Where are you hiding the pangolins? screening tools to detect illicit contraband at international borders and their adaptability for illegal wildlife trafficking.

The illegal movement of wildlife poses a public health, conservation and biosecurity threat, however there are currently minimal screening tools available at international ports of entry to intercept wildlife trafficking efforts. This review first aimed to explore the screening tools available or under development for the detection of concealed wildlife contraband at international ports, including postal services, airlines, road border crossings and maritime routes. Where evidence was deficient, publications detailing the use of methods to uncover other illicit substances, such as narcotics, weapons, human trafficking, explosives, radioactive materials, or special nuclear material, were compiled and assessed for their applicability to the detection of wildlife. The first search identified only four citations related to the detection of wildlife, however the secondary search revealed 145 publications, including 59 journal articles and 86 conference proceedings, describing screening tools for non-wildlife illicit contraband detection. The screening tools uncovered were analysed for potential fitness for purpose for wildlife contraband detection, to evaluate the feasibility of their implementation and their ease of use. The deficiencies evident in terms of resource availability and research efforts targeting wildlife trafficking highlights a potentially substantial national and international security threat which must be addressed.

Assessing the emergence time of SARS-CoV-2 zoonotic spillover.

Understanding the evolution of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) and its relationship to other coronaviruses in the wild is crucial for preventing future virus outbreaks. While the origin of the SARS-CoV-2 pandemic remains uncertain, mounting evidence suggests the direct involvement of the bat and pangolin coronaviruses in the evolution of the SARS-CoV-2 genome. To unravel the early days of a probable zoonotic spillover event, we analyzed genomic data from various coronavirus strains from both human and wild hosts. Bayesian phylogenetic analysis was performed using multiple datasets, using strict and relaxed clock evolutionary models to estimate the occurrence times of key speciation, gene transfer, and recombination events affecting the evolution of SARS-CoV-2 and its closest relatives. We found strong evidence supporting the presence of temporal structure in datasets containing SARS-CoV-2 variants, enabling us to estimate the time of SARS-CoV-2 zoonotic spillover between August and early October 2019. In contrast, datasets without SARS-CoV-2 variants provided mixed results in terms of temporal structure. However, they allowed us to establish that the presence of a statistically robust clade in the phylogenies of gene S and its receptor-binding (RBD) domain, including two bat (BANAL) and two Guangdong pangolin coronaviruses (CoVs), is due to the horizontal gene transfer of this gene from the bat CoV to the pangolin CoV that occurred in the middle of 2018. Importantly, this clade is closely located to SARS-CoV-2 in both phylogenies. This phylogenetic proximity had been explained by an RBD gene transfer from the Guangdong pangolin CoV to a very recent ancestor of SARS-CoV-2 in some earlier works in the field before the BANAL coronaviruses were discovered. Overall, our study provides valuable insights into the timeline and evolutionary dynamics of the SARS-CoV-2 pandemic.

Integrating Fréchet distance and AI reveals the evolutionary trajectory and origin of SARS-CoV-2.

A genome, composed of a precisely ordered sequence of four nucleotides (ATCG), encompasses a multitude of specific genome features like AAA motif. Mutations occurring within a genome disrupt the sequential order and composition of these features, thereby influencing the evolutionary trajectories and yielding variants. The evolutionary relatedness between a variant and its ancestor can be estimated by assessing evolutionary distances across a spectrum of genome features. This study develops a novel, alignment-free algorithm that considers both the sequential order and composition of genome features, enabling computation of the Fréchet distance (Fr) across multiple genome features to quantify the evolutionary status of a variant. Integrating this algorithm with an artificial recurrent neural network (RNN) reveals the quantitative evolutionary trajectory and origin of SARS-CoV-2, a puzzle unsolved by alignment-based phylogenetics. The RNN generates the evolutionary trajectory from Fr data at two levels: genome sequence mutations and organism variants. At the genome sequence level, SARS-CoV-2 evolutionarily shortens its genome to enhance its infectious capacity. Mutating signature features, such as TTA and GCT, increases its infectious potential and drives its evolution. At the organism level, variants mutating a single biomarker possess low infectious potential. However, mutating multiple markers dramatically increases their infectious capacity, propelling the COVID-19 pandemic. SARS-CoV-2 likely originates from mink coronavirus variants, with its origin trajectory traced as follows: mink, cat, tiger, mouse, hamster, dog, lion, gorilla, leopard, bat, and pangolin. Together, mutating multiple signature features and biomarkers delineates the evolutionary trajectory of mink-origin SARS-CoV-2, leading to the COVID-19 pandemic.

