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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.

Bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) has been shown to enhance vaccine effectiveness and antitumor immunity. In our previous study, we reported that co-administration of BCG-CWS with the encapsidated dengue antigens, UV-inactivated DENV2 and DENV2 NS1, synergistically induced DENV-specific adaptive immune responses in mice. As dendritic cells (DCs) are key immune players that mediate innate and adaptive immunity, we, here, asked how well the response of DCs to this adjuvant aligns with the immune responses elicited in vivo . The responses of primary monocyte-derived DCs to BCG-CWS-adjuvanted encapsidated dengue immunogens compared with the unadjuvanted vaccine were investigated. DCs stimulated by BCG-CWS and the dengue nanoparticle vaccine exhibited a superior response. This response correlated well with the stronger immune response observed in mice. This was evidenced by the marked elevation in expression levels of DC activation markers, such as CD80, CD83, CD86, and HLA-DR, and various innate immune cytokines. Additionally, this adjuvant markedly elevated the expression levels of miRNAs related to DC function, such as miR-146a, miR-147, miR-223, and miR-155. These immune components could suppress DENV2 multiplication in bystander skin cells. BCG-CWS exerted an adjuvant effect on DC responses by enhancing antigen-processing activity and activating several innate immune cytokines and immune-related miRNAs.

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 IS 6110 -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-IS 6110 -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 IS 6110 -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, r 2 = 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.

The wild-born long-tailed macaques ( Macaca fascicularis ) were recently recruited and used as breeders for the National Primate Research Center of Thailand, Chulalongkorn University (NPRCT-CU), and changes in their in-depth gut microbiota profiles were investigated. The Oxford Nanopore Technology (ONT) was used to explore full-length 16S rDNA sequences of gut microbiota in animals once captured in their natural habitat and 1-year following translocation and housing in a hygienic environment at NPRCT-CU. Our findings show that the gut microbiota of macaques after 1 year of hygienic housing and programmed diets feeding was altered and reshaped. The prevalent gut bacteria such as Prevotella copri and Faecalibacterium prausnitzii were enriched after translocation, causing the lower alpha diversity. The correlation analysis revealed that Prevotella copri , Phascolarctobacterium succinatutens , and Prevotella stercorea , showed a positive correlation with each other. Significantly enriched pathways in the macaques after translocation included biosynthesis of essential amino acids, fatty acids, polyamine and butanoate. The effects of microbiota change could help macaques to harvest the energy from programmed diets and adapt their gut metabolism. The novel probiotics and microbiota engineering approach could be further developed based on the current findings and should be helpful for captive animal health care management.

Background Asymptomatic infections with sub-microscopic Plasmodium serve as a silent reservoir of disease, critical to sustaining a low level of remanent malaria in the population. These infections must be effectively identified and targeted for elimination. The sensitivity of light microscopy, the traditional method used for diagnosing Plasmodium infections, is frequently insufficient for detecting asymptomatic infections due to the low density of parasitaemia. The objective of this study was to explore the current prevalence of asymptomatic sub-microscopic Plasmodium carriages to evaluate the parasite reservoir amongst residents from 7 hamlets in Tak Province in northwestern Thailand using a highly sensitive molecular method. Methods Malaria infection was screened in a real-world setting from 3650 finger-prick blood specimens collected in a mass cross-sectional survey using light microscopy and loop-mediated isothermal amplification (LAMP). LAMP results were later confirmed in a laboratory setting in Bangkok using nested PCR, restriction enzyme digestion and DNA sequencing. The association of malaria infection with demographic factors was explored. Results Parasite prevalence was 0.27% (10/3650) as determined by microscopy. Sub-microscopic infection prevalence was 2.33% (85/3650) by LAMP. Of these, 30.6% (26/85) were infected with Plasmodium falciparum , 52.9% (45/85) with Plasmodium vivax , 2.4% (2/85) with Plasmodium malariae , 4.7% (4/85) with mixed P. falciparum and P. vivax , and 9.4% (8/85) had parasite densities too low for species identification. Asymptomatic carriages (T < 37.5  ° C) accounted for 95% (76/80) of all sub-microscopic cases with the highest prevalence occurring in the subjects 31–45 years of age ( p  ≤ 0.035). Participants working on plantations or as merchants had an increased infection risk. Evaluation by microscopy identified 10.53% (10/95) of all Plasmodium infected participants. Conclusion Participants carrying asymptomatic Plasmodium infections with sub-microscopic parasite densities are considerable in this area. These findings provide the true disease burden and risk factors in this region. This information helps to direct policy makers towards better schemes and delivery of targeted interventions. Moreover, this is the first study to use LAMP in mass screening for sub-clinical and sub-microscopic infections in a field setting in Thailand. LAMP proves to be a sensitive and field-deployable assay suitable for national malaria control screening campaigns.

