Plasmodium knowlesi is an emerging zoonotic malaria of increasing public health importance in Southeast Asia. This study aimed to characterise serum protein alterations associated with infection using the Macaca fascicularis model. Serum samples collected from wild and laboratory macaques before and after infection were analysed using a gel-based proteomic workflow comprising two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry (MALDI-TOF/TOF), and two-dimensional immunoblotting. Comparative protein expression maps generated within the pH 4–7 range revealed multiple differentially expressed protein spots between pre-infection and post-infection samples. Proteomic analysis identified several host-derived serum proteins associated with immune, inflammatory, and metabolic responses, including complement component 3 (C3), haptoglobin (HP), α1-antitrypsin (A1AT), serotransferrin (TF), apolipoprotein A-I (APOA1), vitamin D-binding protein (VTDB), and immunoglobulin-related proteins. Notably, a parasite-derived Plasmodium knowlesi infection protein was detected exclusively in infected serum samples, demonstrating the ability of the proteomic platform to identify circulating parasite molecules in host blood. Immunoblotting further identified serotransferrin as the principal IgM-reactive protein in normal macaque serum. In conclusion, serum proteomic profiling successfully revealed both host-response proteins and parasite-derived proteins during Plasmodium knowlesi infection. These findings highlight the value of serum proteomics for investigating host–parasite interactions and support future biomarker discovery for zoonotic malaria.

Sambar deer (Rusa unicolour) are native to most parts of Asia, including Malaysia, Taiwan, Indonesia, and India. Listed as “vulnerable” by the International Union for Conservation of Nature’s Red List, the animal has recently been introduced into the United States, Australia, and New Zealand. Although they can easily adapt to a wide range of habitats, the population of Sambar deer in the wild has dramatically declined, and this may be attributed to poaching, illegal wildlife trade and habitat loss when jungles are cleared for development. This article provides the status and distribution of Sambar deer in Peninsular Malaysia and protection efforts to conserve this species in the country. It also highlights the potential of Sambar deer commercial farming in Peninsular Malaysia as part of the conservation programme and meeting human demand.

Pangolins are considered the most trafficked mammals in the world with all eight species listed on CITES Appendix I. Despite this pervasive threat to their survival, there remains a limited understanding of genetic diversity and connectivity among populations of Asian pangolin species, hampering effective conservation management. We analysed mitogenome sequences of the Sunda pangolin (Manis javanica) from across their Southeast Asia continental distribution, as well as Borneo. Phylogenetic reconstruction revealed six lineages, with clear separation north and south of the Kangar-Pattani biogeographic line in southern Thailand, revealing clear differentiation between Sundaland and Indochinese Sunda pangolin lineages. Further divergence across an east–west divide was observed in central and northern Thailand, extending northwards towards China. Our results provide new insights into the evolutionary relationships among Sunda pangolin populations in Southeast Asia, building on other recent research in this field and helping to establish the species’ baseline phylogeography. These inferences will aid conservation planning and support the genetic traceability of the illegal pangolin trade.

Understanding the prey preference of Malayan tiger (Panthera tigris jacksoni) in Malaysia is important to guide conservation planning initiatives. The utilisation of DNA metabarcoding provides valuable insights, particularly in the field of carnivora diet research. This technique has been proven to be effective for identifying various species within complex mixtures such as scat materials, where visual identification is challenging. The Cytochrome c oxidase subunit I (COI) locus has been selected as it is a widely used as an effective non-invasive approach for diet studies. Hence, given this advance approach, Malayan tiger scats were collected on the basis of existing records of their presence in two types of habitats, namely, protected areas (PA) and human–tiger conflict (HTC) areas. This study aimed to identify prey species in Peninsular Malaysia, based on Malayan tiger scat samples using DNA metabarcoding. Based on the partial mitochondrial COI region, DNA metabarcoding led to the taxonomic resolution of prey DNA remnants in scats and the identification of prey species consumed by Malayan tiger, which were predominately small-to-medium-sized prey, including livestock. The dominant DNA prey detected belongs to the family Canidae, followed by Bovidae, Vespertilionidae, Homonidae, Felidae, Phasianidae and Muridae. A significant difference (p < 0.05) was observed in alpha and beta diversity using the Shannon index and PERMANOVA with regard to prey richness and evenness in two different habitat groups, namely, PA and HTC. Our finding provides insights into Malayan tiger dietary requirements, which can be used to develop conservation plans and strategies for Malayan tiger, particularly for habitat priorities.

