The research team, led by Tamandra H. D’Ombrain of La Trobe University, focused on sambar deer, the most abundant introduced deer species in Victoria, where an estimated 123,061 animals roam public land. Sambar are elusive creatures, and managing them, alongside five other established deer species, has long been hampered by the difficulty of monitoring populations that are widely dispersed, wary of people and expensive to track. Traditional genetic studies have relied almost entirely on tissue samples from culling and recreational hunting, meaning data collection has been opportunistic and restricted to dead animals, precluding repeated sampling of living individuals for mark-recapture population estimates, movement tracking, or before-and-after comparisons of control programs.

To break that bottleneck, the team turned to Genotyping-in-Thousands by sequencing, or GT-seq, a method that uses custom amplicon sequencing of hundreds of targeted DNA loci via a single multiplexed PCR reaction. Unlike reduced-representation sequencing approaches such as DArTseq or RADseq, which can generate thousands of markers but demand high-quality DNA and substantial budgets, GT-seq is designed for exactly the kind of short, degraded, contaminated DNA that faecal samples contain. Species-specific primers and relatively short fragments make it well suited to non-invasive material, and it has already been applied successfully to coyote scats, polar bear faeces, tiger dung and even archival fish scales.

Building the panel required careful balancing. The researchers drew on existing DArTseq data from 739 tissue and blood samples of sambar, rusa and fallow deer across Australia, deliberately selecting markers that could be co-analysed with a decade of existing sambar genetic data. Simulations indicated that at least 200 highly variable SNPs would be needed for reliable individual identification in a species with low genetic diversity stemming from the small number of founding individuals. After iterative rounds of primer design, testing and optimisation, anchored to the closest available reference genome, the red deer mCerEla1.1 assembly, the final panel contained 371 autosomal SNP loci plus three newly designed sex markers, all amplified in a single PCR. Twenty-one loci were deliberately fixed between sambar and rusa, allowing hybridisation, a genuine management concern in southeastern Australia, to be detected.

The heart of the study, and its most striking contribution, was a degradation experiment. Colon sections containing faecal material were collected by Parks Victoria during control operations in northeast Victoria, giving the researchers paired tissue and dung from the same individuals and a uniform starting point. Pellets were distributed across three conditions: fully exposed outdoors, protected from rain and ultraviolet light beneath a 99 percent UV-blocking polycarbonate sheet, and kept indoors at room temperature. Samples were swabbed for DNA after zero, two, four, seven and fourteen days, during a period in October 2023 that included 35.6 millimetres of rainfall and typical spring temperatures. In total, 12.8 million paired-end reads were generated, with 56 percent landing squarely on target loci.

The results were unambiguous. Fresh faecal samples performed as well as, or better than, colon tissue, genotyping an average of 300 of 374 loci. Pellets kept indoors showed virtually no decline, and even fourteen days later the indoor samples still yielded more than 300 loci on average. Protected pellets likewise remained viable throughout the study, with no statistically significant loss of genotyping rate or increase in error. But fully exposed pellets told a different story. Their genotyping rate declined steeply over time, dropping to an average of just 76 loci by day fourteen, while their error rate climbed from 1.4 percent at day two to 13.1 percent at day fourteen. With rain striking on six days of the trial, the researchers concluded that rainfall, rather than sunlight, is the primary driver of DNA loss, consistent with earlier microsatellite work but contrasting with a handful of SNP-based studies conducted under more sheltered, simulated conditions.

The practical implications for field sampling are considerable. Pellets exposed to rain and UV remained usable for roughly four days on average, whereas sheltered samples survived well beyond two weeks. The team recommends sampling during dry spells, prioritising fresh-looking pellets or those protected by vegetation, and avoiding collection immediately after rain, when rehydrated pellets can appear deceptively fresh. Crucially, hard, dry pellets should not be discarded: desiccation itself preserves DNA, and a pellet that looks ancient may still genotype beautifully. The study also showed that visual appearance alone is an unreliable guide, reinforcing calls for research into whether pellet characteristics such as colour and texture can predict genotyping success.

Accuracy, not just yield, was the study’s second battleground. Without filtering, degraded samples produced genetic distances that could scramble individual identities, making different animals appear alike or the same animal appear different. But with a simple two-step filtering threshold, excluding samples with more than 50 percent missing data and then loci with more than 20 percent missing, the panel achieved 100 percent accurate individual assignment using a genetic distance cut-off of 0.1. The three new sex markers, designed from conserved regions between the X and Y chromosomes of deer and other even-toed ungulates, correctly assigned sex in every fresh, indoor and protected sample, with errors appearing only in heavily degraded exposed samples, and never in individuals whose genotyping rate exceeded 0.5.

