Clostridial Enteric Disease Unit

Clostridium perfringens Genotyping Traditionally, production of one or more of four exotoxins (alpha, beta, epsilon and iota toxins) has allowed division of C. perfringens into five toxigenic types. The major diseases caused by these types are listed below.

Type	Major Toxins	Diseases
Type A	Alpha	Myonecrosis, food poisoning, necrotic enteritis in fowl, enterotoxemia in cattle and lambs, necrotizing enterocolitis in piglets; possibly equine colitis, canine hemorrhagic gastroenteritis
Type B	Alpha, Beta, Epsilon	Dysentery in newborn lambs, chronic enteritis in older lambs ("pine"), hemorrhagic enteritis in neonatal calves and foals, hemorrhagic enterotoxemia in adult sheep
Type C	Alpha, Beta	Enteritis necroticans (pigbel) in humans, necrotic enteritis in fowl, hemorrhagic or necrotic enterotoxemia in neonatal pigs, lambs, calves, goats, foals, acute enterotoxemia ("struck") in adult sheep
Type D	Alpha, Epsilon	Enterotoxemia in lambs ("pulpy kidney") and calves, enterocolitis in neonatal and adult goats, possibly enterotoxemia in adult cattle
Type E	Alpha, Iota	Enterotoxemia likely in calves and lambs, enteritis in rabbits; host range and disease type unclear
Type AE	Alpha, Enterotoxin	Canine and porcine enteritis; possibly bovine and equine enteritis

While not used in determining the classical toxin phenotype, enterotoxin is the principal toxin involved in human foodborne illness, and is considered by many to be a virulence attribute in animal strains of C. perfringens. The gene for enterotoxin may be present in any C. perfringens toxin type. Recently, beta2, a novel C. perfringens toxin was identified in strains associated with porcine enteritis. However, a definitive role for this toxin in disease has not been established. Beta2 may be present in any C. perfringens toxin type.

Typing of C. perfringens by toxin neutralization tests in mice or guinea pigs is a traditional part of the diagnosis of clostridial enteritis in domestic animals. For a number of reasons, including cost, lack of availability of standard antitoxin, unexplained variability in results, and humanitarian concerns for animal welfare, in vivo testing is infrequently used at present.

As an alternative to in vivo typing, we have developed and evaluated a genotyping system, based upon detection by polymerase chain reaction (PCR) of the genes for alpha, beta, epsilon, iota and beta2 toxins, and enterotoxin. A description of the multiplex method is available in pdf.

WHAT TO SUBMIT:

Print a copy of the submission form and additional test form if applicable and send it with your cultures.

Under Clinical History/disease please indicate the age of the animal, sex, breed, number affected in the herd, date of death, duration of illness, and possible cause of death fill in the information as completely as possible. This information will be used later to survey sample received.

Note that the preferred specimen is an agar plate or non-thiogycollate broth cultures, confirmed to be a pure culture of Clostridium perfringens. You may submit tissues, but we have a addtional charge for anaerobic culture of these specimens. Cultures should ALWAYS be sealed with parafilm, packed with wet ice packs, and shipped for overnight delivery. Second day delivery is acceptable during cooler times of the year (in Arizona, from mid-November through mid-March).

If your culture will be required for future use, such as for the production of an autogenous vaccine, this should be noted at the time of submission. Please include a request for culture preservation under the section “Clinical History/Disease” on the Submission Form. This service is subject to a charge, plus the cost of return shipping. Cultures will be discarded after 2 weeks if no request for culture preservation is made at the time of submission. Requests for the culture preservation service should be made on the submission form.

Please remember that this genotyping service is for C. perfringens ONLY. We occasionally receive cultures of other clostridia, but we cannot type them.

WHERE TO SUBMIT:

Specimens should be submitted directly to us for genotyping.

Clostridial Enteric Disease Unit
Department of Veterinary Science and Microbiology
University of Arizona
1117 East Lowell Street
Building 90, Room 214
Tucson, AZ 85721-0090
Phone: 520-621-2745
Fax: 520-621-6366

Should any other tests be require, we are associated with the Arizona Veterinary Diagnostic Laboratory (AVDL) and samples can be forwarded. Please go their website for more information. http://microvet.arizona.edu/AzVDL/index.shtml

TURNAROUND TIME:

Isolates will be genotyped and reported by fax within a week of receiving the specimen.

INTERPRETATION OF RESULTS:

Several points should be noted when interpreting results of genotyping.

Correlation of genotype (generated by PCR) with phenotype (determined by mouse or guinea pig inoculation) is nearly 100%. Therefore, detection of the gene for a given toxin is as useful, diagnostically, as detecting the toxin.

We can not state with certainty the proportion of C. perfringens colonies on a primary isolation plate which are "disease related" as opposed to normal flora. Fore example, a single colony isolated from a case of necrotic enteritis or enterotoxemia may be genotype A and originate from the normal gut.

Data from necropsy, bacteriologic culture, and genotyping on cases of possible clostridial enteritis, particularly in pigs and cattle, strongly supports an etiologic role for type A strains in enteritis and enterotoxemia. A finding of genotype A should not be automatically disregarded as normal flora.

If large numbers of spores are seen in gut sections or in direct smears, you may want to consider a heat- or ethanol-shock before anaerobic culture. This MAY allow enrichment for enterotoxigenic C. perfringens.

FOR QUESTIONS OR MORE INFORMATION:

Do not hesitate to call (520-621-2745) or email (jcoombs@email.arizona.edu) if you want to discuss the service.

Last modified April 20, 2006

	Correlation of genotype (generated by PCR) with phenotype (determined by mouse or guinea pig inoculation) is nearly 100%. Therefore, detection of the gene for a given toxin is as useful, diagnostically, as detecting the toxin.
	We can not state with certainty the proportion of C. perfringens colonies on a primary isolation plate which are "disease related" as opposed to normal flora. Fore example, a single colony isolated from a case of necrotic enteritis or enterotoxemia may be genotype A and originate from the normal gut.
	Data from necropsy, bacteriologic culture, and genotyping on cases of possible clostridial enteritis, particularly in pigs and cattle, strongly supports an etiologic role for type A strains in enteritis and enterotoxemia. A finding of genotype A should not be automatically disregarded as normal flora.
	If large numbers of spores are seen in gut sections or in direct smears, you may want to consider a heat- or ethanol-shock before anaerobic culture. This MAY allow enrichment for enterotoxigenic C. perfringens.