Predicting current and future habitat of Indian pangolin (Manis crassicaudata) under climate change.

Climate change is among the greatest drivers of biodiversity loss, threatening up to 15-30% of described species by the end of the twenty-first century. We estimated the current suitable habitat and forecasted future distribution ranges of Indian pangolin (Manis crassicaudata) under climate change scenarios. We collected occurrence records of Indian pangolin using burrow counts, remote camera records and previously published literature in Pakistan during 2021-2023. We downloaded bioclimatic data for current (1970-2000) and future (2041-2060, 2061-2080, 2081-2100) climate scenarios from the WorldClim database using the Hadley Global Environment Model (HadGEM3-GC31-LL). We used MaxEnt software to predict current and future distributions of Indian pangolin, then computed the amount of habitat lost, gained, and unchanged across periods. We obtained 560 Indian pangolin occurrences overall, 175 during the study, and 385 from our literature search. Model accuracy was very good (AUC = 0.885, TSS = 0.695), and jackknife tests of variable importance showed that the contribution of annual mean temperature (bio1) was greatest (33.4%), followed by the mean temperature of the coldest quarter (bio-12, 29.3%), temperature seasonality (bio 4, 25.9%), and precipitation seasonality (bio 15, 11.5%). The maxent model predicted that during the current time period (1970-2000) highly suitable habitat for Indian pangolin was (7270 km2, 2.2%), followed by moderately suitable (12,418 km2, 3.7%), less suitable (49,846 km2, 14.8%), and unsuitable habitat (268,355 km2, 79.4%). Highly suitable habitat decreased in the western part of the study area under most SSPs and in the central parts it declined under all SSPs and in future time periods. The predicted loss in the suitable habitat of the Indian pangolin was greatest (26.97%) under SSP 585 followed by SSP 126 (23.67%) during the time 2061-2080. The gain in suitable habitat of Indian pangolin was less than that of losses on average which ranged between 1.91 and 13.11% under all SSPs during all time periods. While the stable habitat of the Indian pangolin ranged between 64.60 and 83.85% under all SSPs during all time periods. Our study provides the current and future habitat ranges of Indian pangolin in the face of a changing climate. The findings of our study could be helpful for policymakers to set up conservation strategies for Indian pangolin in Pakistan.

Isolation and characterization of a pangolin-borne HKU4-related coronavirus that potentially infects human-DPP4-transgenic mice.

We recently detected a HKU4-related coronavirus in subgenus Merbecovirus (named pangolin-CoV-HKU4-P251T) from a Malayan pangolin1. Here we report isolation and characterization of pangolin-CoV-HKU4-P251T, the genome sequence of which is closest to that of a coronavirus from the greater bamboo bat (Tylonycteris robustula) in Yunnan Province, China, with a 94.3% nucleotide identity. Pangolin-CoV-HKU4-P251T is able to infect human cell lines, and replicates more efficiently in cells that express human-dipeptidyl-peptidase-4 (hDPP4)-expressing and pangolin-DPP4-expressing cells than in bat-DPP4-expressing cells. After intranasal inoculation with pangolin-CoV-HKU4-P251, hDPP4-transgenic female mice are likely infected, showing persistent viral RNA copy numbers in the lungs. Progressive interstitial pneumonia developed in the infected mice, characterized by the accumulation of macrophages, and increase of antiviral cytokines, proinflammatory cytokines, and chemokines in lung tissues. These findings suggest that the pangolin-borne HKU4-related coronavirus has a potential for emerging as a human pathogen by using hDPP4.

Molecular characterization of a novel Spiruromorpha species in wild Chinese pangolin by mitogenome sequence analysis.

