Tuberculosis and Other Mycobacteria

Test codes:

B0015 - Ultrasensitive qualitative detection of Mycobacterium tuberculosis complex by real time polymerase chain reaction. Assay detects but does not differentiate M. tuberculosis, M. bovis, M. africanum and M. microti. Assay does not detect other mycobacteria.

B0029 - Ultrasensitive qualitative detection of Mycobacterium avium, subspecies avium by real time polymerase chain reaction. Assay does not detect subspecies paratuberculosis or other mycobacteria species.

B0030 - Ultrasensitive qualitative detection of Mycobacterium avium, subspecies paratuberculosis (Johne's disease) by real time polymerase chain reaction. Assay does not detect subspecies avium or other mycobacteria species.

B0031 - Ultrasensitive qualitative detection of Mycobacterium intracellulare by real time polymerase chain reaction. Assay does not detect other mycobacteria species.

P0006 - Ultrasensitive qualitative mycobacteria screen by nested polymerase chain reaction. Assay detects but does not differentiate a wide range of mycobacteria, including M. tuberculosis, M. bovis, M. microti, M. intracellulare, M. avium, M. gastri, M. africanum, M. scrofulaceum, M. ulcerans, M. simiae, M. kansasii, M. chelonae and M. fortuitum.

P0007 - Ultrasensitive qualitative mycobacteria species identification by nested polymerase chain reaction and Restriction Fragment Length Polymorphism. This 2-stage assay detects and differentiates a wide range of mycobacteria, including M. tuberculosis/bovis/microti, M. intracellulare, M. avium, M. gastri, M. africanum, M. scrofulaceum, M. ulcerans, M. simiae, M. kansasii, M. chelonae and M. fortuitum.

Many different mycobacteria can cause disease in primates and other species. Primates acquire classic tuberculosis (TB) by contact with other infected nonhuman primates or humans through inhalation or the digestive route (Moreland, 1970). These infected animals can become reservoirs, causing outbreaks of disease. TB infections have also been reported in many other captive and wildlife species.

The main etiologic agents of TB in primates are Mycobacterium tuberculosis, M. bovis, M. africanum and M. microti, and infection by these mycobacteria usually results in pulmonary manifestations and occasionally disseminated disease. Mycobacteria other than tuberculosis (MOTT) have also been implicated in monkey disease, mainly acute and chronic enteropathies and pulmonary infections. Asymptomatic infections by M. avium, M. intracellulariae, M. scrofulaceum and M. simiae have also been reported (Calmette et al., 1923; Smith et al., 1973; Renquist and Potkay, 1979; Brammer et al., 1995). Other saprophyte MOTT have also been isolated from primates but are usually not associated with disease.

Clinical diagnosis of TB in primates can be difficult because infected monkeys may only show mild behavioral changes like anorexia and lethargy. Occasionally, infected monkeys may suddenly die while appearing to be in good condition. The use of skin tests to identify infected monkeys is somewhat unreliable because mycobacteria-infected primates, even within the same species, can have a wide range of responses to tuberculin injection, from negative to strong positive reactions. In addition, skin tests perform inconsistently across closely-related primate species, notably the various species of macaques commonly kept in captivity.

Detection of TB and other mycobacterial infections in primates and other species has relied on tuberculin skin response, serological testing, histopathology, microscopy and culture identification. Among these, the most frequently used methods are culture identification and the tuberculin skin test (also known as the PPD, for Purified Protein Derivative), the latter being a routine test in quarantine and preventive medicine protocols (Fowler, 1993). However, the PPD test is not adequately sensitive or specific in many species and the rate of false negatives is high. Culture and associated biochemical tests for the identification of mycobacteria species are slow and painstaking procedures, and require careful collection and preservation of specimens in order to obtain accurate results.

PCR detection of mycobacterial DNA is highly sensitive when proper specimens are carefully collected. Sample types and collection techniques vary by species; deep respiratory samples obtained using bronchial lavage are often preferred for primates. Gastric lavage can also be a useful sampling technique. Pathology samples should be taken from foci most likely to contain the pathogen -- typically lung or other organ lesions, or enlarged lymph nodes. Trunk washes are used to obtain samples from elephants (National Tuberculosis Working Group for Zoo and Wildlife Species, 2003).

In addition to the detection of a number of mycobacterial species by real time PCR, identification of mycobacteria to the species level can be accomplished rapidly through sequence analysis of PCR products using a restriction fragment length polymorphism (RFLP) technique. Ultrasensitive detection of mycobacteria by PCR and subsequent restriction digest analysis not only allows reliable detection of various species of mycobacteria but in many cases also enables identification of mycobacteria at the species level.

Utilities:

• Confirm the disease causing agent
• Ensure that animal groups and populations are free of tuberculosis or other disease-causing mycobacteria
• Early prevention of spread of mycobacteria among a population
• Minimize human exposure to disease-causing mycobacteria

References:
Brammer, D.W., O’Rourke, C.M., Heath, L.A., Chrisp, C.E., Peter, G.K. and Hofing, G.L. (1995) Mycobacterium kansaii infection in squirrel monkeys (Saimiri sciureus sciureus). J. Med. Primatol. 24: 231-235.
Calmette, A., Smith, G.H. and Soper, W.B.(1923) Tubercle Bacillus Infection and Tuberculosis in Man and Animals, Processes of Infection and Resistance, vol. Xxiii. Williams and Wilkins Company, Baltimore, 689 pp.
Fowler, M.E. (1993) Zoo & Animal Medicine: Current Therapy, 3rd ed., vol. xxv. Saunders, Philadelphia, 617 pp.
Moreland, A.F. (1970) Tuberculosis in New World primates. Lab. Anim. Care 20: 262-264.
Renquist, D.M. and Potkay, S. (1979) Mycobacterium scrofulaceum infection in Erythrocebus patas monkeys. Lab. Anim. Sci. 29: 97-101.
Smith, E.K., Hunt, R.D., Garcia, F.G., Fraser, C.E., Merkal, R.S., Karlson, A.G. (1973) Avian tuberculosis in monkeys. A unique mycobacterial infection. Am. Rev. Respir. Dis. 107: 469-471.
National Tuberculosis Working Group for Zoo and Wildlife Species (2003). Guidelines for the Control of Tuberculosis in Elephants. USDA-APHIS: http://www.aphis.usda.gov/ac/TBGuidelines2003.pdf

Preferred specimens: 1 ml bronchoalveolar lavage, trunk wash (elephants), gastric lavage, lesion or lymph node tissue, shipped overnight at room temperature; or 1 ml frozen tissue shipped frozen.

For specimen types other than those listed here, please call 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:
B0015, B0029, B0030, B0031 and P0006 - 2 business days
P0007 - 3 business days

Methodologies:
B0015, B0029, B0030 and B0031: Qualitative real time PCR
P0006: Qualitative nested PCR
P0007: Qualitative nested PCR and RFLP

Normal range: Nondetected