The gut microbiome refers to the microorganism community living within the digestive tract. The environment plays a crucial role in shaping the gut microbiome composition of animals. The gut microbiome influences the health and behavior of animals, including the critically endangered Malayan tiger (Panthera tigris jacksoni). However, the gut microbiome composition of Malayan tigers, especially those living in their natural habitats, remains poorly understood. To address this knowledge gap, we used next-generation sequencing DNA metabarcoding techniques to analyze the gut microbiome of wild Malayan tigers using fecal samples collected from their natural habitats and in captivity. Our aim was to determine the gut microbiota composition of the Malayan tiger, considering the different types of habitat environments. The results revealed a diverse microbial community within the gut microbiome of Malayan tigers. The prominent phyla that were observed included Firmicutes, Proteobacteria, Actinobacteriota, Fusobacteriota and Bacteroidota. Beta diversity analysis revealed significant differences in gut microbiome composition of Malayan tigers that inhabited oil palm plantations, in villages and protected areas. Diversity analysis also revealed significant difference in the gut microbiome between wild and captive Malayan tigers. However, the distinctions of gut microbiome between wild and captive alpha diversity did not yield significant differences. The differences in microbiome diversity resulted from the interplay of dietary intake and environmental factors. This information will facilitate the establishment of focused conservation approaches and enhance our understanding of the effect of microbiome composition on Malayan tiger health.

The illegal trade in tigers (Panthera tigris) and their derivatives, such as bones, teeth and pelts, is a major threat to the species' long-term persistence. As wild tiger populations have dwindled, a large proportion of trafficked tiger products now derive from captive breeding facilities found throughout Asia. Moreover, wild tigers have been poached and laundered into captive facilities, then falsely designated as captive-bred. The establishment of a DNA registration system is recognized as a key tool to monitor compliance of captive facilities, support tiger trade investigations and improve prosecution outcomes. Here, we present a standardised wildlife forensic DNA profiling system for captive tigers called TigerBase. TigerBase has been developed in four South-East Asia countries with captive tiger facilities: Malaysia, Vietnam, Thailand and Lao PDR. TigerBase DNA profile data is based on 60 single nucleotide polymorphism (SNP) markers, genotyped using two different TaqMan®-based approaches: OpenArray® chip (capable of genotyping 60 SNPs for 48 samples in a single chip), and singleplex TaqMan® assays (capable of genotyping one SNP for one sample per reaction). Of the 60 SNPs, 53 are autosomal nuclear markers, suitable for individualisation and parentage applications, two are sex-linked markers, suitable for sexing, and five are mtDNA markers, suitable for maternal subspecies identification. We conducted a series of validation experiments to investigate the reliability and limitations of these SNP genotyping platforms. We found that the OpenArray® chip platform is more appropriate for generating reference data given its greater throughput, while the singleplex TaqMan® assays are more appropriate for genotyping lower quality casework samples, given their higher sensitivity and throughput flexibility. Only 19 autosomal nuclear markers were validated as singleplex TaqMan® assays, which generally provides ample power for individualisation analysis (probability of identity among siblings was <6.9 ×10-4), but may lack power for specific parentage questions, such as determining parentage of an offspring when one of the parent's genotypes is missing. Further, we have developed pipelines to support standardised SNP calling and decrease the chance of genotyping errors through the use of analytical workflows and synthetic positive controls. We expect the implementation of TigerBase will enhance enforcement of tiger trafficking cases and encourage compliance among captive tiger facilities, together contributing to combatting the illegal tiger trade.

