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Molecular investigation of Anaplasma spp. in domestic and wildlife animals in Peninsular Malaysia

Koh FX, Panchadcharam C, Sitam FT, Tay ST

Vet Parasitol Reg Stud Reports, 2018 08;13:141-147.
PMID: 31014863 DOI: 10.1016/j.vprsr.2018.05.006

Anaplasma spp. are Gram-negative obligate intracellular, tick-borne bacteria which are of medical and veterinary importance. Little information is available on Anaplasma infection affecting domestic and wildlife animals in Malaysia. This study investigated the presence of Anaplasma spp. in the blood samples of domestic and wildlife animals in Peninsular Malaysia, using polymerase chain reaction (EHR-PCR) assays targeting the 16S rRNA gene of Anaplasmataceae. High detection rates (60.7% and 59.0%, respectively) of Anaplasma DNA were noted in 224 cattle (Bos taurus) and 78 deer (77 Rusa timorensis and one Rusa unicolor) investigated in this study. Of the 60 amplified fragments obtained for sequence analysis, Anaplasma marginale was exclusively detected in cattle while Anaplasma platys/Anaplasma phagocytophilum was predominantly detected in the deer. Based on sequence analyses of the longer fragment of the 16S rRNA gene (approximately 1000 bp), the occurrence of A. marginale, Anaplasma capra and Candidatus Anaplasma camelii in cattle, Candidatus A. camelii in deer and Anaplasma bovis in a goat was identified in this study. To assess whether animals were infected with more than one species of Anaplasma, nested amplification of A. phagocytophilum, A. bovis and Ehrlichia chaffeensis DNA was performed for 33 animal samples initially screened positive for Anaplasmataceae. No amplification of E. chaffeensis DNA was obtained from animals investigated. BLAST analyses of the 16S rDNA sequences from three deer (R. timorensis), a buffalo (Bubalus bubalis) and a cow (B. taurus) reveal similarity with that of Candidatus Anaplasma boleense strain (GenBank accession no.: KX987335). Sequence analyses of the partial gene fragments of major surface protein (msp4) gene from two deer (R. timorensis) and a monitor lizard (Varanus salvator) show the detection of a strain highly similar (99%) to that of A. phagocytophilum strain ZJ-China (EU008082). The findings in this study show the occurrence of various Anaplasma species including those newly reported species in Malaysian domestic and wildlife animals. The role of these animals as reservoirs/maintenance hosts for Anaplasma infection are yet to be determined.
Fulltext Rickettsial infections in monkeys, Malaysia

Tay ST, Koh FX, Kho KL, Sitam FT

Emerg Infect Dis, 2015 Mar;21(3):545-7.
PMID: 25695615 DOI: 10.3201/eid2103.141457
Fulltext Little influence of DNA quality on the direct sequencing output of non-human primates' faecal samples

Lee FCH, Sitam FT, Tan LP

J Virol Methods, 2024 Nov 22;332:115074.
PMID: 39580121 DOI: 10.1016/j.jviromet.2024.115074

DNA samples selected for long read sequencing (LRS) are routinely required to be 'pure' with high DNA concentration. Hence the usefulness of samples with substandard DNA quality for LRS is unknown. We aim to perform de-novo assembly of Adenovirus sequenced from non-human primate (NHP) faeces using the Oxford Nanopore technologies (ONT), an LRS platform. Guided by initial conventional PCR screening, we performed ONT sequencing on 34 Adenovirus positive DNA samples, without prior selection based on faeces freshness level or DNA quality. Non-parametric correlation analysis showed that ONT sequencing outputs is not significantly associated (p > 0.05) with DNA concentrations, faeces freshness levels and the OD ratios of A260/A280 and A260/A230. This indicated that conventional DNA quality parameters may not be the most critical factors in determining the suitability of samples for ONT sequencing. A total of 61.76 % (21/34) of the positive-by-PCR-screening samples yielded Adenovirus reads while 38.24 % (13/34) did not in the PCR-free ONT workflow, although rarefaction analysis showed that sequencing saturation was achieved by all samples. Among the 21 samples with adenovirus reads, ten resulted in at least one Adenovirus contig by the Flye assembler while nine did not and two samples had only a single Adenovirus read. Identity similarity above 90 % in conventional PCR screening may help in selecting ONT positive samples.
Fulltext Expediting the sampling, decalcification, and forensic DNA analysis of large elephant ivory seizures to aid investigations and prosecutions