Pangolins are susceptible to a variety of gastrointestinal nematodes due to their burrowing lifestyle and feeding habits, and few parasitic nematodes have been reported. Here, a Chinese pangolin with old wounds on its leg and tail was rescued from the Heyuan City, Guangdong Province. The cox1 and SSU rRNA of the worms from the intestine of the Chinese pangolin had the highest sequence identity of 89.58% and 97.95% to the species in the infraorder Spiruromorpha. The complete mitogenome of the worm was further assembled by next-generation sequencing, with a size of 13,708 bp and a GC content of 25.6%. The worm mitogenome had the highest sequence identity of 78.56% to that of Spirocerca lupi, sharing the same gene arrangement with S. lupi and some species in other families under Spiruromorpha. However, the mitogenome between the worm and S. lupi showed differences in codon usage of PCGs, sequences of NCR, and tRNA secondary structures. Phylogenetic analysis showed that the worm mitogenome was clustered with S. lupi in the family Thelaziidae to form a separate branch. However, it is still difficult to identify the worm in the family Thelaziidae because the species in the family Thelaziidae are confused, specifically S. lupi and Thelazia callipaeda in the family Thelaziidae were separated and grouped with species from other families. Thus, the parasitic nematode from the Chinese pangolin may be a novel species in Spiruromorpha and closely related to S. lupi. This study enriches the data on gastrointestinal nematodes in the Chinese pangolin.

Recombination-aware phylogenetic analysis sheds light on the evolutionary origin of SARS-CoV-2.

SARS-CoV-2 can infect human cells through the recognition of the human angiotensin-converting enzyme 2 receptor. This affinity is given by six amino acid residues located in the variable loop of the receptor binding domain (RBD) within the Spike protein. Genetic recombination involving bat and pangolin Sarbecoviruses, and natural selection have been proposed as possible explanations for the acquisition of the variable loop and these amino acid residues. In this study we employed Bayesian phylogenetics to jointly reconstruct the phylogeny of the RBD among human, bat and pangolin Sarbecoviruses and detect recombination events affecting this region of the genome. A recombination event involving RaTG13, the closest relative of SARS-CoV-2 that lacks five of the six residues, and an unsampled Sarbecovirus lineage was detected. This result suggests that the variable loop of the RBD didn't have a recombinant origin and the key amino acid residues were likely present in the common ancestor of SARS-CoV-2 and RaTG13, with the latter losing five of them probably as the result of recombination.

Genetic assessment of captive breeding program of Indian Pangolin: implications for conservation and management.

BACKGROUND: Captive breeding programs play a vital role in conservation of threatened species, necessitating an understanding of genetic diversity among captive individuals to ensure long-term genetic viability, appropriate mate selection, and successful reintroduction to native habitats. METHODS AND RESULTS: We did not observe any recent genetic bottleneck, and population showed moderate genetic diversity. The estimated effective population size, representing individuals capable of contributing genetically to future generations, was estimated as 18.6 individuals (11.4-35.1 at 95% CI). Based on the genetic make-up and allelic diversity, we found seventeen pangolins (11 females and 6 males) were genetically unrelated and relatively more potent than others. CONCLUSION: In this study, we evaluated the captive breeding program of the Indian pangolin population at the Pangolin Conservation Breeding Centre in Nandankanan Zoological Park, Bhubaneswar, Odisha. We highlight the significance of genetic monitoring within the captive population of Indian pangolin for preserving genetic diversity and ensuring the long-term survival of the species. We established the genetic profiles of all 29 pangolins and identified 17 pangolins to be prioritized for enhanced breeding and future zoo exchange programs. We appreciate the zoo authorities for promoting genetic assessment of pangolin for better and more effective monitoring of the captive breeding of the endangered Indian pangolin.

An RNA-Seq analysis of coronavirus in the skin of the Pangolin.

Protection of the Critically Endangered East Asian Pangolin species is hampered by the vulnerability of captive individuals to infection. Studies have previously shown the pangolin to have a unique pseudogenisation of many immunity genes (including IFNE, IFIH1, cGAS, STING, TLR5, and TLR11), and we suspected that these losses could account for this vulnerability. Here we used RNA-Seq data to show the effect of these gene losses on the transcriptional response to a viral skin infection in a deceased pangolin. This virus is very closely related to the one causing the current COVID-19 pandemic in the human population (SARS-CoV2), and we found the most upregulated pathway was the same one previously identified in the lungs of SARS-CoV2-infected humans. As predicted, we found that the pathways downstream of the lost genes were not upregulated. For example, the pseudogenised interferon epsilon (IFNE) is known to be particularly important in epithelial immunity, and we show that interferon-related responses were not upregulated in the infected pangolin skin. We suggest that the pangolin's innate gene pseudogenisation is indeed likely to be responsible for the animal's vulnerability to infection.

Infantile hemangioma in a subadult Chinese pangolin: a case report.