Complete mitochondrial genome sequences have significant relevance in the study of phylogenetic relationships, evolution, and population genetics. In this paper, we present the complete mitochondrial genome of the red junglefowl ( Gallus gallus spadiceus ) from Peninsular Malaysia, inferred using next-generation sequencing (NGS). The mitogenome is 16,785 bp in length with the structural organization of an avian mitochondrial arrangement compromising 13 protein-coding regions, 22 tRNAs, 2 rRNAs, and 1 control region. No internal stop codon was found in the protein-coding genes. Overall base composition is A: 30.3%, C: 32.5%, G: 13.5%, and T: 23.7%, indicating a high A + T content of 54.0%. Phylogenetic tree analysis revealed that red junglefowl from Peninsular Malaysia is grouped together with other members of Gallus gallus specifically from Southeast Asia, with 89% bootstrap value support. These research findings might be beneficial for red junglefowl genetic identification, molecular systematic studies, and conservation management interest in the future.

Background The Malayan pangolin (Manis javanica) is a placental mammal and is listed as Critically Endangered on the IUCN Red List of Threatened Species. Most previous attempts to breed pangolins in captivity have met with little success because of dietary issues, infections, and other complications, although a previous study reported breeding pangolins in captivity to the third generation. In our previous pangolin genome sequencing data analysis, we obtained a considerable amount of bacterial DNA from a pregnant female Malayan pangolin (named “UM3”), which was likely infected by Paraburkholderia fungorum—an agent of biodegradation and bioremediation in agriculture. Methodology Here, we further confirmed and characterized this bacterial species using PCR, histological staining, whole-genome sequencing, and bioinformatics approaches. PCR assays with in-house designed primer sets and 16S universal primers showed clear positive bands in the cerebrum, cerebellum, lung, and blood of UM3 suggesting that UM3 might have developed septicaemia. Histological staining showed the presence of Gram-negative rod-shaped bacteria in the pangolin brain and lungs, indicating the colonization of the bacteria in these two organs. In addition, PCR screening of UM3’s fetal tissues revealed the presence of P. fungorum in the gastrocnemius muscle, but not in other tissues that we examined. We also sequenced and reconstructed the genome of pangolin P. fungorum, which has a genome size of 7.7 Mbps. Conclusion Our study is the first to present detailed evidence of the presence of P. fungorum in a pangolin and her fetus (although preliminary results were presented in our previous article). Here, we raise the concern that P. fungorum may potentially infect humans, especially YOPI (young, old, pregnant, and immunocompromised) people. Therefore, caution should be exercised when using this bacterial species as biodegradation or bioremediation agents in agriculture.

Intro: Adenovirus has often been used as a health risk indicator due to its ubiquitous presence in the environment, humans and wildlife. However, a wide range of prevalence values have been reported. We hereby address the prevalence of Adenovirus specifically among non-human primate populations. Methods: Primate faeces were collected from the ground of the habitat of nine primate populations in Selangor and Kuala Lumpur. Faeces samples were scored on a scale of 1 to 5 based on freshness, subjected to bead-beating and magnetic beads-based DNA extraction, followed by PCR amplification. Locations that reported positive samples or had abundant samples were sampled again, within the next day to two months, to determine the detection rate of Adenovirus. Findings: Four out of nine primate populations reported zero detection on the first sampling, whereby 8, 11, 23, and 16 samples were collected respectively. These sites were not sampled further. The detection of Adenovirus among the other five primate populations ranged from 4.8% to 35.3% (1/21 to 6/17) at any individual sampling events. Adenovirus detection per population over two sampling events fluctuated between -12.2%% and +2.1%. This brought to an overall prevalence of 23.3% (17/73) if only primate population reporting positive cases were considered, and 13.0% (17/131) if all of the nine populations were considered. Conclusion: The prevalence of Adenovirus reported by our study seems to fall within the range of reported prevalence in the literature - when only positive populations were considered. It is critical to take note that the ‘actual’ prevalence could be lower, if non-positive populations were to be included. Thus, the prevalence reported in the literature needs to be verified cautiously from this aspect. This fundamental information would aid resource planning for monitoring or research activities, especially if costly next generation sequencing crucial for emerging pathogen surveillance and zoonotic diseases were to be implemented.