Ewart KM, Lightson AL, Sitam FT, Rovie-Ryan JJ, Mather N, McEwing R

Forensic Sci Int Genet, 2020 01;44:102187.
PMID: 31670244 DOI: 10.1016/j.fsigen.2019.102187

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.
Molecular detection of Anaplasma spp. in pangolins (Manis javanica) and wild boars (Sus scrofa) in Peninsular Malaysia

Koh FX, Kho KL, Panchadcharam C, Sitam FT, Tay ST

Vet Parasitol, 2016 Aug 30;227:73-6.
PMID: 27523941 DOI: 10.1016/j.vetpar.2016.05.025

Anaplasma spp. infects a wide variety of wildlife and domestic animals. This study describes the identification of a novel species of Anaplasma (Candidatus Anaplasma pangolinii) from pangolins (Manis javanica) and Anaplasma bovis from wild boars (Sus scrofa) in Malaysia. Based on 16S rRNA gene sequences, Candidatus Anaplasma pangolinii is identified in a distinct branch within the family Anaplasmataceae, exhibiting the closest sequence similarity with the type strains of Anaplasma bovis (97.7%) and Anaplasma phagocytophilum (97.6%). The sequence also aligned closely (99.9%) with that of an Anaplasma spp. (strain AnAj360) detected from Amblyomma javanense ticks. The nearly full length sequence of the 16S rRNA gene derived from two wild boars in this study demonstrated the highest sequence similarity (99.7%) to the A. bovis type strain. Partial 16S rRNA gene fragments of A. bovis were also detected from a small population of Haemaphysalis bispinosa cattle ticks in this study. Our finding suggests a possible spread of two Anaplasma species in the Malaysian wildlife and ticks. The zoonotic potential of the Anaplasma species identified in this study is yet to be determined.
Fulltext Simian malaria in wild macaques: first report from Hulu Selangor district, Selangor, Malaysia

Akter R, Vythilingam I, Khaw LT, Qvist R, Lim YA, Sitam FT, et al.

Malar J, 2015 Oct 05;14:386.
PMID: 26437652 DOI: 10.1186/s12936-015-0856-3

BACKGROUND: Malaria is a vector-borne parasitic disease which is prevalent in many developing countries. Recently, it has been found that Plasmodium knowlesi, a simian malaria parasite can be life-threatening to humans. Long-tailed macaques, which are widely distributed in Malaysia, are the natural hosts for simian malaria, including P. knowlesi. The aim of the present study was to determine the prevalence of simian malaria parasites in long-tailed macaques in the district of Hulu Selangor, Selangor, Malaysia.
METHODS: A total of 70 blood samples were collected from Macaca fascicularis dwelling in the forest of Hulu Selangor by the Department of Wildlife and National Parks Peninsular Malaysia, Kuala Lumpur, Malaysia. DNA was extracted using PureLink™ Genomic DNA Kits. Conventional and nested PCR were used to detect the genus and species of Plasmodium parasites respectively. In addition, phylogenetic analysis was carried out to confirm the species of Plasmodium parasites.
RESULTS: Thirty-five (50 %) of the 70 samples were positive for Plasmodium using genus-specific primers. These positive samples were then subjected to nested PCR targeting the 18S ribosomal RNA genes to detect all five simian malaria parasites: namely, P. knowlesi, Plasmodium inui, Plasmodium cynomolgi, Plasmodium fieldi, and Plasmodium coatneyi. All five species of simian malaria parasites were detected. Of these, P. inui was the predominant (65.7 %), followed by P. knowlesi (60 %), P. cynomolgi (51.4 %) P. coatneyi (45.7 %) and P. fieldi (2.9 %). A total of nine macaques had mono-infection with P. knowlesi (four), P. cynomolgi (two), P. coatneyi (two) and P. fieldi (one). Eleven of the macaques had dual infections while 12 had triple infections. Three macaques were infected with four species of Plasmodium. Molecular and phylogenetic analysis confirmed the five species of Plasmodium parasites.
CONCLUSION: This study has provided evidence to elucidate the presence of transmission of malaria parasites among the local macaques in Hulu Selangor. Since malaria is a zoonosis, it is important to determine the new control strategies for the control of malaria.
Fulltext The complete mitochondrial genome of Malayan Gaur (Bos gaurus hubbacki) from Peninsular Malaysia