BACKGROUND: Hemangiomas are a relatively common type of tumor in humans and animals. Various subtypes of hemangiomas have been described in the literature. The classification methods for hemangiomas differ between human and veterinary medicine, and the basis for tumor classification can be found in the literature. CASE PRESENTATION: This study describes a tumor in the subcutaneous tissue of the right dorsum of an artificially rescued juvenile Chinese pangolin. Computed tomography (CT) examination yielded the preliminary diagnosis of a vascular malformation, and surgery was performed to resect the tumor. Histopathological examination showed that the tumor mainly was consisted of adipose tissue, capillaries, and spindle cells in the fibrous stroma. Immunohistochemistry showed the positive expression of CD31, CD34, α-SMA, GLUT1 and WT-1 in the tumor tissue, and the tumor was eventually diagnosed as an infantile haemangioma. CONCLUSION: The final diagnosis of infantile hemangioma was depended on the histopathological immunohistochemical and CT examination of the neoplastic tissue. This is the first report of infantile hemangioma in a critically endangered species Chinese pangolin.

Visualisation and detection of latent DNA deposited by pangolin scales onto plastic packaging materials.

We report on the detection and visualisation of latent DNA from pangolin scales deposited onto a plastic packaging material through the use of a nucleic acid staining dye. This latent DNA deposited by pangolin scales was subsequently isolated and analysed using DNA barcoding method. Pangolins are the most illegally traded mammalian species due to the demand for their scales and meat. The demand for their scales were mostly fuelled by its use in traditional medicines. The scales are usually packed into bags and transported globally via sea routes. This is the first report detailing the detection of trace latent DNA from processed wildlife products, on surfaces of bags that they were packaged in. Prior to this report, it was not known if the dried pangolin scales contained transferable quantities of biological material for DNA analyses. To address this, scales were removed from a roadkill Sunda pangolin (Manis javanica), processed by drying and packaged into one of five plastic bags. The presence of pangolin latent DNA was detected on the surface of the plastic bags and visualised using Diamond™ nucleic acid dye. Swabs were then used to recover the stained biological material from various locations in the five bags. The DNA was isolated and quantified using a newly designed quantitative PCR (qPCR) specific to M. javanica to amplify a fragment of the mitochondrial DNA cytochrome b gene. There was a positive correlation between the number of stained particles and DNA quantity, and a greater number of stained particles were found at the bottom of the bag than were found at the top. Conventional PCR targeting part of the cyt b gene amplified a product from all 30 samples taken from the bags and in all cases, sequence data generated matched that of the Sunda pangolin, as expected. All negative controls yielded no results. The method described here is the very first use of a nucleic acid staining dye to detect latent DNA from a mammalian species, other than humans, and highlights the opportunity for further use of Diamond™ nucleic acid dye in wildlife forensic science. It is anticipated that this method will be invaluable in retrieving latent DNA deposited by wildlife products from the environment in which they were contained, to determine the presence of these illegal wildlife products even when previously hidden, inaccessible, or no longer present physically. Further research is required to understand if the use on non-human mammalian wildlife species is feasible.

Vulnerability of pangolin SARS-CoV-2 lineage assignment to adversarial attack.

Pangolin is the most popular tool for SARS-CoV-2 lineage assignment. During COVID-19, healthcare professionals and policymakers required accurate and timely lineage assignment of SARS-CoV-2 genomes for pandemic response. Therefore, tools such as Pangolin use a machine learning model, pangoLEARN, for fast and accurate lineage assignment. Unfortunately, machine learning models are susceptible to adversarial attacks, in which minute changes to the inputs cause substantial changes in the model prediction. We present an attack that uses the pangoLEARN architecture to find perturbations that change the lineage assignment, often with only 2-3 base pair changes. The attacks we carried out show that pangolin is vulnerable to adversarial attack, with success rates between 0.98 and 1 for sequences from non-VoC lineages when pangoLEARN is used for lineage assignment. The attacks we carried out are almost never successful against VoC lineages because pangolin uses Usher and Scorpio - the non-machine-learning alternative methods for VoC lineage assignment. A malicious agent could use the proposed attack to fake or mask outbreaks or circulating lineages. Developers of software in the field of microbial genomics should be aware of the vulnerabilities of machine learning based models and mitigate such risks.

Genomic analyses reveal poaching hotspots and illegal trade in pangolins from Africa to Asia.

