Full-length 16S rDNA sequencing based on Oxford Nanopore Technologies revealed the association between gut-pharyngeal microbiota and tuberculosis in cynomolgus macaques.

Tuberculosis (TB) is an infectious disease caused by the Mycobacterium tuberculosis complex (Mtbc), which develops from asymptomatic latent TB to active stages. The microbiome was purposed as a potential factor affecting TB pathogenesis, but the study was limited. The present study explored the association between gut-pharyngeal microbiome and TB stages in cynomolgus macaques using the full-length 16S rDNA amplicon sequencing based on Oxford Nanopore Technologies. The total of 71 macaques was divided into TB (-) control, TB (+) latent and TB (+) active groups. The differential abundance analysis showed that Haemophilus hemolyticus was decreased, while Prevotella species were increased in the pharyngeal microbiome of TB (+) macaques. In addition, Eubacterium coprostanoligenes in the gut was enriched in TB (+) macaques. Alteration of these bacteria might affect immune regulation and TB severity, but details of mechanisms should be further explored and validated. In summary, microbiota may be associated with host immune regulation and affect TB progression. The findings suggested the potential mechanisms of host-microbes interaction, which may improve the understanding of the role of microbiota and help develop therapeutics for TB in the future.

Advancing tuberculosis diagnosis and management in cynomolgus macaques using Xpert MTB/RIF ultra assay.

The detection and management of Mycobacterium tuberculosis complex (MTBC) infection, the causative agent of tuberculosis (TB), in macaques, including cynomolgus macaques (Macaca fascicularis), are of significant concern in research and regions where macaques coexist with humans or other animals. This study explored the utility of the Xpert MTB/RIF Ultra assay, a widely adopted molecular diagnostic tool to diagnose tuberculosis (TB) in humans, to detect DNA from the Mycobacterium tuberculosis complex in clinical samples obtained from cynomolgus macaques. This investigation involved a comprehensive comparative analysis, integrating established conventional diagnostic methodologies, assessing oropharyngeal-tracheal wash (PW) and buccal swab (BS) specimen types, and follow-up assessments at 3-month, 6-month, and 12-month intervals. Our results demonstrated that the Xpert MTB/RIF Ultra assay was able to detect MTBC in 12 of 316 clinical samples obtained from cynomolgus macaques, presenting a potential advantage over bacterial culture and chest radiographs. The Xpert MTB/RIF Ultra assay exhibited exceptional sensitivity (100%) at the animal level, successfully detecting all macaques positive for M. tuberculosis as confirmed by traditional culture methods. The use of PW samples revealed that 5 positive samples from 99 (5.1%) were recommended for testing, compared to 0 samples from 99 buccal swab (BS) samples (0.0%). In particular, the definitive diagnosis of TB was confirmed in three deceased macaques by MTB culture, which detected the presence of the bacterium in tissue autopsy. Our findings demonstrate that the implementation of the Xpert MTB/RIF Ultra assay, along with prompt isolation measures, effectively reduced active TB cases among cynomolgus macaques over a 12-month period. These findings highlight the advance of the Xpert MTB/RIF Ultra assay in TB diagnosis and its crucial role in preventing potential outbreaks in cynomolgus macaques. With its rapidity, high sensitivity, and specificity, the Xpert MTB/RIF Ultra assay can be highly suitable for use in reference laboratories to confirm TB disease and effectively interrupt TB transmission.

Influence of COVID-19 on the emergence of stone-tool use behavior in a population of common long-tailed macaques (Macaca fascicularis fascicularis) in Thailand.

Stone tool use is a rare behavior across nonhuman primates. Here we report the first population of common long-tailed macaques (Macaca fascicularis fascicularis) who customarily used stone tools to open rock oysters (Saccostrea forskali) on a small island along the Thai Gulf in Koh Ped (KPE), eastern Thailand. We observed this population several times during the past 10 years, but no stone-tool use behavior was observed until our survey during the coronavirus disease 2019 (COVID-19) pandemic in July 2022. KPE is located in Pattaya City, a hotspot for tourism in Thailand. Tourists in this area frequently provided large amounts of food for the monkeys on KPE. During the COVID-19 curfew, however, tourists were not allowed to access the island, and monkeys began to face food scarcity. During this time, we observed stone-tool use behavior for the first time on KPE. Based on our observations, the first tool manipulation was similar to stone throwing (a known precursor of stone tool use). From our observations in March 2023, we found 17 subadult/adult animals performing the behavior, 15 of 17 were males and mostly solitary while performing the behavior. The M. f. fascicularis subspecies was confirmed by distribution, morphological characteristics, and mtDNA and SRY gene sequences. Taken together, we proposed that the stone tool use behavior in the KPE common long-tailed macaques emerged due to the COVID-19 food scarcity. Since traveling is no longer restricted many tourists have started coming back to the island, and there is a high risk for this stone tool-use behavior to disappear within this population of long-tailed macaques.

Development of Quantitative Real-Time PCR and Loop-Mediated Isothermal Amplification Assays for the Surveillance and Diagnosis of Herpes B Virus Infection.