Aim: The Sunda pangolin (Manis javanica) is the most widely distributed Asian pangolin species. It is one of the most trafficked mammals in the world, which not only negatively impacts wild Sunda pangolin populations, but also poses a potential disease risk to other species, including humans and livestock. Despite the imminent threat to the species’ survival and its prevalence in the wildlife trade, the phylogeography and evolution of the Sunda pangolin is not well understood. We aimed to investigate the species’ phylogeography across its distribution to improve our understanding of the species’ evolutionary history, elucidate any taxonomic uncertainties and enhance the species’ conservation genetic management and wildlife forensics applications. Location: Southeast Asia and southern China. Methods: We sequenced mtDNA genomes from 23 wild Sunda pangolins from Borneo and Peninsular Malaysia. We used these data in conjunction with previous generated mtDNA and nuclear datasets from across the species’ range to perform various phylogenetic and population genetic analyses. Results: We identified an evolutionarily distinct mtDNA lineage in north Borneo, which was estimated to be ~1.6 million years divergent from lineages in west/south Borneo and the mainland, comparable to the divergence time of the Palawan pangolin. There appeared to be mitonuclear discordance, with no apparent genetic structure across Borneo based on analysis of nuclear SNPs. Main conclusions: These findings are consistent with the ‘out of Borneo hypothesis’, whereby Sunda pangolins diversified in Borneo before subsequently migrating throughout Sundaland, and/or a secondary contact scenario between mainland and Borneo. We have elucidated possible taxonomic issues in the Sunda/Palawan pangolin complex, and highlight the critical need for additional georeferenced samples to accurately apportion its range-wide genetic variation into appropriate taxonomic and conservation units. Additionally, these data have improved forensic species identification testing involving these species and permit the implementation of geographic provenance testing in some scenarios.

Conservation translocation and reintroduction for the purpose of repopulating and reinforcing extirpated or depleted populations has been recognised as an important conservation tool, particularly for gibbon conservation in the immediate future. Feasibility assessments involving multiple factors, including taxonomic and genetic assessment of rescued and captive gibbons, are imperative prior to translocation and reintroduction programmes. In this study, we attempt to determine the subspecies and origin of captive Hylobateslar, White-handed gibbons, from Peninsular Malaysia to assist in future translocation and reintroduction programmes. A total of 12 captive and rescued H.lar samples were analysed using the control region segment of mitochondrial DNA. Sequence analyses and phylogenetic trees constructed using neighbour-joining, maximum likelihood, Bayesian inference, and network methods congruently differentiate all 12 captive individuals used in this study from other H.lar subspecies suggesting that these individuals belong to the H.larlar subspecies. In addition, two populations of H.l.lar were observed: (1) a southern population consisting of all 12 individuals from Peninsular Malaysia, and (2) a possible northern population represented by three individuals (from previous studies), which might have originated from the region between the Isthmus of Kra, Surat Thani-Krabi depression, and Kangar-Pattani. Our findings suggest that the complete control region segment can be used to determine the subspecies and origin of captive H.lar.

Tigers are killed to supply a demand for many wildlife products despite a ban on commercial international trade. As populations decrease, products from substitute species (i.e. lions and leopards) have been fraudulently sold as tiger. DNA forensic techniques are needed to definitively identify tiger in order to secure prosecutions although this is complicated by the presence of numts. Therefore, we have developed and validated a CO1 genetic marker that preferentially amplifies the mtDNA CO1 region and excludes the nuclear CO1 pseudogene, which we expect to be of use in tiger forensic casework.

Pangolins are the most trafficked mammal in the world, and all eight species are listed under CITES Appendix I. DNA-based wildlife forensic techniques are recognized as an important component of investigating a pangolin seizure. In particular, determining the species of pangolin in a seizure will 1) confirm the presence of pangolin to establish the legality of any trade, and 2) ensure appropriate laws are applied to their fullest extent in a prosecution. Furthermore, valuable intelligence data, such as determining the geographic provenance of samples, can be produced through analysis of pangolin seizures. Despite the immense scale of the pangolin trade, standardized wildlife forensic techniques for testing pangolin seizures are in their infancy. To address this, here, we present a standardized genetic marker suitable for species identification of all eight pangolin species, and outline practical strategies for sampling large-volume pangolin scale seizures. We assessed the repeatability, reproducibility, robustness, sensitivity and phylogenetic resolution of this species identification test. Critically, the assay was tested in four wildlife forensic laboratories involved in testing pangolins. Additionally, we demonstrated the test’s utility to conduct geographic provenance analysis of Phataginus tricuspis samples. We analysed five large-volume pangolin scale seizures in Malaysia, which elucidated key target species, poaching hotspots, and trafficking routes. Phataginus tricuspis was the most commonly identified species (88.8%) from the seizure samples, and 84.3% of these P. tricuspis individuals were likely sourced from western central Africa. We expect the implementation of the techniques presented in this paper will improve enforcement of pangolin trafficking crimes.

