Figures
Abstract
The traditional concept of “tonic food” and demand for traditional Chinese medicine make pangolins the largest population of illegally smuggled mammals in the world. Illegal hunting and trade are not only responsible for the sharp decline in pangolin populations but also provide conditions for pathogenic transmission. In 2021, we rescued 21 confiscated unhealthy Malayan pangolins, none of which survived. This study aimed to investigate the reasons for their unexpected deaths and the potential pathogens that may be transmitted during smuggling. Physical examination found that more than 80% pangolins were parasitized with A. javanense ticks. Autopsy and pathological staining analysis revealed multiple organ damage in the deceased pangolins. Pathogens nucleic acid detection of 33 tick samples showed that the positive rate of Rickettsia spp., Anaplasma spp., Ehrlichia spp. Babesia spp., and Colpodella spp. were 90.91%, 6.06%, 6.06%, 15.15% and 18.18%, respectively. Furthermore, pangolin samples were positive for Rickettsia spp. (42.86%, 9/21), Ehrlichia sp. (4.76%, 1/21), and Babesia sp. (4.76%, 1/21). This study confirmed that spotted fever triggered by Rickettsia spp. from A. javanense might accelerate the most death of confiscated pangolins, while Ehrlichia sp., and Babesia sp. infection potentially accelerating a few deaths. Of note, A. javanense ticks carrying Colpodella spp. were detected for the first time in Malayan pangolins. However, whether Colpodella spp. are pathogenic to pangolins is unknown. Further research on the diagnosis, treatment, surveillance, and elimination of ticks and tick-borne diseases in humans, livestock, and wildlife should provide insight into wildlife conservation and zoonotic disease prevention.
Author summary
As a globally distributed obligate bloodthirsty ectoparasite, ticks are the vector second only to mosquitoes, capable of transmitting a variety of pathogenic viruses, bacteria, and protozoa to humans, livestock, and wildlife. Illegal poaching and smuggling pose a serious threat to the survival of pangolins and are also a potential route for the spread of tick-borne diseases. The present study was conducted to investigate the reasons for the unexpected death of Malayan pangolins confiscated in 2021, as well as to explore the potential pathogens that may be transmitted during smuggling. Based on the clinical symptoms, autopsy, and molecular detection results, we speculate that the primary cause was the spotted fever triggered by Rickettsia spp. from A. javanense, which accelerated the death of smuggled pangolins. Ehrlichia sp. or Babesia sp. infection might be associated with the death of a few pangolins. Moreover, A. javanense carrying Colpodella spp. were detected for the first time in Malayan pangolins, and whether Colpodella spp. are pathogenic to pangolins awaits further study. The findings suggest that more attention should be paid to the diagnosis and treatment of tick-borne diseases in the rescue operation of pangolins and other wildlife.
Citation: Li B, Zhai J-Q, Wu Y-J, Shan F, Zou J-J, Hou F-H, et al. (2024) Molecular identification of tick-borne Rickettsia, Anaplasma, Ehrlichia, Babesia, and Colpodella in confiscated Malayan pangolins. PLoS Negl Trop Dis 18(11): e0012667. https://doi.org/10.1371/journal.pntd.0012667
Editor: Pradeep J., Sri Balaji Vidyapeeth (Deemed to be University): Sri Balaji Vidyapeeth, INDIA
Received: April 4, 2024; Accepted: October 30, 2024; Published: November 22, 2024
Copyright: © 2024 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and the GenBank accession numbers of the gene sequences obtained in this study have been provided in Table 3.
