Quantitation of Cytokine Gene Expression: A Molecular Approach for Evaluating Protective Immunity Against Tuberculosis
American Association of Zoo Veterinarians Conference 1997
Suzanne Kennedy-Stoskopf, DVM, PhD
College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA


Tuberculosis continues to be a problem in non-domestic species. Following exposure to Mycobacterium spp., most healthy individuals mount a vigorous and effective cell-mediated immune response to curtail the infection. The protective immune response depends upon interactions between infected macrophages, activated T lymphocytes, natural killer (NK) cells, and induced cytokines, which are regulatory proteins and glycoproteins that mediate interactions between cells of the immune system. A dysfunctional cell-mediated response can lead to progressive primary disease or reactivation of endogenous foci of mycobacteria.

A positive tuberculin reaction reflects delayed-type hypersensitivity to mycobacterial antigens. The test cannot distinguish between mere exposure and active disease. Isolation of the mycobacterial organism remains the definitive method for confirming infection. In some species, radiographic findings can support a diagnosis of active tuberculosis, but for other species, radiographs may not even be a realistic option. Numerous ancillary tests have been developed to aid in the diagnosis of clinical tuberculosis, particularly for hoofstock. The blood tuberculosis battery (BTB) assay, validated for deer, evaluates cell-mediated and humoral immunity in addition to inflammatory parameters.1 The bovine interferon-gamma (IFN-y) assay is an ELISA designed to detect IFN-y in whole blood incubated with bovine PPD.4 Because the reagents used in the ELISA to detect the presence of IFN-y are monoclonal antibodies directed against bovine IFN-y, there is some concern how effective this assay will be for non-bovid ungulates. Nonetheless, both the BTB and IFN-y assays are designed to evaluate the effectiveness of an individual’s immune response to mycobacterial antigens.

In particular, production of IFN-y, a cytokine associated with cell-mediated immune responses, is depressed in cultures of M. tuberculosis-stimulated peripheral blood mononuclear cells from humans with tuberculosis.5 An ELISA is also used to measure IFN-y in humans and again there is low probability that this assay would be applicable to species other than perhaps some nonhuman primates.

Recently, investigators have evaluated the expression of IFN-y mRNA in humans with tuberculosis and found levels to be low compared to healthy tuberculin reactors.2,5 Similar results were obtained for mRNA levels of interleukin-2, another cytokine important for the generation of cell-mediated immunity. The molecular sequences of cytokine genes are fortunately well conserved phylogenetically, making the use of consensus sequence primers to measure levels of cytokine mRNA by reverse transcription quantitative competitive polymerase chain reaction (RT-qcPCR) feasible.

Consensus primers derived from mouse and human sequences have been used successfully to measure mRNA levels of IFN-y and IL-2 in cats, dogs, pigs, cattle, and horses.3 In theory, this technique should be readily adaptable to a wide variety of non-domestic species which are exposed to mycobacteria and develop a positive tuberculin reaction. Exposed healthy individuals would be predicted to have elevated levels of mRNA for IFN-y and IL-2 compared to animals with active replication of the organism and clinical lesions.

Literature Cited

1.  Griffin, J.F.T. and J.P. Cross. 1989. Diagnosis of tuberculosis in New Zealand farmed deer: an evaluation of intradermal skin testing and laboratory techniques. Irish Vet. J. 42:101–107.

2.  Johnson, B.J. and D.N. McMurray. 1994. Cytokine gene expression by cultures of human lymphocytes with autologous Mycobacterium tuberculosis-infected monocytes. Infect. Immun. 62:1444–1450.

3.  Rottman, J.B., W.A.F. Tompkins, and M.B. Tompkins. 1996. A reverse transcription-quantitative competitive polymerase chain reaction (RT-qcPCR) technique to measure cytokine gene expression in domestic mammals. Vet. Pathol. 33:242–248.

4.  Wood, P.R., and J.S. Rothel. 1994. In vitro immunodiagnostic assays for bovine tuberculosis. Vet. Micro. 40:125–135.

5.  Zhang, M., Y. Lin, D.V. Iyer, J. Gong, J.S. Abrams, and P.F. Barnes. 1995. T-cell cytokine responses in human infection with Mycobacterium tuberculosis. Infect. Immun. 63:3231–3234.


Speaker Information
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Suzanne Kennedy-Stoskopf, DVM, PhD
College of Veterinary Medicine
North Carolina State University
Raleigh, NC, USA

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