Tuberculosis is highly contagious disease that can be transmitted between humans and animals. Asian elephants ( Elephas maximus ) in captivity live in close contact with humans in many Asian countries. In this study, we developed an interferon gamma release assay (IGRA) for elephant TB detection using antigens from the MTB complex (MTBC) and nontuberculous mycobacteria (NTM) as stimulating antigens (PPD, ESAT6, CFP10) to elicit a cell-mediated immune response (CMIR). The developed assay was applied to an elephant herd of more than 60 animals in Thailand, and the results were compared with those obtained through serological detection. IGRA has sufficient sensitivity for detecting elephant interferon gamma (eIFNγ) from specific antigen-stimulated PBMCs. Among 60 animals tested, 20 samples (33.3%) showed negative results for both MTBC and NTM infection. Eighteen samples (30%) showed positive responses against PPD from M. bovis and/or ESAT6 and CFP10, indicating MTBC infection. In contrast, only 15.6% showed seropositivity in a commercial serological test kit for elephant TB. The discrepancies between serological and CMIR highlight that the two methods may detect different stages of elephant TB. Therefore, employing both tests may enable them to complement each other in correctly identifying elephants that have been exposed to MTBC.

Tuberculosis (TB) is the first infectious disease to be screened-out from specified pathogen-free cynomolgus macaques ( Macaca fascicularis ; Mf) using in human pharmaceutical testing. Being in either latent or active stage after exposure to the Mycobacterium tuberculosis complex (MTBC), the monkey gamma-interferon release assay (mIGRA) was previously introduced for early TB detection. However, a notable incidence of indeterminate results was observed. In this study, we compared two positive mitogen references, phytohemagglutinin (PHA) that is used in the QuantiFERON-TB Gold Plus kit (QFT-PHA) and a combination of Concanavalin A and Pokeweed mitogen (ConA+PWM), in a cohort of 316 MTBC-exposed Mf. Following a 29-month follow-up of 100 selected animals, we established a new mIGRA interpretation algorithm that demonstrated a significant shift in the negative and indeterminate cases regardless of whether the QFT-PHA or ConA+PWM was used as a mitogen. That is, if the OD NIL value was ≤0.18, OD MIT-NIL > OD NIL , and the OD TB1/2-NIL were ≥0.05 and ≥25% of individual OD NIL , the mIGRA result was interpreted as ‘positive’. If the OD NIL value was ≤0.18, OD MIT-NIL > OD NIL , and the OD TB-NIL was <0.05, the mIGRA result was interpreted as ‘negative’. If the OD NIL value was >0.18 or the OD of mitogen references [OD (QFT-PHA) and OD (ConA+PWM) ] were ≤0.18, the mIGRA result was interpreted as ‘indeterminate’. As a result, negative cases increased by 10–50%, indeterminate cases decreased by 40–80%, and the number of TB-positive cases remained unchanged. Our findings highlight the critical role of mitogens as positive controls in mIGRA interpretation. This study provides the mIGRA value for the TB screening of cynomolgus macaques that enables the identification of true positive and suspicious TB cases for quarantine programs.

Tuberculosis (TB) is an infectious disease caused by the Mycobacterium tuberculosis complex ( Mtb c), 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.

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.

Multi-stage tuberculosis (TB) vaccines composed of active- and dormancy-associated antigens are promising to trigger the immune protection against all TB stages. However, scientists are still in quest of the suitable vaccine candidates. In this study, we identified the potential targets for this vaccine in a high TB burden country, Thailand. Peptide microarray was applied to gauge IgA and IgG antibodies specific to 16,730 linear epitopes of 52 dormancy-associated Mycobacterium tuberculosis ( M. tb ) proteins in three study groups: active tuberculosis (ATB), latent tuberculosis infection (LTBI) and endemic healthy control (EHC). Preferential IgA recognition against epitopes of dormancy-associated proteins was identified in LTBI group. Validation of these findings revealed that LTBI subjects exhibited the greater levels of Rv2659c- and Rv1738-specific IgA than those of household contacts, but less than did ATB subjects. Frequencies of IFNγ-producing CD4 + and CD8 + T cells induced by proteins Rv2659c and Rv1738 were higher in LTBI than ATB individuals. The results indicated that LTBI group in a high TB burden country demonstrated cell-mediated immune response to proteins Rv2659c and Rv1738 stronger than those of ATB. These immune responses likely contribute to natural protection against dormant M. tb and might be potential targets for a multi-stage TB vaccine.