General information
The sensitivity of the assay is 10 organisms per 1 gram of feces. The PCR will amplify DNA that is directly extracted from fecal material.

We need approximately 0.5 - 1 gram of fecal material. The sample should be taken from the abnormal loose stool and must be free of cat litter.

For storage and shipping, add approximately 4-5 volumes of rubbing alcohol (70 % isopropyl alcohol) to the tube. This way, no refrigeration or freezing is necessary during the shipment. If no alcohol is available, place the sample in the freezer, and ship with a frozen gel ice pack.

Our stability studies have shown that DNA in fecal samples is stable for up to 72 hours under refrigeration and for at least four weeks when frozen at -20°C.

Turnaround: 1-3 business days after receipt of samples. This time may vary depending upon day and time the samples are received. See assay schedule.

General information about enterotoxigenic Clostridium perfringens
Diarrhea is one of the most common reasons for presentation of dogs and cats to a veterinarian, and bacterial causes always need to be considered as a differential diagnosis. Clostridium perfringens, a gram-positive, anaerobic, spore-forming rod, has been commonly associated with bacterial diarrhea in both dogs and cats. One of the main virulence factors believed to be associated with C. perfringens-associated diarrhea is the Clostridium perfringens enterotoxin (CPE), which is encoded by the cpe gene. CPE, which is produced during sporulation and released after cell lysis, has been shown to induce mucosal damage, increase intestinal permeability, and reduce water absorption, thus leading to diarrhea.

Clinical signs:
In general, any dog or cat with small and/or large bowel diarrhea may have C. perfringens-associated diarrhea. The diarrhea may be acute or chronic and may or may not be accompanied by the presence of mucus, blood, or tenesmus. The disease might range from mild and self-limiting to severe, but systemic signs are uncommon. C. perfringens is considered to be a major cause of nosocomial diarrhea, which often occurs a few days after admission of a patient to a veterinary hospital. As clinical signs of C. perfringens-associated diarrhea are not specific, a diagnosis can not be based on clinical signs alone. Microscopic examination of fecal smears and enumeration of fecal endospores has been shown to be of little use for the diagnosis of C. perfringens-associated diarrhea, because sporulation occurs in both healthy and diarrheic animals. Similarly, anaerobic fecal culture should not be used for the diagnosis of C. perfringens-associated diarrhea, because this organism is a normal constituent of the intestinal microflora of healthy dogs and cats.

Diagnosis:
Immunodetection of CPE in feces (using an ELISA or RPLA) is commonly used for the diagnosis of C. perfringens-associated diarrhea. However, assays currently used in veterinary medicine are intended for human use, and their performance has not been validated for dogs or cats. Also, there are some concerns regarding the sensitivity and specificity of these assays.
Sensitivity is considered extremely important, because the effects of CPE on the intestinal mucosa are believed to be concentration-dependent. We have recently developed and validated a PCR-based test for the detection of the cpe gene in canine and feline fecal samples. The principle behind this diagnostic test is that C. perfringens organisms harboring the cpe gene
are considered to produce CPE. It should be noted that the cpe gene, like the enterotoxin itself, has been identified in feces from healthy animals, so a combination of clinical signs and detection of the cpe gene increases the accuracy of the diagnosis. This PCR assay is performed directly on DNA extracted from canine and feline fecal samples. The sensitivity of this PCR- based assay for the detection of the cpe gene is very high and the detection limit is less than 10 organisms/sample. Specificity of the PCR assay was verified by sequencing of PCR amplicons. Due to the non-specific clinical signs associated with C. perfringens- diarrhea, any dog or cat with diarrhea may have C. perfringens-associated diarrhea. Patients that develop diarrhea after being hospitalized should be tested. Also, patients with bloody diarrhea, in which another cause can not be identified, are candidates for testing. In order to effectively manage C. perfringens-associated diarrhea, accurate diagnosis is crucial.

Therapy:
Metronidazole, tylosin, erythromycin, and ampicillin are considered effective in treating diarrhea associated with Clostridium perfringens. However, strains resistant to these antibiotics may occur. Increased prevalence of resistance to antibiotics is an emerging issue of major public health importance. This underscores the importance of refraining from indiscriminate use of antibiotics in diarrheic patients and supports the use of specific diagnostics and effective treatments against the specific pathogens identified.

 

Further reading
Marks SL, Kather EJ. Clostridium perfringens- and Clostridium difficile-associated diarrhea. In Green CE, editor: Infectious diseases of the dog and cat, 3rd ed, 2006, St. Louis, Saunders, pp 363-369.

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