Funding: This study was funded by the Innovation and Enhancement Youth Engineering Team Project of Guangdong Pharmaceutical University (Project Number: 2024QZ13) awarded to BL. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Pangolins are toothless mammals that feed on termites and are covered by distinctive overlapping keratinized scales. They have no natural predators, but their scales are highly valued in traditional Chinese medicine, making them one of the most illegally smuggled mammals in the world. Between August 2000 and July 2019, the international trafficking of pangolins and their derivatives involved an estimated 895 000 animals [1]. To strengthen their protection, the eight extant pangolin species were added to the International Union for Conservation of Nature (IUCN) Red List of threatened species in 2019 [2]. China also upgraded pangolins from a National Level II protected wild species to National Level I protected wild species in 2020. To increase numbers, many researchers have also attempted to initiate artificial breeding programs but have thus far been unsuccessful. Difficulties in captive breeding may be related to their specialized diets and poor stress responses, e.g., abnormalities in the immune, respiration, and digestive systems. In addition, the decrease in pangolin populations may be related to the occurrence of disease, with wild pangolins reported to be highly susceptible to infection by viruses, bacteria, and blood parasites [3].
Pangolins prey on insects and are also preyed upon by other wild animals, so protecting pangolins is crucial for maintaining food chain stability. Some countries in Asia and Africa have a tradition of hunting, consuming, and utilizing pangolins. Overhunting in Asia has led to a sharp decrease in the population of pangolins, which has also promoted illegal smuggling of African pangolins to Asia. Since the outbreak of COVID-19, the hidden dangers of the wildlife trade to public health security have attracted more considerable attention. Pangolins, bats, and other wildlife were considered as potential intermediate hosts of zoonotic pathogens such as Ebola virus, SARS-CoV-2, and HKU4-CoV-like viruses [4, 5], and other viruses which transmitted between wildlife and domestic animals, including paramyxovirus, astrovirus, pseudorabies virus, porcine circovirus 2 [5]. Therefore, illegal trade of pangolins not only destroys biodiversity and the environment, but also provides an important route for the transmission of new and recurrent infectious diseases.
Ticks are a diverse group of hematophagous ectoparasites found on mammals, birds, reptiles, and amphibians. While sucking blood, ticks also serve as pathogen vectors, transmitting viruses, bacteria, and protozoa, causing tick-borne diseases (TBDs) [6]. TBDs impose a significant threat to human healthy, livestock production, and wildlife survival, especially in temperate, tropical, and subtropical regions of the world [7,8]. Although analysis of ticks and TBDs in pangolins has deep significance for conservation and public health, relevant research remains limited. Rickettsia africae, the causative pathogen of African tick-bite fever, has been detected in Amblyomma javanense ticks on Malayan pangolins in Malaysia [9], Thailand [3], and southern China [10], and in Amblyomma compressum ticks on giant pangolins from the Congo [11]. Haemaphysalis hystricis, Haemaphysalis formosensis, and Amblyomma testudinarium have been reported in Formosan pangolins (M. p. pentadactyla) from Taiwan in China, with many of the H. hystricis ticks infected with Rickettsia conorii subsp., Anaplasma spp., Ehrlichia spp., and Cytauxzoon spp. [12]. Candidatus Borrelia javanense has been detected in A. javanense ticks on pangolins (Manis javanica) seized in anti-smuggling operations in southern China [13] and Babesia spp. have also been found in confiscated pangolins (M. javanica) in Thailand [14].
The present study was conducted to investigate the reasons for the unexpected death of confiscated Malayan pangolins and the potential pathogens that may be transmitted during smuggling. Our research not only provides a reference for the diagnosis of pangolin-related diseases and conservation but also for the prevention and control of zoonotic diseases.
Materials and methods
Ethics statement
This study was approved by the Guangzhou Zoo (Guangzhou Wildlife Research Center) Ethics Committee (approval number GZZOO2020031001). All procedures used during the research were in accordance with relevant guidelines and regulations.
Pangolins
The 21 live Malayan pangolins studied here were rescued and treated by customs and the Department of Forestry of Guangdong Province in March 2021, then raised in a dark and quiet environment for further health assessment and rehabilitation by the Guangdong Provincial Wildlife Rescue Center at Guangzhou Zoo and the Guangdong Institute of Applied Biological Resources (China). Two mornings after the rescue, pangolins were anesthetized for physical examination and ectoparasites check, these operations complied with ethics approval (Wild Animal Treatment Regulation No. [2011] 85). All procedures used during the research were approved by the Guangzhou Zoo (Guangzhou Wildlife Research Center) Ethics Committee (approval number GZZOO2020031001).
