA obtained from fecal specimens. Sensitivitiesof PCR detection methods have been reported to be between 96% and 100% whencompared with the cytotoxicity assay [7].The aim of this study was to define the prevalence of Clostridium spp. in95 fecal samples (36 diarrhoeic and 59 non-diarrhoeic) collected from dogs at theVeterinary Faculty of Parma. Especially, we payed attention to C. perfringens andC. difficile isolation.148
Ann. Fac. Medic. Vet. di Parma (Vol. XXVII, 2007) pag. 143 - pag. 156Materials and methodsFecal samples were collected from 95 diarrhoeic and non-diarrhoeic dogs,41 female and 54 male, over an 8-mo period (20 th July 2006 - 20 th March 2007). Ageof the diarrhoeic dogs (n= 36) ranged between 6 months and 12 years (mean, 4.2 yr;median, 3 yr), and that of the non-diarrhoeic dogs (n= 59) ranged between 3 monthsand 12 years (mean, 3.8 yr; median, 3 yr).Some dogs (n= 38) were kennelled dogs; others belonged to veterinarystudents and staff of the Veterinary Faculty of Parma (n= 47). Ten dogs were admittedto the Didactic Veterinary Hospital.All fecal specimens were naturally voided. Assays were performed on fecescollected within 3 hours. After analysis, samples were immediately stored at -20°C.All fecal samples were cultured onto prereduced blood agar plates,containing vitamine K, sheep blood and haemin (Schaedler agar, Oxoid, Basingstoke,Hampshire, England), and at the same time inoculated in Cooked Meat Broth (Oxoid,England). Samples were also streaked onto prereduced selective medium containingcycloserine-cefoxitin-fructose agar (CCFA) for the isolation of C. difficile. Plateswere incubated anaerobically at 37°C for 48-72 hours. After 72 hours of anaerobicincubation in Cooked Meat Broth, the samples were subjected to heat shock forspore selection and then cultured onto Schaedler agar and/or CCFA to enhance sporegermination and increase culture yield.For C. perfringens identification, colonies showing characteristic dualhaemolytic zones (Figure 1) were picked up and identified by Gram’s stain andbiochemical tests utilizing Rapid ID 32A (bioMérieux SA, Marcy-l’Etoile, France).Preliminary identification of C. difficile was based on colonial appearance (Figure2), odor (horse manure), lack of aerotolerance, cell morphology after Gram stainingand typical chartreuse fluorescence under ultraviolet light. Final identification wasperformed by using a rapid latex slide agglutination test (C. difficile, Oxoid, England)and Rapid ID 32A biochemical profiles (bioMérieux SA, France). The other clostridiawere identified only biochemically (Rapid ID 32A, bioMérieux).All isolates were stored on cryopreservation beads (MAST Diagnostics,D.I.D, Diagnostic International Distribution S.p.A., Italy) at -70°C.Figure 1: Typical colonies of Clostridium perfringens onto Schaedler agar showingcharacteristic dual haemolytic zones149
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Prevalence of Clostridium sPP. in diarrhoeic and healthy dogs ...

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