The legal and illegal trade in wildlife for food, medicine and other products is a globally significant threat to biodiversity that is also responsible for the emergence of pathogens that threaten human and livestock health and our global economy. Trade in wildlife likely played a role in the origin of COVID-19, and viruses closely related to SARS-CoV-2 have been identified in bats and pangolins, both traded widely. To investigate the possible role of pangolins as a source of potential zoonoses, we collected throat and rectal swabs from 334 Sunda pangolins (Manis javanica) confiscated in Peninsular Malaysia and Sabah between August 2009 and March 2019. Total nucleic acid was extracted for viral molecular screening using conventional PCR protocols used to routinely identify known and novel viruses in extensive prior sampling (> 50,000 mammals). No sample yielded a positive PCR result for any of the targeted viral families-Coronaviridae, Filoviridae, Flaviviridae, Orthomyxoviridae and Paramyxoviridae. In the light of recent reports of coronaviruses including a SARS-CoV-2-related virus in Sunda pangolins in China, the lack of any coronavirus detection in our 'upstream' market chain samples suggests that these detections in 'downstream' animals more plausibly reflect exposure to infected humans, wildlife or other animals within the wildlife trade network. While confirmatory serologic studies are needed, it is likely that Sunda pangolins are incidental hosts of coronaviruses. Our findings further support the importance of ending the trade in wildlife globally.

The COVID-19 outbreak has infected over 6 million people across the world. The origin of COVID-19 coronavirus (CoV) remains unknown, although pangolins have been suggested as potential hosts. We investigated two pangolins seized in Guangdong Province, China. Molecular screening revealed CoV in one pangolin ("Dahu"), while another ("Meidong") was infected byEhrlichia ruminantium. Dahu exhibited difficulty breathing, infections of lung, intestines, and nostrils, as revealed by computed tomography imaging and necropsy. Previous phylogenetic analyses showed bat coronavirus RaTG13 is closer to COVID-19 CoV compared to pangolin coronavirus. Over 20 caregivers have had close physical contact with CoV-positive Dahu, but none became infected with CoV. Our data suggest that pangolins are unlikely the natural reservoir or secondary hosts of COVID-19 CoV. Pangolins seems to be victims infected by CoV carried by a not yet unidentified natural reservoir host species, perhaps due to their weakened immune system.

Genetic and geographical proximity between Macaca fascicularis, Macaca namestrina and humans are posing a dangerous threat in terms of disease transmission to humans.The objective is to determine the fungal species in actively shedding anatomical sites of these macaques inhabiting forest fragment of different matrix types in Kemasul Forest Reserve, Pahang State, Malaysia.Matrix of Chemomoi (CM) had higher coverage of acacia (Acacia mangium), whereas JambuRias (JR) encompassed oil palm plantation.Swab samples were obtained from throat, anus and penis/vagina of three individual of macaques from each species and geographic location (N=12).The swabs were then streaked onto Potato Dextrose Agar plate containing 25 mg/ml chloramphenicol to obtain pure colony.The isolated fungal species were identified morphologically, and molecular identification was carried out in some of the isolates.The results reveal 18 fungal species from 10 genera identified, with JR showing 13 species compared to CM with only 7 species, but significant difference was observed between fungal distribution in different geographical locations and macaques species (p<0.05).Fungal species from both macaque species were higher in JR compared to CM but no significant difference in different anatomical sites.The most common fungi species found in JR is Aspergillus fumigatus, as it was found in all anatomical sites of both macaques but it was absent from samples in CM.Only two fungi species shared between both geographic locations which are A. ruber and A. flavus.This study reveals potential risk to both human-macaque, thus relocating the human settlements adjacent to the forests and proper waste management system are essential.

