avian & livestock assay data sheet

Clostridium species

Test code: B0037 - Qualitative detection of Clostridium difficile bacteria by polymerase chain reaction.  Assay also detects and differentiates C. dif toxin-producing genes A and B.

Test code: B0042 - Ultrasensitive qualitative detection of Clostridium perfringens alpha toxin and enterotoxin by real time PCR

Test code: B0043 - Ultrasensitive qualitative detection of Clostridium piliforme (Tyzzer's disease) bacteria by real time PCR

Test code: B0061 - Qualitative detection but not differentiation of several common Clostridium species, including C. difficile, C. piliforme and C. perfringens, by polymerase chain reaction. Assay DOES NOT detect Clostridium botulinum.

Clostridium difficile
Clostridium difficile is a gram positive, anaerobic, spore forming motile rod bacterium that commonly inhabits the intestinal tract of many mammalian species, reptiles and birds. It is also found in the environment. The bacterium is a highly diverse organism, with more than 400 unique types, and has several virulence factors. Exotoxin A and B are the most significant factors, and bacterial production of exotoxins is correlated with pathogenicity of individual strains of C. difficile. Toxin A is an enterotoxin, promoting fluid exudation from the intestinal mucosa, and acts synergistically with the cytotoxic toxin B through attachment to specific receptors on the surface of enterocytes. The combined action of these toxins results in necrosis of superficial epithelium and edema in affected areas of intestine.

The organism is an important cause of enteric disease in laboratory rodents and horses. Hamsters, guinea pigs and mice may be affected by pseudomembranous colitis induced by antimicrobial therapy. In neonatal foals, C. difficile has been associated with hemorrhagic necrotizing enterocolitis and diarrhea. The lack of an established intestinal microflora may make foals more susceptible to colonization by this bacterium. Adult horses may develop typhlocolitis and outbreaks of nosocomially acquired diarrhea have been reported (Donaldson and Palmer, 1999; Madewell et al., 1995; Perrin et al., 1993).

C. difficile has also recently been implicated as a cause of typhlocolitis in nursing piglets, chronic diarrhea in dogs and enterotoxemia in ostriches.

In clinically normal patients, an established intestinal microflora is thought to competitively prevent proliferation of C. difficile and subsequent toxin attachment. Alteration of intestinal microbial balance with antibiotic use and increased exposure to the organism in a hospital setting allows C. difficile to colonize the gut in susceptible individuals.

Bacterial culture of C. difficile is not highly sensitive and does not differentiate the pathogenic and non-pathogenic strains. Specific tests for C. difficile toxins used in the diagnostic laboratory include cell culture, which relies on the presence of biologically active toxin, and an ELISA assay which detects immunologically active toxin that may or may not be biologically active.

PCR detection of C. difficile is highly sensitive and can discriminate between toxigenic and nontoxigenic strains of the organism by detecting its toxin producing genes.

Clostridium perfringens
Clostridium perfringens is a Gram-positive, rod-shaped, anaerobic, spore-forming bacterium found as a normal component of decaying vegetation, marine sediment, the intestinal tract of humans and other vertebrates, insects and soil.

Infections due to C. perfringens can result in tissue necrosis, bacteremia, emphysematous cholecystitis and gas gangrene. The bacteria can secrete α-toxin which results in gangrene formation. If patients ingest the bacteria, colic, diarrhea and sometimes nausea can result.

Food poisoning due to C. perfringens bacteria is one of the common causes of food-borne illness. Poorly prepared meat and poultry are commonly the sources of food poisoning. The enterotoxin (CPE) secreted by the bacteria, which mediates the food poisoning, is heat-resistant and cannot be destroyed easily. Furthermore, the bacteria themselves form spores that can withstand cooking temperatures. If these spores are then left at room temperature, germination may begin and infective bacterial colonies develop. Generally, the incubation time of these spores is 6 to 24 (commonly 10 to 12) hours after ingestion of contaminated food. Since meat and poultry are often prepared in advance of consumption, this allows good opportunities for the spores to germinate.

