Abstract

There seems to be an increasing number of reports of TB in pinnipeds. The reasons for this world-wide trend are far from clear.

The diagnosis of tuberculosis in pinnipeds in a clinical situation is not easy. Interpretation of intradermal tuberculin testing (IDT) is frustrating because of possible sensitization of the seals by non-pathogenic mycobacteria.

Careful, accurate and repeatable methods of IDT need to be applied. A standard interpretation chart is suggested for adoption to enable uniform interpretation of IDTs. It is also necessary to be able to correlate IDT results with ELISA tests and where possible with cultures. New techniques of testing and diagnosis should be examined and evaluated as they are developed.

Introduction

Tuberculosis (TB) is generally associated with animals or humans being infected with Mycobacterium tuberculosis or M. bovis.

Mycobacteriosis (MB) refers to mammals infected with "Atypical mycobacteria", i.e. non-mammalian mycobacteria which can cause tuberculosis-like lesions in mammals (Reddacliff & Lim 1990).

Isolated cases of TB in pinnipeds have recently been reported in the literature (Forshaw and Phelps, 1991; Cousins, pers. comm.). The TB outbreak in the captive pinnipeds at Atlantis Marine Park, Western Australia yielded isolates originally identified as M. bovis.

Subsequent testing has revealed that while these and other more recent pinniped isolates most closely resemble M. bovis, the seal strains appear to lack the major protein antigen MPB70, produced by other M. bovis isolates. The DNA patterns produced by Restriction Endonuclease Analysis and studies using Southern Blotting and hybridization with two DNA probes indicate that the seal strain may be unique (Cousins et al 1990).

Isolates from captive pinnipeds (1985-1986), wild pinnipeds (1989-1991) and an infected seal keeper (1988) are all identical.

These TB isolates can best be described as members of the M. tuberculosis complex (the recognised members of this complex being: M. tuberculosis, M. bovis. M. microti and M. africanum.) Further taxonomic studies are being conducted (Cousins pers. comm.).

Mycobacteriosis in marine mammals has been reported on several occasions (Boever, 1978; Gutter et al, 1987; Lewis, 1987; Morales et al, 1985; Stoskopf et al, 1987; Wells et al, 1990). Isolates from these marine mammal cases included: M. fortuitum, M. marinum, M. chelonii, M. smegmatis. Generally, these cases occur in individual animals often in poor or stressed condition. The causative organisms involved are ubiquitous in marine and freshwater environments and are common pathogens of fish.

A problem may arise for veterinarians when testing pinnipeds for TB as the presence of MB organisms may sensitize the animals to the injection of tuberculin during intradermal tuberculin testing (IDT). Difficulties can be experienced in the reading and interpretation of the IDTs. A uniform approach to the application and interpretation of tuberculin tests for pinnipeds is suggested.

Intradermal Tuberculin Testing (IDT)

The IDT measures the cellular response of the immune system to mycobacterial antigens. The single, IDT requires 0.1 ml. of Tuberculin (Bovine Purified Protein Derived (PPD)) 3mg/ml to be injected intradermally and observed for any reaction 72 hours later.

With pinnipeds it is suggested that a comparative IDT utilizing Bovine and Avian PPD become the standard method. This will help distinguish mycobacterial infection of mammalian origin from infection with other mycobacteria.

Method: A. Injection

1.  The animal is restrained (preferably with sedation) and the lateral neck area clipped. An area of 100 mm x 100 mm is sufficient.

2.  The two injection sites should be well separated and marked with a felt pen marker. Diagonal placement makes for easier injection and reading.

3.  Using calipers, the double fold skin thickness at each injection site is measured in mm. and recorded.

4.  Each site is swabbed with alcohol prior to the intradermal injection of:

a.  0.1 ml of Avian PPD tuberculin (25,000 units/ml).

b.  0.1 ml of Bovine PPD tuberculin (3 mg/ml).

5.  The time of each injection is recorded.

Method: B. Reading

1.  The pinnipeds are again restrained, so that the double fold skin thickness can be measured with the calipers and recorded at 72 hours post injection.

2.  It is important that a consistent method of caliper operation is employed and that the same person measures the skin thickness on each occasion. Varying results can be obtained with a change in the amount of restraint, different muscle or skin tension or with the animal in a different position. The firmness of any swelling and the associated oedema may also make reading difficult.