Herpes B virus (BV) is a zoonotic virus which can be transmitted from macaques to humans, which is often associated with high mortality rates. Because macaques often exhibit asymptomatic infections, individuals who come into contact with these animals face unexpected risks of BV infections. A serological test is widely performed to investigate BV infections. However, the assay's sensitivity and specificity appeared to be inadequate, and it does not necessarily indicate ongoing viral shedding. Here, we developed LAMP and qPCR assays aiming to detect BVs with a high sensitivity and specificity in various macaque species and validated them using oral swab samples collected from 97 wild cynomolgus macaques living in Thailand. Our LAMP and qPCR assays detected more than 50 and 10 copies of the target sequences per reaction, respectively. The LAMP assay could detect BV within 25 min, indicating its advantages for the rapid detection of BV. Collectively, our findings indicated that both assays developed in this study exhibit advantages and usefulness for BV surveillance and the diagnosis of BV infections in macaques. Furthermore, for the first time, we determined the partial genome sequences of BVs detected in cynomolgus macaques in Thailand. Phylogenetic analysis revealed the species-specific evolution of BV within macaques.

Non-invasive specimen collections for Mycobacterium tuberculosis detection in free-ranging long-tailed macaques (Macaca fascicularis).

Surveillance of infectious diseases in free-ranging or wild animals has been widely conducted in many habitat-range countries after the COVID-19 episode. Thailand is located in the center of the distribution range of long-tailed macaques (Macaca fascicularis; Mf) where the animals have both frequent human contact and a high prevalence of human tuberculosis. For the large-scale detection of Mycobacterium tuberculosis complex (MTBC) using IS6110-nested PCR in free-ranging Mf, non-invasive sampling was developed using oral (via rope bait) and fecal (direct swabs of fresh feces) specimen collection. Firstly, the MTBC-IS6110-nested PCR was validated in non-invasively collected specimens, in terms of its specificity and sensitivity, and then compared with those of the invasively collected oral and rectal swabs in 24 captive MTBC-suspected Mf. After validation, these methods were applied to survey for the prevalence of shed MTBC (MTBCS) in four previously reported MTBC-infected populations. A total of 173 baited rope specimens and 204 freshly defecated excretions were collected. The limit of detection of the IS6110-nested PCR technique was 10 fg/µL and the 181-bp PCR amplicon showed 100% sequence similarity with the MTB H37Rv genome sequence. Comparing the MTBCS detection between the invasive and non-invasive collected specimens in captive suspected Mf revealed a significant correlation between the two types of oral specimens (oral swabs and baited ropes; n = 24, r2 = 1, p-value < 0.001), but fresh fecal swabs showed higher MTBCS frequencies than the rectal swabs. Moreover, the proportion of MTBCS-positive free-ranging Mf were significantly higher in the fresh fecal swabs (8.82%; 95% CI; 4.9-12.7%) than in the baited ropes (5.20%; 95% CI; 1.9-8.5%). This result indicates that oral sampling via baited ropes and fecal sampling via defecated excretion swabs can serve as ancillary specimens for MTBCS detection in free-ranging non-human primates.

Prevalence of Mycobacterium tuberculosis Complex among Wild Rhesus Macaques and 2 Subspecies of Long-Tailed Macaques, Thailand, 2018-2022.

We identified tuberculosis in 1,836 macaques from 6 wild rhesus (Macaca mulatta), 23 common long-tailed (M. fascicularis fascicularis), and 6 Burmese long-tailed (M. fascicularis aurea) macaque populations in Thailand. We captured, anesthetized, and collected throat, buccal, and rectal swab specimens from the macaques. We screened swabs for Mycobacterium tuberculosis complex (MTBC) using insertion sequence 6110-specific nested PCR. We found higher MTBC prevalence at both population and individual levels among M. mulatta than M. fascicularis fascicularis macaques; all 3 M. fascicularis aurea macaque populations were positive for tuberculosis. We found that throat swab specimens provided the best sample medium for detecting MTBC. Our results showed no difference in MTBC prevalence between male and female animals, but a higher percentage of adults were infected than subadults and juveniles. Although we detected no association between frequency of human-macaque interaction and MTBC prevalence, bidirectional zoonotic transmission should be considered a possible public health concern.

Tuberculosis detection in nonhuman primates is enhanced by use of testing algorithms that include an interferon-γ release assay.

OBJECTIVE: To develop a testing algorithm that incorporates multiple assays to evaluate host cellular and humoral immunity and antigen detection concerning Mycobacterium tuberculosis complex (MTBC) infection in captive nonhuman primates. ANIMALS: Cohorts of captive-bred and wild-caught macaques from 5 different geographic regions. PROCEDURES: Macaques were tested for MTBC infection by use of a γ interferon tuberculosis (GIFT) assay, an interferon-γ release assay, and other assays. In the first 2 cohorts (n = 15 and 181), initial validation of the GIFT assay was performed by use of experimentally infected and unexposed control macaques. In the next 3 cohorts (n = 59, 42, and 11), results were obtained for opportunistically collected samples from macaques exposed during spontaneous outbreaks. RESULTS: Sensitivity and specificity of the GIFT assay in the control cohorts were 100% and 97%, respectively, and were variable but enhanced by incorporating results from multiple assays in spontaneous outbreaks. CLINICAL RELEVANCE: The detection and management of MTBC infection in captive nonhuman primate populations is an ongoing challenge, especially with animal imports and transfers. Despite standardized practices of initial quarantine with regular intradermal tuberculin skin testing, spontaneous outbreaks continue to be reported. Since infection encompasses a range of disease manifestations over time, a testing algorithm that incorporates multiple assays, such as the GIFT assay, to evaluate host cellular and humoral immunity in addition to agent detection is needed. Testing a combination of samples from controlled studies and spontaneous outbreaks of MTBC infection in nonhuman primates would advance the development and validation of a functional algorithm that incorporates promising tools such as the GIFT assay.