Test code: B0082
- Qualitative Assay
B0082: Ultrasensitive qualitative detection of Clostridium
tetani by real time PCR.
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).
has also recently been implicated as a cause of typhlocolitis in
nursing piglets, chronic diarrhea in dogs and enterotoxemia in
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
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 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
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
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)
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,
The organism is an obligate gram-negative bacteria found in
necrotic foci in spore forms. Transmission is mainly through the
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).
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
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.
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
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.
Help confirm the
disease causing agent
Shorten the time required to confirm a clinical
diagnosis of Clostridium
infection to the species level
Help ensure that colonies, populations and
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
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.
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.
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
Rectal swab, or 0.2 ml feces, or food
swab, or lesion swab, or environmental surface swab; or 0.2 ml
EDTA whole blood, serum or plasma; or wound culture; or fresh,
frozen or fixed tissue.
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
2 business days