Helicobacter PCR tests for rodents

P0010 - Ultrasensitive Helicobacter species screen by real time polymerase chain reaction. This screen detects but does not differentiate H. pylori, H. heilmannii, H. bilis, H. hepaticus, H. rappini, H. felis, H. salomonis and other Helicobacter species.

P0011 - Ultrasensitive Helicobacter species identification by real time polymerase chain reaction and PCR product sequence analysis. This 2-stage assay detects and differentiates H. pylori, H. heilmannii, H. bilis, H. hepaticus, H. rappini, H. felis, H. salomonis and other Helicobacter species.

Many species of the genus Helicobacter have been identified in mammals and their pathogenicity varies. Some species can induce significant disease while others appear to merely colonize the gastrointestinal tract.

Helicobacter species that have been identified in mice and rats include H. muridarum, H. rappini, H. hepaticus, and H. bilis, all of which colonize the gastrointestinal tract. Among them, H. muridarum and H. rappini are generally not considered to be pathogenic. However, in susceptible strains of mice H. hepaticus causes acute focal, nonsuppurative, necrotizing hepatitis which progresses to chronic, active hepatitis characterized by minimal necrosis, hepatocytomegaly, oval cell hyperplasia and cholangitis. Typically, relatively few infectious organisms are apparent on routine histologic examination. Mouse strains A/JCr, B6C3F1, BALB/cAnCr, C3H/HeNCr, SJL/NCr, and SCID/NCr have been shown to develop hepatitis due to H. hepaticus infection, and infected A/JCr and male B6C3F1 mice show increased incidence of hepatocellular neoplasms.

H. bilis can be found in the bile, liver, and intestines of mice. Infection of both rats and mice with this bacterium is associated with chronic hepatitis. It is not known whether H. bilis causes liver tumors in mice. However, infection of rat or mouse liver by H. hepaticus can significantly affect the interpretation of carcinogenicity bioassays and other research using rodents.

In the past, definitive diagnosis of H. hepaticus- or H. bilis-associated liver disease in mice was based on the presence of histologic alterations as well as culture of the infectious agent. However, Helicobacter identification by culture from tissue or feces is very difficult due to the organism’s fastidious growth requirements. Sensitivity of the culture method is low.

H. hepaticus organisms have been detected in lesioned liver by methods such as Steiner and Warthin-Starry histochemical stains, immunoflourescence, in situ hybridization, immunohistochemistry and ultrastructural analysis. A serum ELISA test for circulating IgG antibodies against H. hepaticus has also been used to provide information for determining in vivo infection status in pathogenesis studies. However, none of these methods are very useful for large-scale screening. PCR-based diagnostic assays for H. hepaticus and H. bilis offer greater specificity, higher sensitivity and lower cost compared to culture, histochemical stains, or ultrastructural analysis. Recently, the growing use of real time PCR technology has further improved sensitivity and specificity of molecular detection of Helicobacter species, making this method the most important approach for routine colony screening. Real time PCR detection also allows retrospective studies of paraffin-embedded tissue and other archived tissue biopsies.

Helicobacter pylori is a gram-negative spiral bacterium found in gastric mucosa and associated with active and chronic gastritis. H. pylori can establish a chronic, persistent infection, which may lead to gastric or duodenual ulcers, gastric cancer and gastric lymphomas. Studies have revealed that approximately 50% of the world’s human population is infected with H. pylori.

Biochemically, the bacterium produces catalase, oxidase and urease enzymes. The urease enzyme permits the bacterium to metabolize urea present in the gastric mucosa and establish a microenvironment favorable to the organism. H. pylori is a highly motile organism with multiple unipolar flagella. Both the urease enzyme and the flagella are considered to be important virulence factors.

Diagnosis of Helicobacter pylori infection in humans relies on upper endoscopy or the 13C-urea breath test (see review by Nakamura, 2001). Although the endoscopy procedure permits culture of the bacterium from biopsy specimens (the gold standard for diagnosis), demonstration of urease activity and histological detection of the germ, the procedure is expensive and invasive. The 13C-urea breath test is a well-established, relatively sensitive, specific and noninvasive method. Molecular tests, such as PCR, can also offer precise diagnosis of H. pylori infections. In fact, molecular testing by PCR can complement other diagnostic tests because it can be applied to archival fixed tissue, environmental samples, gastric fluid, oral secretions, and stool samples, in which traditional diagnostic tests do not have sensitivity and perform poorly. Studies have shown than PCR detection of H. pylori in gastric fluid specimens can reach a sensitivity of 96% and a specificity of 100% (Westblom et al., 1993; Yoshida et al., 1999). This capability is especially useful in monitoring active H. pylori infection in rodents and other animals, as the breath test is difficult to conduct for these animals.

Helicobacter heilmannii (previously known as Gastrospirillum hominis) is a 4-10 µm long, spiral-shaped, motile bacterium with three to eight coils, a wavelength of about 1 µm, up to 14 uni- or bipolar flagella, and no periplasmic filaments. In humans, gastric infection with H. heilmannii is associated with the development of chronic gastritis (found in the stomachs of 0.2 to 4% of patients with gastritis) and low-grade mucosa-associated lymphoid tissue lymphoma in humans. Eradication of H. heilmannii by antibiotic treatment of patients can result in complete remission of MALT lymphoma, indicating a causal relationship between H. heilmannii infection and MALT lymphoma. Unlike H. pylori infections, gastric infections with H. heilmannii or Gastrospirillum-like organisms are not restricted to humans. A broad range of animals including dogs, cats, pigs, and cattle are naturally infected, with frequencies ranging from 80% to 100%. It has been suggested that H. heilmannii infection in humans may be a zoonosis and that animals may serve as a reservoir for transmission to humans.

Definitive culture of H. heilmannii has not been achieved to date (Anderson et al., 1996) and diagnosis of H. heilmannii infection is usually made on the basis of its distinct spiral morphology, compared with H. pylori, on silver- stained tissue sections. However, there are a number of large, gastric, spiral organisms such as H. felis, H. salomonis, and H. bizzozeronii which are indistinguishable from H. heilmannii on routine light microscopy, and H. pylori grown in a broth culture can also adopt a morphology identical to that of H. heilmannii (Fawcett et al., 1999). Molecular detection methods are required for more definitive identification (Trebesius et al., 2001). PCR is the most sensitive and specific tool for detecting Helicobacter infection (Whary and Fox, 2006).

Utilities:

Help confirm the disease causing agent
Shorten the time required to confirm a clinical diagnosis of Helicobacter infection.
Help ensure that rodent colonies are free of Helicobacter species
Early prevention of spread of these bacteria among a colony
Minimize personnel exposure to these bacteria
Safety monitoring of biological products that derive from rodents

Specimen requirements: 1 fecal pellet, or 0.2 ml fresh, frozen or fixed tissue. For H. pylori and H. heilmannii assays only, 0.2 ml gastric wash is the preferred specimen type.

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.

References:
Whary, M.T. and Fox, J.G. (2006). Detection, eradication, and research implications of Helicobacter infections in laboratory rodents. LabAnimal, Vol. 35 No. 7:25-36.

Turnaround time: 2 business days (1 week for P0011)

Methodologies:
B0021, B0023, B0039 and P0010 - Qualitative real time PCR
P0011 - Qualitative real time PCR + PCR product sequence analysis

Normal range: Nondetected