equine 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.

B0037 is included in P0015 - equine GI/diarrhea panel

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.

Test code: B0082 - Qualitative Assay B0082: Ultrasensitive qualitative detection of Clostridium tetani by real time PCR.

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.

Clostridium tetani
Clostridium tetani is a rod-shaped gram-positive bacterium that is commonly found in soil. It cannot grow in the presence of oxygen and the best temperature for its growth is 33 to 37°C. When growth conditions become adverse, the bacteria will turn into spores. C. tetani spores are extremely hardy and are resistant to many antiseptics and even to heat unless boiled for several minutes. The spores are long-lived and are distributed worldwide in soils and in the gut of various livestock and companion animals.

C. tetani causes the severe disease tetanus when spores enter the body through wounds. In deep wounds, such as those from a puncture or contaminated needle injection, the combination of tissue death and limited exposure to surface air can result in a very low-oxygen environment, allowing C. tetani spores to germinate and grow. As the bacteria grow in the wounds, they can release the toxins tetanolysin and tetanospasmin. While the function of tetanolysin is still not certain, tetanospasmin ("tetanus toxin") is one of the most potent toxins known, with an estimated lethal dose less than 2.5 nanograms per kilogram of body weight, and is responsible for the symptoms of tetanus. This tetanus toxin acts on the nervous system by blocking the release of certain neurotransmitters. It causes muscle spasm and respiratory failure. The gene encoding tetanospasmin is found on a plasmid carried by many strains of C. tetani; strains of bacteria lacking the plasmid are unable to produce toxin. Tetanus vaccine, also called tetanus toxoid, is prepared by inactivation of tetanospasmin by formaldehyde.

Diagnosis of wound tetanus relies on clinical observation assisted by laboratory confirmation, including detection of toxin in body fluids and wound tissues. Detection of toxin and the isolation and identification of toxigenic C. tetani traditionally relies on the use of time-consuming mouse bioassays. PCR detection of the toxin producing plasmid is rapid, specific and sensitive; it is now the most important tool to confirm the diagnosis of C. tetani infection.

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 herds 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

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.
Akbulut, D., Grant, K.A., McLauchlin, J. (2005) Improvement in laboratory diagnosis of wound botulism and tetanus among injecting illicit-drug users by use of real-time PCR assays for neurotoxin gene fragments. J Clin Microbiol. 43:4342–4348.
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