wildlife and zoo assay data sheet

Mycoplasma species

Test codes:

B0002 - Qualitative detection of Mycoplasma pneumoniae by polymerase chain reaction.

B0009 - Ultrasensitive Mycoplasma screen by real time PCR.  This screen detects but does not differentiate M. arginini, M. fermentans, M. hominis, M. hyorhinis, M. orale, M. pirum, M. salivarium, M. agassizii, M. cynos and others. This screen does not detect M. pneumoniae or M. pulmonis.

B0041 - Ultrasensitive qualitative detection of Mycoplasma pulmonis by real time polymerase chain reaction

B0096 - Ultrasensitive qualitative detection of Mycoplasma mustelae by real time polymerase chain reaction

Mycoplasma species are the smallest free-living prokaryotic microorganisms of the class Mollicutes. These bacteria do not have cell walls and are thought to have devolved by genome reduction from gram-positive bacteria. Most Mycoplasma species are host-specific facultative anaerobes. Because of their stringent growth requirements, they do not usually replicate in the environment and are obligate parasites. Their complex growth requirements include cholesterol, fatty acids, and amino acids.

Mycoplasmas are gram-negative bacteria due to their absence of a cell wall. Because of this characteristic, these bacteria are not responsive to many common antibiotics such as penicillin or other beta-lactam antibiotics that target cell wall synthesis. There are more than 100 recognized species in the genus Mycoplasma. Some species cause disease in humans, such as M. pneumoniae which causes atypical pneumonia and other respiratory disorders, and M. genitalium which is linked to pelvic inflammatory diseases.

A number of Mycoplasma species can cause respiratory diseases in animals and humans. In the respiratory tract, Mycoplasma bacteria attach to ciliated epithelial cells and cause their destruction.

Many Mycoplasma species are found in research laboratories as contaminants in cell culture due to contamination from lab staff or contaminated cell culture medium ingredients. Contamination rates by Mycoplasma species in established cell cultures have been reported at 15-35%, with even higher occurrence cited in some populations.  Mycoplasmas may induce cellular changes, including chromosome aberrations and changes in metabolism and cell growth. Severe Mycoplasma infections may even destroy a cell line. Therefore biological products derived from these cultures must be screened routinely to help ensure product stability and integrity. A number of techniques have been used to detect Mycoplasma, including DNA probe, enzyme immunoassays, PCR, plating on sensitive agar and staining with a DNA stain including DAPI or Hoechst. Culture detection of Mycoplasma is especially difficult because of these organisms' stringent growth requirements. For many applications, PCR is the most commonly used method of detection due to its speed, high sensitivity and specificity.

Mycoplasma pneumoniae
M. pneumoniae was first linked to respiratory infection more than one hundred years ago. M. pneumoniae can be transmitted through close contact via respiratory droplets. M. pneumoniae has a surface receptor which enables attachment to the host cell surface. This receptor can attach to a number of different cell types such as respiratory tract epithelia and red blood cells. At high concentrations, M. pneumoniae can inhibit ciliary action within the respiratory tract and cause cell necrosis. This damage is caused by cytotoxins from M. pneumoniae as well as indirectly from the host immune response.

Mycoplasma pneumoniae is very difficult and slow to grow in culture media. However, molecular detection of M. pneumoniae by PCR is highly sensitive and specific, with fast turnaround time.

Mycoplasma pulmonis
Murine respiratory mycoplasmosis in mice and rats is caused by Mycoplasma pulmonis. In addition to exhibiting high morbidity and reduced birth rate, mice and rats infected with M. pulmonis often develop imperceptible infections. These can cause problems in research studies using these animals, as physiological mechanisms and the immune system may be affected. Experimental results obtained with infected animals can be misleading.

Diagnosis of M. pulmonis infection can be done using serological methods such as microbial isolation or enzyme linked immunosorbent assay (ELISA) (Cassell et al., 1981). However, in vitro isolation is time-consuming, and serological methods often give incorrect results due to cross-reactivity between different species of rodent mycoplasmas (Cassell et al., 1981; Davis et al., 1987; Davidson et al., 1994). Serological assays also lack sensitivity because a low level of antibody -- early in an infection or in immunodeficient animals -- may not be detected.

Mycoplasma mustelae
In 1983, a new strain of fast growing, glucose-fermenting Mycoplasma bacteria was isolated from the lungs of healthy mink kits 1–2 months of age in Denmark (Salih et al., 1983). This species was named M. mustelae. Currently little is known about this Mycoplasma species, but it can be detected by PCR.

Utilities:

• Help confirm the disease causing agent
• Shorten the time required to confirm a clinical diagnosis of Mycoplasma infection.
• Help ensure that animal populations are free of these mycoplasmas
• Early prevention of spread of these mycoplasmas
• Minimize personnel exposure to these mycoplasmas
• Preservation of cell cultures
• Safety monitoring of biological products and cultures

References:
Armstrong, S.E., Mariano, J.A. and Lundin, D.J. (2010) The scope of Mycoplasma contamination within the biopharmaceutical industry.  Biologicals, 38:211-213.
Maczyñska, B., Matusiewicz, K., Chiciak, J., Stankiewicz, M.,  Sozañska, B. and Boznañski, A. (2002) Comparison of detectability of Mycoplasma pneumoniae infections in children, using PCR-test and serological methods: indirect immunofluorescence and immunoenzymatic assay. Clin.  Microbiol. and Infect. 8 (Suppl. 1):  P1346.
Cassell, G.H., Lindsey, J.R., Davis, J.K., Davidson, M.K., Brown, M.B. and Mayo, J.G. (1981) Detection of natural Mycoplasma pulmonis infection in rats and mice by an enzyme linked immunosorbent assay (ELISA). Lab Anim Sci 31: 676–682.
Davis, J.K., Cassell, G.H., Gambill, G., Cox, N., Watson, H. and Davidson, M. (1987) Diagnosis of murine mycoplasmal infections by enzyme-linked immunosorbent assay (ELISA). Isr J Med Sci 23: 717–722.
Davidson, M.K, Davis, J.K., Gambill, G.P., Cassell, G.H. and Lindsey, J.P. (1994) Mycoplasmas of laboratory rodentsMycoplasmas in Animals: Laboratory Diagnosis, Iowa State University Press, Ames p. 97–133.
Salih, M.M., Friis, N.F., Arseculeratne, S.N., Freundt, E.A. and Christiansen, C. (1983) Mycoplasma mustelae, a new species from mink. Int. J. Syst. Bacteriol. 33:476–479.

Specimen requirements:

B0002, B0009 or B0096 - 0.2 ml whole blood in EDTA (purple top) tube, or 0.2 ml serum, CSF or bronchial lavage, or 0.2 ml fresh, frozen or fixed tissue, or nasopharyngeal swab, throat swab or vaginal swab, or 0.2 ml culture.

B0041 - Nasopharyngeal swab or tracheal wash.

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 (B0002); qualitative real time PCR (B0009, B0041, B0096)

Normal range: Nondetected