Tick collection and morphological identification
After careful physical examination, we found that 17 of the 21 pangolins were parasitized with ticks. The ticks were gently removed from the pangolins with tweezers and identified to species, life stage, and sex according to morphological criteria [15]. Finally, 16 male and 17 female ticks were stored at −80°C for DNA & RNA isolation and molecular detection.
Pangolin tissue collection
Although active treatment methods were implemented, all 21 confiscated unhealthy pangolins ultimately died. Diagnostic necropsy was performed within six hours of all 21 pangolins death [16]. Carefully observed and recorded the gross changes on the body surface, external opening, and major organs. Tissues with typical lesions, including heart, lung, liver, kidney, spleen, were removed from pangolin carcass, which stored in 4% paraformaldehyde fix solution (Beyotime Biotechnology, Nantong, China) in sterile tubes and kept at room temperature, or directly stored in sterile tubes and kept at −80°C for further investigation, respectively.
Hematoxylin and eosin (HE) staining
Tissues of dead pangolins stored in 4% paraformaldehyde fix solution at room temperature for at least 48 h were embedded in paraffin, cut into sections of 5 μm in thickness and sticked onto the glass slide. After deparaffinized and rehydrated, the sections on slides were stained with H&E staining kit (Abcam, Cambridge, UK) according to the guidance, mounted with coverslips and observed using a suit of Olympus equipment (BX53 with PM-C 35 digital camera).
Nucleic acid extraction of ticks and pangolin samples
Tick or pangolins tissues homogenates were prepared using a frozen tissue homogenizer (SCIENTZ, Ningbo, China) after adding twice the volume of phosphate-buffered saline (PBS). The homogenate or pangolins blood DNA and RNA were extracted using Tissue/Blood DNA/RNA Kit (OMEGA, Norcross, Georgia, USA) as described by the manufacturer respectively. Extracted DNA and RNA were stored at ˗80°C for further pathogens detection.
Molecular detection of ticks and pathogens
The total RNA of tick samples was used as template for species identified by the species primer pairs (Table 1) targeted 16S rRNA [17]. The RT-PCR reaction was conducted using the TaKaRa PrimeScript One Step RT-PCR Kit (TaKaRa, Shiga, Japan) according to the product information.
For the detection of RNA viral pathogens including avian influenza virus (AIV), coronavirus (CoVs), canine distemper virus (CDV), encephalomyocarditis virus (ECMV), parainfluenza virus type 5 (PIV5). The total RNA of ticks, pangolins tissue or blood samples was used as template for RT-PCR reaction and specifically amplified using primers as described above. The DNA extracted from ticks or pangolins samples was used as template for DNA viral pathogens detection (canine herpes virus (CHV) and canine parvovirus (CPV)), procaryotic pathogens detection (Rickettsia, Ehrlichia and Anaplasma) [18], and protozoon pathogens detection (Babesia, Theileria and Hepatozoon) [19]. Primers used in this study were listed in Table 1.
The PCR products were run in 3% agarose gel (Thermo Fisher Scientific, Waltham, MA, US) with FluoroVue Nucleic Acid Gel Stain (SMOBIO, Beijing. China) and electrophoresed at 160 V for 25 min to separate DNA fragments. Nucleic acid bands were visualized under ultraviolet (UV) light, and positive bands were excised and purified with a TIANamp Genomic DNA Kit (Tiangen, Beijing, China) and sequenced (Ruibiotech, Beijing, China). The obtained nucleotide sequences were compared with sequences published in GenBank using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to search for sequence homology.