Purpose: PCR and serological-based approaches have been used to identify henipavirus and filovirus (e.g. Nipah virus, Ebola virus) exposure or infection in bat populations in Bangladesh, China, and Southeast Asia. Our understanding of the diversity of these viruses in bat reservoirs, and the frequency of spillover to other animals or people, is extremely limited. We hypothesized that henipaviruses and filoviruses were circulating within multiple bat reservoirs and that spillover of these viruses to non-human primate populations has occurred in Malaysia. Methods & Materials: We utilized a multiplex serological assay to screen sera for reactive IgGs that bound to antigens from henipaviruses and filoviruses. We produced virus attachment glycoproteins (GP) from sixteen virus species in the families Paramyxoviridae and Filoviridae. As part of our ongoing collaboration, we conducted in-country assay training and sera testing at the Department of Wildlife and National Parks, National Wildlife Forensic Laboratory. We screened sera from eight bat genera and sera from Macaca fascicularis populations. Results: Sera samples from Pteropus hypomelanus (n = 56) were reactive with Nipah virus GP (25%) and cross-reactive with GPs from closely-related henipaviruses. In these same P. hypomelanus samples we detected reactivity with Ebola virus and Sudan virus GPs (10% and 5%, respectively). Sera from several Hipposideros species and P. hypomelaus reacted with the Henipavirus species, Mojiang virus GP. Two M. fascicularis sera samples reacted with GPs from Ebola virus and Bundibugyo virus and M. fascicularis sera samples specifically reacted with Mojiang virus; all four samples exhibited median fluorescence intensity values > 10,000. We also detected reactivity to Ebola virus and Sudan virus GPs in sera samples collected from Hipposideros, Cynopterus and Rhinolophus species. We did not detect any samples that were reactive with Reston virus GPs. Conclusion: We detected evidence of past exposure to virus(es) most antigenically-similar to Ebola virus, Bundibugyo virus, and Sudan virus in sera samples from bat and non-human primate populations collected in Malaysia. This is also the first sero-survey for Mojiang virus and the first evidence of exposure to Mojiang virus in bats and non-human primates. Our results suggest that there are different but antigenically related henipaviruses and filoviruses circulating in bats in Malaysia.

Here, we present the first complete mitochondrial genome of Malayan Gaur (Bos gaurus hubbacki) inferred using next-generation sequencing. The mitogenome is 16,367 bp in length with the structural organization of a typical bovine mitochondrial arrangement comprising 13 protein-coding genes, 21 tRNAs, and 2 rRNAs. No internal stop codon was found in the protein-coding genes. Phylogenetic tree analysis revealed that Malayan gaur is more closely related to Burmese banteng instead of gaur.

The illegal ivory trade continues to drive elephant poaching. Large ivory seizures in Africa and Asia are still commonplace. Wildlife forensics is recognised as a key enforcement tool to combat this trade. However, the time and resources required to effectively test large ivory seizures is often prohibitive. This limits or delays testing, which may impede investigations and/or prosecutions. Typically, DNA analysis of an ivory seizure involves pairing and sorting the tusks, sampling the tusks, powdering the sample, decalcification, then DNA extraction. Here, we optimize the most time-consuming components of this process: sampling and decalcification. Firstly, using simulations, we demonstrate that tusks do not need to be paired to ensure an adequate number of unique elephants are sampled in a large seizure. Secondly, we determined that directly powdering the ivory using a Dremel drill with a high-speed cutter bit, instead of cutting the ivory with a circular saw and subsequently powdering the sample in liquid nitrogen with a freezer mill, produces comparable results. Finally, we optimized a rapid 2 -h decalcification protocol that produces comparable results to a standard 3-day protocol. We tested/ optimised the protocols on 33 raw and worked ivory samples, and demonstrated their utility on a case study, successfully identifying 94% of samples taken from 123 tusks. Using these new rapid protocols, the entire sampling and DNA extraction process takes less than one day and requires less-expensive equipment. We expect that the implementation of these rapid protocols will promote more consistent and timely testing of ivory seizures suitable for enforcement action.