Rosli N, Sitam FT, Rovie-Ryan JJ, Gan HM, Lee YP, Hartini Ithnin, et al.

Mitochondrial DNA B Resour, 2019 Jul 13;4(2):2535-2536.
PMID: 33365614 DOI: 10.1080/23802359.2019.1640085

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.
Fulltext Taxonomic and genetic assessment of captive White-Handed Gibbons (Hylobateslar) in Peninsular Malaysia with implications towards conservation translocation and reintroduction programmes

Gani M, Rovie-Ryan JJ, Sitam FT, Kulaimi NAM, Zheng CC, Atiqah AN, et al.

Zookeys, 2021;1076:25-41.
PMID: 34975272 DOI: 10.3897/zookeys.1076.73262

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.
Fulltext Genome sequence analysis of Malayan pangolin (Manis javanica) forensic samples reveals the presence of Paraburkholderia fungorum sequences

Tan KY, Deng S, Tan TK, Hari R, Sitam FT, Othman RY, et al.

PeerJ, 2023;11:e16002.
PMID: 37810781 DOI: 10.7717/peerj.16002

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.
Habitat shapes the gut microbiome diversity of Malayan tigers (Panthera tigris jacksoni) as revealed through metabarcoding 16S rRNA profiling

Gani M, Mohd-Ridwan AR, Sitam FT, Kamarudin Z, Selamat SS, Awang NMZ, et al.

World J Microbiol Biotechnol, 2024 Feb 28;40(4):111.
PMID: 38416247 DOI: 10.1007/s11274-023-03868-x

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.
Fulltext Little Evidence of Zika Virus Infection in Wild Long-Tailed Macaques, Peninsular Malaysia

Chua CL, Chan YF, Andu ESGS, Rovie-Ryan JJ, Sitam FT, Verasahib K, et al.

Emerg Infect Dis, 2019 02;25(2):374-376.
PMID: 30666941 DOI: 10.3201/eid2502.180258

We tested a sample of 234 wild long-tailed macaques (Macaca fascicularis) trapped in Peninsular Malaysia in 2009, 2010, and 2016 for Zika virus RNA and antibodies. None were positive for RNA, and only 1.3% were seropositive for neutralizing antibodies. Long-tailed macaques are unlikely to be reservoirs for Zika virus in Malaysia.
Fulltext Chikungunya virus in macaques, Malaysia

Sam IC, Chua CL, Rovie-Ryan JJ, Fu JY, Tong C, Sitam FT, et al.

Emerg Infect Dis, 2015 Sep;21(9):1683-5.
PMID: 26291585 DOI: 10.3201/eid2109.150439
Fulltext No Evidence of Coronaviruses or Other Potentially Zoonotic Viruses in Sunda pangolins (Manis javanica) Entering the Wildlife Trade via Malaysia

Lee J, Hughes T, Lee MH, Field H, Rovie-Ryan JJ, Sitam FT, et al.

