Abstract

Orangutans (Pongo pygmaeus) are susceptible to Mycobacterium tuberculosis (M. tuberculosis) infection and infected animals pose a health risk to other animals, caretakers, and the public. Knowledge of the tuberculosis status of an individual is crucial to prevent spread of infection. The intradermal tuberculin test is the traditional method used for screening captive primates for M. tuberculosis infection. However, this test is problematic in orangutans because interpretation of results is subjective and there is a high incidence of false positive or nonspecific reactors.² Although mycobacterial culture is considered the gold standard for determining tuberculosis status, false negatives can occur and results often take many weeks to obtain. New testing modalities promise rapid turn-around time with increased sensitivity and specificity.

In this study of 17 captive orangutans, baseline values were determined for several diagnostic tests that are designed to detect and identify mycobacterial infections. We also attempted to assess the potential value of these tests as screening tools for M. tuberculosis infection in orangutans. These tests included a M. tuberculosis gamma interferon test, a multiple antigen serum ELISA, an Antigen 85 (Ag 85) immunoassay, and the mammalian old tuberculin intradermal test. The results of these tests were compared to the tuberculosis status of each animal which was evaluated using individual and group histories, physical examinations, complete blood counts (CBC), serum biochemistry profiles, and thoracic radiographs, as well as acid-fast staining and mycobacterial cultures of tracheal and gastric lavage samples.

The M. tuberculosis gamma interferon immunoassay quantifies and compares the amount of gamma interferon that is produced by sensitized lymphocytes after incubation of blood with bovine purified protein derivative (PPD), avian PPD, and a nil antigen, thereby measuring a component of cell-mediated immune reactivity to mycobacteria.^1,5 The serum ELISA uses multiple antigens to detect antibody responses to infection with mycobacteria, thereby measuring a component of humoral immune reactivity. The optical density values are then compared to positive control values and the ratio of these results is reported.⁷ Antigen 85 complex proteins are major secretory products of actively replicating mycobacteria and are typically produced in the early stages of infection.⁸ Measurement of serum Ag 85 by monoclonal antibody immunoassay could provide a method for identifying active mycobacterial infections that is less dependent on host immunity. This test has previously been shown to detect serum Ag 85 in orangutans, although correlation with clinical disease or mycobacterial infection has not been demonstrated.⁶

In this study, physical examination findings, CBC results, biochemistry panel results, thoracic radiographic findings, and clinical signs of the study animals were not suggestive of infection with M. tuberculosis. No tracheal or gastric lavage cultures grew M. tuberculosis. Eight of the 17 animals reacted to intradermal tuberculin tests. Five animals in the study had current or historic positive atypical mycobacterial cultures and all of these animals had positive intradermal tests.

Based on cut-off levels determined for macaques,¹ gamma interferon test results were positive for Mycobacterium avium (M. avium) complex in nine of the 17 animals. Of these nine, one had an historic gastric culture of a M. avium complex bacterium, but the other eight had no positive mycobacterial cultures. One of the 17 animals was positive for M. tuberculosis based on the gamma interferon test; this animal had a historic positive gastric culture for Mycobacterium fortuitum, which shares antigenic determinants with M. tuberculosis.⁴

The optical density ratios for the multiple antigen ELISA were highly variable between individuals. There was generally high seroreactivity to PPD and Ag 85, and less seroreactivity to modified protein 70, purified from M. bovis strain AN5 (MPB), early secreted antigen target of M. tuberculosis (ESAT), antigen 64 secretory protein of M. tuberculosis (Ag 64), and antigen 32 secretory protein of M. tuberculosis (Ag 32). Optical density ratio values showed no significant differences when animals with positive intradermal tests were compared to those with negative skin tests. Similarly, there were no significant differences when animals with a history of positive mycobacterial cultures were compared to those with no such history, and there were no differences between those that were positive for M. avium on the gamma interferon test when compared to those that were negative.

All 17 of the orangutans in the study showed reactivity in the Ag 85 immunoassay. Most reactions were mild to moderate, but three had relatively strong reactions. Two of these three had positive atypical mycobacterial cultures and all three had positive intradermal tests. On the gamma interferon assay, one was negative, one was M. avium complex positive, and one was M. tuberculosis complex positive.

This study established baseline values for the gamma interferon, multiple antigen ELISA, and Ag 85 immunoassay for orangutans that were presumed to be negative for M. tuberculosis infection. However, interpretation of these results is difficult since no animals in the study were found to have M. tuberculosis. In a clinical trial involving 135 macaques, the gamma interferon test was reported to be 92% sensitive and 100% specific for M. tuberculosis complex infection.¹ The gamma interferon test may show some promise for ruling out M. tuberculosis infection in orangutans because only one animal in our study was defined as positive on this test. However, validation of the gamma interferon assay will require positive results with blood from an M. tuberculosis-infected orangutan.

A previous study using a different ELISA protocol with M. avium and Mycobacterium bovis antigens showed an association between ELISA reaction and evidence of mycobacterial infection on gastric lavage, but an inability to differentiate between responses to different mycobacterial species.³ Results from the multiple-antigen ELISA in our study appeared highly variable and non-specific for infection; serologic reactions to PPD and Ag85 appeared to be particularly non-specific. There was generally less reactivity to MPB, ESAT, Ag64, and Ag32, so these antigens may be useful for detecting M. tuberculosis infection, assuming that an infected animal would have markedly greater seroreactivity than the orangutans in this study.

Previous investigators have questioned the specificity of the dot blot Ag85 immunoassay for orangutans,⁶ and the results here seem to suggest a high background reactivity to this antigen that may make it poorly specific for detecting M. tuberculosis infections in this species.

In order to further evaluate the effectiveness of all of these tests at determining true M. tuberculosis infection status, they will need to be evaluated on orangutans found to be culture positive for M. tuberculosis.

Literature Cited

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