3.  An increase of 4mm or more in skin thickness is regarded as a positive reaction. However, the measured skin thicknesses should be used as a guide and interpretation based on the Standard Interpretation Chart (see Figure 1.)

4.  It is a disadvantage of this procedure that the pinnipeds need to be subjected to a second restraint within 72 hours but so far no complications have been observed.

Figure 1. Interpretation of the Comparative Tuberculin Test National Bovine Brucellosis and Tuberculosis Eradication Campaign, Department of Primary Industry, Australia.

Method: C. Interpretation

1.  The measured skin thickness for each type of tuberculin is plotted on the relevant axis of the chart and the result read from the section of the chart where the plots intersect.

2.  Three categories of reaction are provided: Positive, Negative and Suspect.

ELISA Testing

An enzyme linked immunosorbent assay (ELISA) aims to detect circulating antibodies to TB. A modified ELISA assay was used by Cousins (1987) to test pinnipeds reacting to the IDT. Serum samples were collected and tested against both Bovine and Avian PPD antigens.

There was good correlation between the results of ELISA and IDTs in the pinnipeds sampled. The 10 Western Australian animals were sampled only 7-22 days after IDTs and it is possible that a serologic anamnestic response may have been stimulated in these animals.

However, a further sample of 18 pinnipeds were tested from a South Australian population at the time of IDT and more than 90 days after a previous IDT. The ELISA results were all negative, the comparative IDTs revealing 3 suspect reactions (as interpreted from the Standard Interpretation Chart). Subsequently the 3 suspect animals were euthanized and underwent a detailed necropsy with some 90 lymph nodes and tissue samples cultured for mycobacteria over a period of 3 months. No mycobacterial organisms were cultured.

The ELISA assay seems to have some merit for the detection of TB in pinnipeds and could be especially useful in conjunction with IDTs.

ELISA testing has the advantages of only requiring a single restraint of the animals and the serum, once collected, can be stored frozen for later laboratory assay.

ELISA could well prove to be a practical field test but the validity of a single ELISA assay needs more evaluation.

References

1.  Boever, W.J. (1978). Mycobacterium chelonei infections in three Natterer's Manatees, In: Mycobacterial infections in Zoo Animals, R. Montali (ed), pp 251-252.

2.  Cousins, D.V. (1987). ELISA for detection of tuberculosis in seals, Veterinary Record, 121, p 305.

3.  Cousins, D.V., Francis B.R., Gow B.L., Collins D.M., McGlashan, Gregory A., Mackenzie R.M., (1990). Tuberculosis in captive seals; bacteriological studies on an isolate belonging to the Mycobacterium tuberculosis complex. Research in Veterinary Science, 48, pp 196-200.

4.  Forshaw D and Phelps G.R. (1989). Tuberculosis due to Mycobacterium bovis in a captive colony of Pinnipeds. (Manuscript submitted to J.Wildlife Diseases).

5.  Gutter A.E., Wells S.K., and Spraker T.R. (1987). Generalised mycobacteriosis in a California sea lion (Zalophus californicus). J. Zoo Animal Medicine, 18, pp 118-120.

6.  Lewis, J.C.M. (1987). Cutaneous mycobacteriosis in a Southern sea lion. Aquatic Mammals, 13, pp 105-108.

7.  Morales P., Madin S.H., and Hunter A. (1985). Systemic Mycobacterium marinum infection in an Amazon manatee. JAVMA, 187, pp 1230-1231.

8.  Reddacliff G. and Lim L. (1990). Infectious Disease Monitoring in Captive and Wild Pinnipeds, Final Report : Project No. 8889/23, Australian National Parks & Wildlife Service.

9.  Stoskopf, M.K., Meench T., Thoen C., Charache P. (1987). Tuberculosis in pinnipeds. Proc. Annual Meet. Am. Assoc. Zoo Vet., p 393.

10. Wells, S.K., Gutter A. and Van Meter K. (1990). Cutaneous mycobacteriosis in a harbour seal : attempted treatment with hyperbaric oxygen. Journal of Zoo and Wildlife Medicine, 21, pp 73-78.