Results
Observations and clinical symptoms of confiscated Malayan pangolins
Upon rescue, all 21 pangolins including four underage female (weight in 1.95 kg to 3.2 kg), ten adult females (weight in 4.0 kg to 5.7 kg) and seven adult males (weight in 7.5 kg to 8.3 kg), exhibited poor vigor, accompanied by various symptoms such as cough, drowsiness, anorexia, and edema of extremities. Rescuers also observed inexplicable wounds on the body surface of pangolins, which may have been caused by hunting or transportation. In total, 17 of the 21 pangolins (80.95%) were parasitized with ticks, and some tick bite wounds were broken and infected. After gently removing all ticks, the wounds were disinfected with normal saline and compound lysozyme disinfectant spray (Kalo, Kunshan, China). Intramuscular injection of Synulox RTU (Zoetis, New York, USA) (0.05 ml/kg body weight) once daily for three consecutive days. Although active antibiotic treatment methods were implemented, clinical symptoms were not relieved, some tick bite and wounds had not recovered, and the pangolins died one after another from an unknown cause. Before death, several pangolins also exhibited bloody stools, hematuria, convulsions, and other neurological symptoms.
Identification and phylogenetic analysis of ticks
Based on the morphological characteristics [15], ticks collected from the confiscated pangolins were initially identified as Amblyomma javanense (A. javanense) (Fig 1A–1D). To further confirm the tick species, specific DNA sequences were amplified from cDNA of two ticks and sequenced for 16S rRNA. BLAST results showed that 16S rRNA gene of ticks (Genbank PQ047839 and PQ048025) exhibit 99% similarity with A. javanense. Genetic evolution showed that the selected sequences of 16S rRNA gene belonged to the same branch as A. javanense (Fig 1E).
(A)&(B) Dorsa view and ventral view of male A. javanense tick. (C)&(D) Dorsa view and ventral view of female A. javanense tick. (E) Phylogenetic tree based on the 16S rRNA of ticks from confiscated Malayan pangolins. Analyses were conducted using MEGA v6.0 with the maximum-likelihood algorithm. Bootstrap values were calculated with 1000 replicates. The number on each branch indicates bootstrap value. Red circle: sequences of ticks obtained in this study.
Autopsy, microscopic lesion and blood tests of dead Malayan pangolins
In the autopsy of pangolins, congestion and edema were observed in most internal organs, especially lung and heart (Fig 2A and 2B). Ascites, flatulence and yellow serous membrane of digestive tract were shown in some pangolins (Fig 2C and 2D). Large hemorrhage region, spots of hemorrhage were observed on the surface of the lung (Fig 2A). Additionally, the cut surface of the lungs showed infiltration with foamy fluid. Myocardial edema, myocardial collapse, and ventricular congestion were evident in the heart. (Fig 2B). Some pangolins showed obvious ascites, intestinal bloating (Fig 2C), and the serosa of gastric and duodenal turned yellow (Fig 2D). The results of the hematologic and serum biochemical test also revealed that pangolins had mostly anemic appearance, inflammation and hepatorenal dysfunction (Fig 2E and 2F).
(A) Congestion and hemorrhage observed in the lung. (B) Myocardial edema, myocardial collapse, and ventricular congestion were evident in the heart. (C) obvious ascites, intestinal bloating. (D) serosa of gastric and duodenal turned yellow. (E)&(F) blood testing showed levels of HGB (hemoglobin), MCHC (mean corpuscular hemoglobin concentration), LY (lymphocyte), ALB (albumin), BUN (blood urea nitrogen), PHOS (phosphate), GLU (glucose), and Na (Natrium) were out of the reference range.