The white-bellied pangolin (Phataginus tricuspis) is the world's most trafficked mammal and is at risk of extinction. Reducing the illegal wildlife trade requires an understanding of its origins. Using a genomic approach for tracing confiscations and analyzing 111 samples collected from known geographic localities in Africa and 643 seized scales from Asia between 2012 and 2018, we found that poaching pressures shifted over time from West to Central Africa. Recently, Cameroon's southern border has emerged as a site of intense poaching. Using data from seizures representing nearly 1 million African pangolins, we identified Nigeria as one important hub for trafficking, where scales are amassed and transshipped to markets in Asia. This origin-to-destination approach offers new opportunities to disrupt the illegal wildlife trade and to guide anti-trafficking measures.

Benchmarking splice variant prediction algorithms using massively parallel splicing assays.

BACKGROUND: Variants that disrupt mRNA splicing account for a sizable fraction of the pathogenic burden in many genetic disorders, but identifying splice-disruptive variants (SDVs) beyond the essential splice site dinucleotides remains difficult. Computational predictors are often discordant, compounding the challenge of variant interpretation. Because they are primarily validated using clinical variant sets heavily biased to known canonical splice site mutations, it remains unclear how well their performance generalizes. RESULTS: We benchmark eight widely used splicing effect prediction algorithms, leveraging massively parallel splicing assays (MPSAs) as a source of experimentally determined ground-truth. MPSAs simultaneously assay many variants to nominate candidate SDVs. We compare experimentally measured splicing outcomes with bioinformatic predictions for 3,616 variants in five genes. Algorithms' concordance with MPSA measurements, and with each other, is lower for exonic than intronic variants, underscoring the difficulty of identifying missense or synonymous SDVs. Deep learning-based predictors trained on gene model annotations achieve the best overall performance at distinguishing disruptive and neutral variants, and controlling for overall call rate genome-wide, SpliceAI and Pangolin have superior sensitivity. Finally, our results highlight two practical considerations when scoring variants genome-wide: finding an optimal score cutoff, and the substantial variability introduced by differences in gene model annotation, and we suggest strategies for optimal splice effect prediction in the face of these issues. CONCLUSION: SpliceAI and Pangolin show the best overall performance among predictors tested, however, improvements in splice effect prediction are still needed especially within exons.

Pangolin HKU4-related coronaviruses found in greater bamboo bats from southern China.

Coronavirus (CoV) spillover originating from game animals, particularly pangolins, is currently a significant concern. Meanwhile, vigilance is urgently needed for coronaviruses carried by bats, which are known as natural reservoirs of many coronaviruses. In this study, we collected 729 anal swabs of 20 different bat species from nine locations in Yunnan and Guangdong provinces, southern China, in 2016 and 2017, and described the molecular characteristics and genetic diversity of alphacoronaviruses (αCoVs) and betacoronaviruses (ßCoVs) found in these bats. Using RT-PCR, we identified 58 (8.0%) bat CoVs in nine bat species from six locations. Furthermore, using the Illumina platform, we obtained two representative full-length genomes of the bat CoVs, namely TyRo-CoV-162275 and TyRo-CoV-162269. Sequence analysis showed that TyRo-CoV-162275 shared the highest identity with Malayan pangolin (Manis javanica) HKU4-related coronaviruses (MjHKU4r-CoVs) from Guangxi Province, whereas TyRo-CoV-162269 was closely related to HKU33-CoV discovered in a greater bamboo bat (Tylonycteris robustula) from Guizhou Province. Notably, TyRo-CoV-162275 has a putative furin protease cleavage site in its S protein and is likely to utilize human dipeptidyl peptidase-4 (hDPP4) as a cell-entry receptor, similar to MERS-CoV. To the best of our knowledge, this is the first report of a bat HKU4r-CoV strain containing a furin protease cleavage site. These findings expand our understanding of coronavirus geographic and host distributions.

Detection of a novel Babesia sp. in Amblyomma javanense, an ectoparasite of Sunda pangolins.