Perhaps most importantly for managers, the panel proved compatible with the existing DArTseq datasets that underpin sambar monitoring. Discordance between faecal and tissue genotypes from the same individual was below one percent, comparable to the best published GT-seq faecal studies, and discordance with DArTseq data, though higher at around three percent, remained low enough for co-analysis. When the 200 most reliably genotyped SNPs were tested against a filtered dataset of 7,412 SNPs, both recovered the same three main population clusters across southeastern Australia, and pairs flagged as close relatives in the small dataset were almost always close in the large one. The authors caution that kinship resolution is coarser with 200 markers and that high background relatedness among sambar complicates precise relationship classification, but first-order kinship and broad population structure remain firmly within reach.

At an estimated AUD 36 per sample for consumables, with a plate of 96 samples processed from extraction to sequencing in one to two weeks, the panel offers a realistic route to large-scale, non-invasive genetic monitoring of living deer. That opens the door to genetic mark-recapture population estimates, evaluation of control programs, dispersal tracking and early detection of hybrids or new populations, all without ever handling an animal. The study is, to the authors’ knowledge, the first to quantify environmental degradation effects on GT-seq faecal genotyping, and its lessons, sample fresh, sample dry, filter stringently, extend far beyond deer, offering a blueprint for non-invasive genetic monitoring of both invasive and threatened species worldwide.

Subject of Research: Development of a GT-seq SNP amplicon panel for non-invasive genotyping of invasive sambar deer faecal pellets and the effects of environmental exposure on faecal DNA degradation and genotyping success.

Article Title: Development of a SNP Amplicon Panel for Invasive Deer Faecal Pellets and the Effects of Environmental Exposure on Genotyping Success

Article References: D'Ombrain, T. H., Harrisson, K. A., Pacioni, C., Hill, E., & Murphy, N. P. (2026). Development of a SNP Amplicon Panel for Invasive Deer Faecal Pellets and the Effects of Environmental Exposure on Genotyping Success. Ecology and Evolution, 16(9), Article e74388. https://doi.org/10.1002/ece3.74388

Image Credits: AI Generated

DOI: 10.1002/ece3.74388

Keywords: sambar deer, faecal DNA, GT-seq, SNP panel, non-invasive genetic sampling, DNA degradation, wildlife monitoring, invasive species, population genetics, genotyping error, individual identification, Australia

Haemaphysalis ticks belonging to the subgenus Kaiseriana are recognized as important vectors of tick-borne pathogens throughout tropical Asia. Yet, despite their medical and veterinary significance, data on which tick species infest captive wildlife in East Malaysia have remained scarce. Captive animals such as Sambar deer, a large cervid native to South and Southeast Asia, can act as amplifying hosts for ticks and the agents they transmit, and facilities like bioparks and teaching farms create conditions in which ticks, wildlife, livestock, and people come into close and repeated contact. Understanding exactly which tick species are present, and which pathogens they harbor, is therefore a prerequisite for any meaningful risk assessment. The new study set out to close that gap by combining classical morphological identification with DNA-based species confirmation and pathogen screening.

Between 2021 and 2022, the researchers collected a total of 60 ticks from five Sambar deer at the two Malaysian sites. Each specimen was first identified morphologically using standard taxonomic keys, an approach that relies on subtle differences in body structures such as the palps, scutum, and spiracular plates. The team then attempted to extract DNA from 49 of the specimens. Here the study encountered a practical constraint familiar to anyone working with field-collected arthropods: specimen degradation. Because the ticks had been preserved and stored under conditions that compromised their genetic material, only seven samples retained DNA of sufficient quality and quantity for downstream molecular analysis. Rather than discarding the morphological work, the researchers treated the seven genetically tractable ticks as a focused molecular window onto the broader collection.

For species identification, the team sequenced the mitochondrial cytochrome c oxidase subunit I gene, known as cox1, a workhorse marker in animal DNA barcoding. The resulting sequences were compared against reference databases and subjected to phylogenetic analysis using maximum likelihood methods, which infer evolutionary trees by finding the branching pattern that best explains the observed genetic differences. Two Haemaphysalis species emerged with strong statistical support. Sequences assigned to Haemaphysalis shimoga showed 97.88 to 99.27 percent identity to reference material, while those assigned to Haemaphysalis bispinosa were even more convincing at 99.5 to 100 percent identity. Crucially, each species resolved in its own well-supported monophyletic clade on the phylogenetic trees, meaning that all sequences of each species descended from a common ancestor to the exclusion of the other, a pattern that reinforces the species-level assignments.

The identification of H. shimoga in Malaysia is particularly noteworthy. The taxon was originally described from southern India as a subspecies of Haemaphysalis cornigera, and its presence on Sambar deer in Malaysian Borneo extends the known geographic footprint of this tick considerably. H. bispinosa, by contrast, has a long and somewhat complicated history in the region. Work dating back to the late 1960s argued, on biogeographic grounds, that H. bispinosa should be considered an introduced species in the Malay Peninsula and Borneo, likely arriving with domesticated animals. Its confirmation here on captive Sambar deer, at sites on both sides of the country, is consistent with that picture of a tick that thrives wherever its preferred hosts and suitable climatic conditions coincide, including in managed wildlife settings.