Histopathological analysis revealed a significant presence of inflammation across multiple organs, including lung, heart, liver, kidney, spleen and Lymph nodes (Fig 3). The microscopic lesions included alveolar collapse, alveolar wall thickening, inflammatory cell infiltration, and capillary dilation and congestion (Fig 3A); myocardial fibers vacuolar denaturation (Fig 3B); hepatic sinus congestion, hepatic cytoplasmic vacuolization and hepatocyte edema (Fig 3C); the glomerulus capillary is expandable and hyperemia; renal tubular epithelial cells exhibit edema, vacuolar degeneration, dilation, and calcification; fibrous tissue hyperplasia, capillary dilation, and inflammatory cell infiltration were observed in renal interstitial (Fig 3D); splenic congestion with hemosiderin deposition in the red pulp (Fig 3E); the thin cortex, absence of lymph nodes, medullary vascular dilation and congestion, and numerous macrophages observed in the spinal cord (Fig 3F). To sum up, the confiscated Malayan pangolins were more likely to die of Multiple Systems Organ Failure.
(A) Lung, alveolar collapse, alveolar wall thickening, inflammatory cell infiltration, and capillary dilation and congestion. (B) Heart, myocardial fibers vacuolar denaturation. (C) Liver, hepatic sinusoid, hepatic cytoplasmic vacuolization and hepatocyte edema. (D) Kidney, the glomerulus capillary is expandable and hyperemia. Renal tubular epithelial cells exhibit edema, vacuolar degeneration, dilation, and calcification. Fibrous tissue hyperplasia, capillary dilation, and inflammatory cell infiltration are observed in renal interstitial. (E) Spleen, splenic congestion with hemosiderin deposition in the red pulp. (F) Lymph nodes, the thin cortex, absence of lymph nodes, medullary vascular dilation and congestion, and numerous macrophages observed in the spinal cord.
Identification and phylogenetic analysis of tick-borne pathogens
Ticks, tissues and blood samples from the confiscated pangolins were collected for pathogens detection. The results of PCR and RT-PCR showed that all samples were negative for viral pathogens (AIV, CDV, CHV, CPV, CoVs, EMCV and PIV5). Furthermore, the DNA samples were amplified to detect Rickettsia, Anaplasma, Babesia, Theileria, and Hepatozoon species (Table 2).
Rickettsiaceae.
Rickettsia sp. was the most common tick-borne pathogen identified in the tick samples and pangolin samples, accounting for 90.91% (30/33) and 42.86% (9/21) respectively. A homology search of the generated sequences (~410 bp) revealed that the sequences of the Rickettsiaceae 17 kDa gene amplified from ticks and pangolins had 77.23%–100.00% similarity to the Rickettsia sequences in GenBank. By comparing sequences, we found considerable differences in homology among sequences (75.50% to 100.00%). Sequences from the tick samples (PT1-2, PT2, PT3-2, PT4, PT6, PT8, PT9-1, PT12-1, PT12-2, PT16-2, and PT20-2) and the pangolin samples (Pangolin1, Pangolin3, Pangolin4, Pangolin6, Pangolin9, Pangolin12, and Pangolin20), showed 96.88%–100.00% identity with Candidatus Rickettsia (GenBank MH932031 and MH932038) (Table 3). Sequences from the tick samples (PT14-2, PT16-4, and PT16-6) and pangolin samples (Pangolin14 and Pangolin16) exhibited 98.49%, 99.03%, 99.49%, 98.52% and 98.75% similarity, respectively, with Rickettsia rhipicephali (GenBank CP003342) (Table 3). Sequences from the PT5-1, PT17-1, and PT20-1 samples showed 87.13% similarity with Rickettsia rickettsii (GenBank CP018914), 82.37% similarity with Rickettsia japonica (CP047359), and 86.63% similarity with unclassified Rickettsia (GenBank KT261767), respectively (Table 3). The PT1-1, PT3-1, PT5-2, PT9-2, PT11-1, PT11-2, and PT17-2 samples also showed 79.08%–79.95% similarity with uncultured Rickettsia sp. (GenBank GQ302893) (Table 3). The remaining sequences amplified from the PT16-1, PT16-3, PT16-5, PT18-1, PT19-1, and PT19-2 samples exhibited 77.23%–78.90% homology to Rickettsia rhipicephali (GenBank CP013133) (Table 3).