Ecohealth, 2020 09;17(3):406-418.
PMID: 33226526 DOI: 10.1007/s10393-020-01503-x

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.
Fulltext De novo sequencing, assembly and analysis of eight different transcriptomes from the Malayan pangolin

Mohamed Yusoff A, Tan TK, Hari R, Koepfli KP, Wee WY, Antunes A, et al.

Sci Rep, 2016 09 13;6:28199.
PMID: 27618997 DOI: 10.1038/srep28199

Pangolins are scale-covered mammals, containing eight endangered species. Maintaining pangolins in captivity is a significant challenge, in part because little is known about their genetics. Here we provide the first large-scale sequencing of the critically endangered Manis javanica transcriptomes from eight different organs using Illumina HiSeq technology, yielding ~75 Giga bases and 89,754 unigenes. We found some unigenes involved in the insect hormone biosynthesis pathway and also 747 lipids metabolism-related unigenes that may be insightful to understand the lipid metabolism system in pangolins. Comparative analysis between M. javanica and other mammals revealed many pangolin-specific genes significantly over-represented in stress-related processes, cell proliferation and external stimulus, probably reflecting the traits and adaptations of the analyzed pregnant female M. javanica. Our study provides an invaluable resource for future functional works that may be highly relevant for the conservation of pangolins.
Fulltext Phylogeography of the Sunda pangolin, Manis javanica: Implications for taxonomy, conservation management and wildlife forensics

Sitam FT, Salgado-Lynn M, Denel A, Panjang E, McEwing R, Lightson A, et al.

Ecol Evol, 2023 Aug;13(8):e10373.
PMID: 37593756 DOI: 10.1002/ece3.10373

The Sunda pangolin (Manis javanica) is the most widely distributed Asian pangolin species, occurring across much of Southeast Asia and in southern China. It is classified as Critically Endangered and 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. Here, 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 potential wildlife forensics applications. We sequenced mtDNA genomes from 23 wild Sunda pangolins of known provenance originating from Malaysia to fill sampling gaps in previous studies, particularly in Borneo. To conduct phylogenetic and population genetic analyses of Sunda pangolins across their range, we integrated these newly generated mitochondrial genomes with previously generated mtDNA and nuclear DNA data sets (RAD-seq SNP data). We identified an evolutionarily distinct mtDNA lineage in north Borneo, estimated to be ~1.6 million years divergent from lineages in west/south Borneo and the mainland, comparable to the divergence time from the Palawan pangolin. There appeared to be mitonuclear discordance, with no apparent genetic structure across Borneo based on analysis of nuclear SNPs. 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 identification testing involving these species and permit the implementation of geographic provenance testing in some scenarios.
Pangolin genomes and the evolution of mammalian scales and immunity

Choo SW, Rayko M, Tan TK, Hari R, Komissarov A, Wee WY, et al.

Genome Res, 2016 10;26(10):1312-1322.
PMID: 27510566

Pangolins, unique mammals with scales over most of their body, no teeth, poor vision, and an acute olfactory system, comprise the only placental order (Pholidota) without a whole-genome map. To investigate pangolin biology and evolution, we developed genome assemblies of the Malayan (Manis javanica) and Chinese (M. pentadactyla) pangolins. Strikingly, we found that interferon epsilon (IFNE), exclusively expressed in epithelial cells and important in skin and mucosal immunity, is pseudogenized in all African and Asian pangolin species that we examined, perhaps impacting resistance to infection. We propose that scale development was an innovation that provided protection against injuries or stress and reduced pangolin vulnerability to infection. Further evidence of specialized adaptations was evident from positively selected genes involving immunity-related pathways, inflammation, energy storage and metabolism, muscular and nervous systems, and scale/hair development. Olfactory receptor gene families are significantly expanded in pangolins, reflecting their well-developed olfaction system. This study provides insights into mammalian adaptation and functional diversification, new research tools and questions, and perhaps a new natural IFNE-deficient animal model for studying mammalian immunity.

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