Development of diagnostic methods and epidermiology of tuberculosis in free-ranging long-tailed macaque macaca fascicularis and rhesus macaque M. mulatta in Thailand

Cross-species infectious disease transmission, especially between humans and wildlife, is increasing during the global warming. Tuberculosis (TB) is an air-borne infectious disease that can be transmitted from humans to non-human primates (NHPs), however, no report of TB prevalence in free ranging NHPs that highly interacted with humans in Thailand was published. Thus, this study was conducted and separated into 3 phases. Phase I, active TB in 1,836 free-ranging macaques including 6 rhesus (Macaca mulatta; Mm), 23 common long-tailed (M. fascicularis fascicularis; Mff), and 3 Burmese long-tailed (M. f. aurea; Mfa) macaque populations throughout Thailand was assesses. Monkeys were captured, anesthetized, and collected oropharyngeal, buccal, and rectal swabs. The swabs were screened for Mycobacterium tuberculosis complex (MTBC), a causative agent of TB, using IS6110-nested PCR technique, which had a limit of detection at 10 fg/μl of MTBC. The 181-bp nucleotide sequence of PCR amplicon showed 100% similarity with the MTB H37Rv genome sequence. The oropharyngeal swabs provided the best medium of specimens for MTBC detection. The MTBC prevalence, at both population and individual levels, in Mm was higher than Mff. All three populations of Mfa were MTBC positive. No difference in MTBC prevalence between males and females, but adults showed a higher infection frequency than subadults and juveniles. The study detected no association between the frequency of human interaction and the MTBC prevalence. Phase II, the non-invasively oral and fecal specimen collections were developed, using a rope bait method and a direct swab of the freshly defecated excretion, for large-scale MTBC surveillance in free-ranging macaques. First, the non-invasively collected specimens were compared with invasively specimens for MTBC detection in captive Mff, using IS6110-nested PCR. A significant correlation was detected between two types of oral specimens (oral swabs and baited ropes; n = 24, r2 = 1, p-value < 0.001), but fresh fecal swabs showed higher MTBC frequencies than the rectal swabs. Later, 1 Mff and 3 Mfa populations that showed MTBC positive results in Phase I were noninvasively collected oral (173 animals) and fecal (204 animals) specimens and repeated for MTBC detection. The proportion of MTBC-positive Mff was significantly higher in the freshly fecal swabs (8.82%) than in the baited ropes (5.20%). Phase 3, the TB-multi antigen print immunoassay (TB-MAPIA) was developed to detect a latent TB infection in 119 plasma samples of 3 Mfa populations that showed positive MTBC test in Phase I. The TB-MAPIA antibody test detected two-fold higher of positive results than the IS6110 nested-PCR antigen test. Thus, this study revealed that the free-ranging macaques in Thailand are potential reservoirs of MTBC that can lead to zoonotic transmission to other macaques or humans, the preventive measure should be established.