People ingesting these bacteria can develop abdominal cramping and diarrhea. Vomiting and fever are unusual. Illness usually resolves within 24 hours. It is also possible that many cases of C. perfringens food poisoning remain subclinical, as antibodies to the toxin are common among humans. This has led to the conclusion that most of the population has experienced food poisoning due to C. perfringens.

Detection of C. perfringens by culture is slow and not very sensitive. PCR detection is the method of choice for rapid, sensitive and specific detection of this pathogen (Abubakar, 2007).

Clostridium piliforme
Infection with Clostridium piliforme results in Tyzzer’s disease, which is characterized by necrotic lesions in the liver, digestive organs and heart. A number of animal species are susceptible to this organism, including mice, rats, rabbits, dogs, cats, primates, and horses.

The organism is an obligate gram-negative bacteria found in necrotic foci in spore forms. Transmission is mainly through the fecal-oral route.

Although Tyzzer’s is a severe disease in many animal species, infected mice often do not exhibit clinical symptoms. These mice become carriers of the disease and spread the pathogen to other mice and other animal species. Interestingly, different mouse strains differ in their susceptibility to the pathogen (Waggie et al., 1981).

Clostridium piliforme cannot be cultivated in artificial media, so diagnosis may be based on microscopic examination of tissues, serological assays or steroid challenge tests; these methods all require blood or necropsy samples. When steroid challenge assays are performed, extreme care must be taken to avoid spreading the pathogen. Moreover, microscopic examination, serology and steroid challenge all suffer from a lack of sensitivity and are labor intensive.

Detection of this pathogen by polymerase chain reaction is highly sensitive and specific. The test can be performed on fecal specimens rather than blood or tissue, resulting in less trauma and risk to animals.

Utilities:

• Help confirm the disease causing agent
• Shorten the time required to confirm a clinical diagnosis of Clostridium infection.
• Identify Clostridium infection to the species level
• Help ensure that flocks or animal facilities are free of these bacteria
• Early prevention of spread of these bacteria
• Minimize personnel exposure to these bacteria
• Safety monitoring of biological products and vaccines that derive from susceptible animals and birds

References:
Abubakar, I., Irvine, L., Aldus, C.F., Wyatt, G.M., Fordham, R., Schelenz, S., Shepstone, L., Howe, A., Peck, M. and Hunter, P.R. (2007) A systematic review of the clinical, public health and cost-effectiveness of rapid diagnostic tests for the detection and identification of bacterial intestinal pathogens in faeces and food. Health. Technol. Assess. 11:1-216.
Donaldson, M.T. and Palmer, J.E. (1999) Prevalence of Clostridium perfringens enterotoxin and Clostridium difficile toxin A in feces of horses with diarrhea and colic. J. Am. Vet. Med. Assoc. 215:358 361.
Madewell, B.R., Tang, Y.J., Jang, S., Madigan, J.E., Hirsh, D.C., Gumerlock, P.H. and Silva, J. (1995) Apparent outbreaks of Clostridium difficile associated diarrhea in horses in a veterinary medical teaching hospital. J. Vet. Diagn. Invest. 7:343 346.
Perrin, J., Cosmetatos, I., Gallusser, A., Lobsiger, L., Straub, R. and Nicolet J. (1993) Clostridium difficile associated with typhlocolitis in an adult horse. J. Vet. Diagn. Invest. 5:99 101.
Waggie, K.S., Hansen, C.T. Ganaway, J.R. and Spencer, T.S. (1981) A study of mouse strains susceptibility to Bacillus piliformis (Tyzzer's disease): the association of B-cell function and resistance. Lab. Anim. Sci. 31:139-142

Specimen requirements: Rectal swab, cloacal swab, or 0.2 ml feces, or food swab, or lesion swab, or environmental surface swab.

Contact Zoologix if advice is needed to determine an appropriate specimen type for a specific diagnostic application. For specimen types not listed here, please contact Zoologix to confirm specimen acceptability and shipping instructions.

For all specimen types, if there will be a delay in shipping, or during very warm weather, refrigerate specimens until shipped and ship with a cold pack unless more stringent shipping requirements are specified. Frozen specimens should be shipped so as to remain frozen in transit. See shipping instructions for more information.

Turnaround time: 2 business days

Methodology: Qualitative PCR

Normal range: Nondetected