wildlife and zoo assay data sheet

PCR panel for mycobacteriosis in amphibians

Panel code: P0030 - Ultrasensitive detection of Mycobacterium species commonly infecting amphibians, by real time PCR.  Panel P0030 includes:

Test code B0070: Mycobacterium xenopi
Test code B0071: Mycobacterium chelonae
Test code B0072: Mycobacterium liflandii / ulcerans / marinum / shottii / pseudoshottii

Panel does not detect other mycobacterial species.

P0030 is included in P0031 - amphibian screening panel

The genus Mycobacterium contains more than 70 species, which can be divided into two groups based upon their growth rate in culture. Slow-growing species require more than 7 days to be visible on culture, whereas the rapid-growing species require less than 7 days. In general, many slow-growing species are pathogenic in humans, whereas rapid-growing species are usually considered nonpathogenic.

Mycobacteriosis in amphibians, especially frogs, is caused by acid-fast bacilli of the genus Mycobacterium. Many mycobacterial species found in the environment can cause infection in amphibians, including M. marinum, M. chelonae, M. ulcerans, M. liflandii and M. xenopi (Mve-Obiang et al., 2005, Trott et al., 2004). All these mycobacerial species are ubiquitous in nature and can be found in water. They are resistant to normal water treatment.

Mycobacteriosis is a serious threat to the health of a frog colony, although tuberculosis usually only results in frogs which are already ill, are stressed or have weakened immune systems. Infected frogs will usually develop cutaneous and visceral milliary granulomas (white millet-like bumps) and ulcerative lesions on the skin. Any of these mycobacteria can also gain entry to a frog’s body through the skin wounds and lesions. Once through the skin, these mycobacteria can also affect internal organs.

Mycobacterium xenopi is a slow-growing, waterborne, scotochromogenic species of Mycobacterium. It was first isolated from skin lesions of the frog, Xenopus laevis (Schwabacher, et al., 1959). The organism is very resistant to most disinfectants used to clean water systems and has been isolated in samples collected from water systems in homes and hospitals.

Mycobacterium chelonae is a fast-growing, nontuberculous mycobacterial species. It is a normal commensal in water and soil that can cause disease in humans, fish and amphibians. Recently, this mycobacterium was isolated from captive South African clawed frogs (Xenopus laevis) showing chronic weight loss and nonhealing ulcerative skin lesions (Green et al., 2000), and also from dolphin showing pyogranulomas in blubber, as well as marked acute multifocal necrosuppurative pneumonia and lymphadenitis (Wünschmann et al., 2008). M. chelonae can cause a mortality rate of 90% in infected immunocompromised human patients such as AIDS or cancer patients. In humans, infection is usually via the pulmonary route.

Mycobacterium liflandii / M. ulcerans / M. marinum / M. shottii / M. pseudoshottii infection of Xenopus tropicalis can also result in coelomitis (infection of the coelom/abdomen) and subsequent general or local edema (bloating). Cutaneous lesions and bloating can occur independently. Once these signs develop, the disease is consistently fatal and affected frogs are usually euthanized.

Diagnosis of infections caused by mycobacteria, especially nontuberculous mycobacteria, remains a difficult task both in microbiology and pathology. Culture identification followed by biochemical analysis is often slow and may not differentiate pathogenic from nonpathogenic mycobacteria species due to their close similarity. False positives occur in this way in both staining and culture, particularly in environmental specimens.  Molecular detection by PCR, on the other hand, is a rapid, sensitive and specific technique for sensitive mycobacterial detection and differentiation.

This panel detects and differentiates M. xenopi and M. chelonae; it detects but does not differentiate the group M. liflandii / M. ulcerans / M. marinum / M. shottii / M. pseudoshottii, as these last five species are genetically nearly identical.

Utilities:

• Help confirm the disease causing agent
• Shorten the time required to confirm a clinical diagnosis
• Help ensure that animal facilities or amphibian populations are free of these mycobacteria
• Early prevention of spread of these mycobacteria in a facility or geographic area
• Minimize human exposure to these mycobacteria
• Safety monitoring of biological products that derive from susceptible animals

References:
Schwabacher H. (1959) A strain of Mycobacterium isolated from skin lesions of a cold blooded animal, Xenopus laevus, and its relation to atypical acid-fast bacilli occurring in man. J. Hygiene, 57:57-67
Mve-Obiang, A., Lee, R.E., Umstot, E.S., Trott, K.A., Grammer, T.C.,  Parker, J.M.,  Ranger, B., Grainger, R., Mahrous, E.A. and Small, P.L.C. (2005) A newly discovered mycobacterial pathogen isolated from lethal infections in laboratory colonies of Xenopus species produces a novel form of the M. ulcerans macrolide toxin, mycolactone. Infect. and Immun. 73: 3307-3312.
Trott, K.A., Stacy, B.A., Lifland, B.D., Diggs, H.E., Harland, R.M., Khokha, M.K., Grammer, T.C. and Parker, J.M. (2004) Characterization of a Mycobacterium ulcerans-like infection in a colony of African Tropical Clawed Frogs (Xenopus tropicalis) Comp. Med. 54: 309-317.
Green, S. L., Lifland, B. D., Bouley, D. M., Brown, B. A., Wallace, R. J., Jr. and Ferrell, J. E., Jr. (2000) Disease attributed to Mycobacterium chelonae in South African clawed frogs (Xenopus laevis). Comp. Med. 50: 675-679.
Wünschmann, A., Armien, A., Beth Harris, N., Brown-Elliott, B.A., Wallace R.J., Jr., Rasmussen, J., Willette, M. and Wolf, T. (2008) Disseminated Panniculitis in a Bottlenose Dolphin (Tursiops truncatus) due to Mycobacterium chelonae Infection. J. Zoo Wildlife Med. 39: 412-420.

Specimen requirement: Skin swab, or environmental surface swab, or 0.2 ml fresh tissue, or 0.2 ml fixed tissue, or biofilm swab from filter media or tank surface. Tissue or swab samples may be placed in 70% ethanol if desired; if so, ethanol volume should be minimized - only use enough ethanol to cover the head of the swab or the piece of tissue.

Please call if advice is needed to decide which sample type is appropriate for the specific diagnostic application. 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 real time PCR

Normal range: Nondetected