BACKGROUND: Babesia is a protozoal, tick-borne parasite that can cause life-threatening disease in humans, wildlife and domestic animals worldwide. However, in Southeast Asia, little is known about the prevalence and diversity of Babesia species present in wildlife and the tick vectors responsible for its transmission. Recently, a novel Babesia species was reported in confiscated Sunda pangolins (Manis javanica) in Thailand. To investigate the presence of this parasite in Singapore, we conducted a molecular survey of Babesia spp. in free-roaming Sunda pangolins and their main ectoparasite, the Amblyomma javanense tick. METHODS: Ticks and tissue samples were opportunistically collected from live and dead Sunda pangolins and screened using a PCR assay targeting the 18S rRNA gene of Babesia spp. DNA barcoding of the cytochrome oxidase subunit I (COI) mitochondrial gene was used to confirm the species of ticks that were Babesia positive. RESULTS: A total of 296 ticks and 40 tissue samples were obtained from 21 Sunda pangolins throughout the 1-year study period. Babesia DNA was detected in five A. javanense ticks (minimum infection rate = 1.7%) and in nine different pangolins (52.9%) located across the country. Phylogenetic analysis revealed that the Babesia 18S sequences obtained from these samples grouped into a single monophyletic clade together with those derived from Sunda pangolins in Thailand and that this evolutionarily distinct species is basal to the Babesia sensu stricto clade, which encompasses a range of Babesia species that infect both domestic and wildlife vertebrate hosts. CONCLUSIONS: This is the first report documenting the detection of a Babesia species in A. javanense ticks, the main ectoparasite of Sunda pangolins. While our results showed that A. javanense can carry this novel Babesia sp., additional confirmatory studies are required to demonstrate vector competency. Further studies are also necessary to investigate the role of other transmission pathways given the low infection rate of ticks in relation to the high infection rate of Sunda pangolins. Although it appears that this novel Babesia sp. is of little to no pathogenicity to Sunda pangolins, its potential to cause disease in other animals or humans cannot be ruled out.

Pangolin Genomes Offer Key Insights and Resources for the World's Most Trafficked Wild Mammals.

Pangolins form a group of scaly mammals that are trafficked at record numbers for their meat and purported medicinal properties. Despite their conservation concern, knowledge of their evolution is limited by a paucity of genomic data. We aim to produce exhaustive genomic resources that include 3,238 orthologous genes and whole-genome polymorphisms to assess the evolution of all eight extant pangolin species. Robust orthologous gene-based phylogenies recovered the monophyly of the three genera and highlighted the existence of an undescribed species closely related to Southeast Asian pangolins. Signatures of middle Miocene admixture between an extinct, possibly European, lineage and the ancestor of Southeast Asian pangolins, provide new insights into the early evolutionary history of the group. Demographic trajectories and genome-wide heterozygosity estimates revealed contrasts between continental versus island populations and species lineages, suggesting that conservation planning should consider intraspecific patterns. With the expected loss of genomic diversity from recent, extensive trafficking not yet realized in pangolins, we recommend that populations be genetically surveyed to anticipate any deleterious impact of the illegal trade. Finally, we produce a complete set of genomic resources that will be integral for future conservation management and forensic endeavors for pangolins, including tracing their illegal trade. These comprise the completion of whole-genomes for pangolins through the hybrid assembly of the first reference genome for the giant pangolin (Smutsia gigantea) and new draft genomes (∼43x-77x) for four additional species, as well as a database of orthologous genes with over 3.4 million polymorphic sites.

DIAGNOSTIC IMAGING AND OROTRACHEAL INTUBATION IN CLINICALLY HEALTHY WILD SUNDA PANGOLINS (MANIS JAVANICA) IN SINGAPORE.

Pangolins are amongst the most overexploited species in the world and all eight species of pangolins are threatened with extinction. These animals are rare in zoological collections and often suffer high mortality rates in captivity. Maintaining healthy populations in captivity has become more important with the declining populations in the wild, but knowledge of veterinary care of these animals is limited. Interpreting radiography and ultrasonography images in a patient can be challenging without knowledge of normal findings. The Wildlife Healthcare and Research Center (WHRC) at Mandai Wildlife Reserve (MWR) admits an average of 25 Sunda pangolins (Manis javanica) annually, which is a Critically Endangered species native to Singapore. All the pangolins are triaged on admission and anesthetized for a health assessment before release into the wild. Endotracheal intubation using an otoscope and stylet is a novel technique in the species that has been developed and is commonly performed with these pangolins. A retrospective study was done on 20 clinically healthy wild pangolins to determine normal ultrasonography and radiography findings in the species. Notable findings include the presence of radio-opaque particles in the stomachs of all pangolins, the presence of free fluid cranial to the left kidney (13/20), a spleen with multifocal hypoechoic regions (6/20), and open epiphyseal plates of long bones even in large individuals weighing as heavy as 6.8 kg. Ultrasonographic images and measurements of kidney, spleen, and adrenal gland sizes as well as intestinal, gallbladder, and urinary bladder wall thickness were also described. These diagnostic imaging findings can advance the veterinary care of captive and wild pangolins.