With the tick species confirmed, the researchers turned to the pathogens. They screened the seven molecularly characterized specimens for bacteria of the genus Anaplasma using sequencing of the 16S ribosomal DNA gene, and for protozoan parasites of the genus Theileria using the 18S ribosomal DNA gene. Both gene targets are standard markers for detecting and differentiating these organisms, and the resulting sequences were again placed into phylogenetic frameworks by maximum likelihood analysis. The results were remarkable for a sample set of just seven ticks. Five of the seven, comprising four H. shimoga and one H. bispinosa, were infected with Theileria species. Sequences matched Theileria capreoli at 98.83 to 100 percent identity and Theileria cervi at 95.07 to 99.74 percent identity, two protozoans associated with cervid hosts in various parts of the world.

The single H. bispinosa specimen proved to be the study’s most consequential finding. It tested positive for Anaplasma phagocytophilum, with sequence identities of 99.53 to 100 percent, and it simultaneously harbored both Theileria cervi and Theileria capreoli, making it a documented triple co-infection in a single tick. Anaplasma phagocytophilum is the agent of human granulocytic anaplasmosis as well as tick-borne fever in ruminants, and it is regarded as a widespread, multi-host pathogen with highly adaptive strategies that allow it to persist across ticks, wild and domestic mammals, and occasionally humans. Its detection in a Haemaphysalis tick infesting captive deer in Malaysia raises the possibility that the ecological circuitry needed to maintain this bacterium, competent tick vectors and susceptible mammalian hosts in close proximity, is already in place in the country.

Co-infections of this kind are increasingly understood to be the rule rather than the exception in tick microbiology. Individual ticks frequently carry multiple pathogens at once, and mixed infections can influence disease severity in hosts, complicate diagnosis, and alter transmission dynamics in ways that single-pathogen models fail to capture. The Malaysian finding, in which one tick carried two protozoan parasites and one bacterial pathogen simultaneously, is a vivid local illustration of that global pattern. It also underscores why molecular screening of even small numbers of well-characterized ticks can be informative: morphological identification alone would have revealed which tick species were present, but only DNA sequencing exposed the pathogen community hiding inside them.

The study’s authors are careful about the limits of their data. With only seven specimens yielding usable DNA, the prevalence estimates that could be calculated are statistically fragile, and the degraded condition of most of the collection means the true infection rates among the 60 ticks remain unknown. Detection of pathogen DNA in a tick also does not by itself demonstrate that the tick can biologically transmit the agent, nor does it confirm that the deer were infected. Those questions would require blood testing of the hosts, experimental vector competence studies, and larger, systematically designed tick surveys. What the study does establish, firmly, is a set of firsts: the first molecular confirmation of H. shimoga and H. bispinosa on captive Sambar deer in Malaysia, and the first molecular detection of A. phagocytophilum alongside Theileria co-infection in ticks from the country.

The practical implications extend in several directions. For wildlife managers and veterinarians responsible for captive deer, the results argue for routine tick surveillance and acaricide-based control at facilities where animals are held in semi-natural enclosures. For public health authorities, the presence of A. phagocytophilum DNA in a local tick vector warrants attention, particularly given that people working at bioparks, farms, and slaughterhouses occupy exactly the interfaces where tick bites are most likely. The researchers have deposited all their sequence data in GenBank, making the cox1, 16S, and 18S sequences available for future comparative studies across Southeast Asia. As climate change, land-use change, and the wildlife trade continue to reshuffle the distributions of ticks and their hosts in the tropics, baseline datasets of this kind, anchored in both morphology and molecular phylogenetics, will become only more valuable for tracking how tick-borne pathogen landscapes evolve in Malaysia and beyond.

Subject of Research: Molecular identification of Haemaphysalis tick species and detection of Anaplasma and Theileria co-infections in captive Sambar deer in Malaysia

Article Title: First Molecular Report of Haemaphysalis shimoga and Haemaphysalis bispinosa Infesting Captive Sambar Deer (Rusa unicolor) in Malaysia, with Detection of Anaplasma phagocytophilum and Theileria Co-infection

Article References: Altwaim, S. A., Kamaludeen, J., Mohammed, M. A., Suif, Z., Mustafa, S., Paul, B. T., Syed-Hussain, S. S., Aziz, N.-A. A., Numan, M., & Ali, A. (2026). First Molecular Report of Haemaphysalis shimoga and Haemaphysalis bispinosa Infesting Captive Sambar Deer (Rusa unicolor) in Malaysia, with Detection of Anaplasma phagocytophilum and Theileria Co-infection. Acta Parasitologica, 71(5), Article 212. https://doi.org/10.1007/s11686-026-01392-0

Image Credits: AI Generated

DOI: 10.1007/s11686-026-01392-0

Keywords: Haemaphysalis shimoga, Haemaphysalis bispinosa, Sambar deer, Anaplasma phagocytophilum, Theileria capreoli, Theileria cervi, tick-borne pathogens, co-infection, Malaysia, cox1 barcoding, tick surveillance, captive wildlife