The phylogenetic trees demonstrated that sequences from the tick samples (PT20-2, PT16-2, PT12-1, PT8, PT6, 4, 3–2, 2, and 1–2) and pangolin samples (Pangolin1, Pangolin2, Pangolin3, Pangolin4, Pangolin6, Pangolin12, and Pangolin20) were in the same clade as Candidatus Rickettsia (GenBank MH932031 and MH932038). Sequences from the PT14-2, PT16-4, PT9-1, PT16-6, Pangolin16, PT14-1, and Pangolin14 samples were close to Rickettsia rhipicephali (GenBank CP013133). Sequences from the PT12-2, PT5-1, PT17-1, and PT20-1 samples appeared in the same clade as, but distinct from, Rickettsia sp. (Genbank KT261767). Sequences from the PT1-1, PT9-2, PT11-1, PT11-2, PT3-1, PT5-2, PT17-2, PT18-1, PT16-1, PT16-3, PT16-5, PT19-1, PT19-2, samples were clustered in the same clade as, but distinct from, uncultured Rickettsia sp. (GenBank GQ302893 and GQ302899) (Fig 4). Significantly, the phylogenetic trees demonstrated that the sequences from the pangolin samples all closely resembled spotted fever group (SFG) members.
Analyses were conducted by using MEGA v6.0 with maximum-likelihood algorithm. Bootstrap values were calculated with 1 000 replicates. Number on each branch indicates bootstrap values. Red circle: sequences amplified from pangolin samples in this study. Red triangle: sequences amplified from tick samples in this study.
Anaplasmataceae.
A 1 458 bp fragment of the 16S rRNA gene of Anaplasmataceae was detected in 12.12% (4/33) of tick samples and 4.76% (1/21) of pangolin samples. Based on phylogenetic analysis, the sequences amplified from the PT1-2, PT12-1 and Pangolin12 showed 99.65%, 99.64% and 99.46 similarity, respectively, to Ehrlichia ruminantium (GenBank NR_074155.1) and were located in the same clade (Table 3 and Fig 5). Sequences from the PT17-1 and PT17-2 samples showed 99.30% and 99.65% similarity, respectively, to uncultured Anaplasma sp. (GenBank KU189193.1) and were also located in the same clade (Table 3 and Fig 5). Therefore, the positivity rate of Ehrlichia spp. and Anaplasma spp. of tick samples were both 6.06%, and the pangolin samples was only positive for Ehrlichia spp., accounting for 4.76% (1/21).
Analyses were conducted using MEGA v6.0 with maximum-likelihood algorithm. Bootstrap values were calculated with 1 000 replicates. Number on each branch indicates bootstrap values. Red circle: sequence amplified from pangolin sample in this study. Red triangle: sequences amplified from tick samples in this study.
Babesia, Theileria, and Hepatozoon.
The positive rate of the BTH primer in tick and pangolin samples were 33.33% (11/33) and 4.76% (1/21), respectively. Sequences (~1 604 bp) of PT1-2, PT2, PT3-1, PT9-1, PT14-2 and Pangolin3 exhibited 94.10%–96.00% similarity to Babesia sp. (GenBank MT256300) and were clustered in the same clade (Table 3 and Fig 6). Sequences of the PT11-1, PT12-1, PT16-2, and PT18-2 samples showed 94.38%–98.99% similarity to Colpodella sp. (GenBank KT600661) and were clustered in the same clade. The PT4 and PT17-1 samples exhibited 100.00% and 99.78% similarity, respectively, to Colpodella sp. (GenBank GQ411073) and were also found in the same clade (Table 3 and Fig 6). Based on the above analysis, the positive rate of Babesia spp. in tick samples was 15.15% (5/33), that of Colpodella spp. was 18.18% (6/33). However, only Babesia spp. was detected in pangolin samples, with a positive rate of 4.76% (1/21). Neither Theileria sp. nor Hepatozoon sp. was detected in tick and pangolin samples.
Analyses were conducted using MEGA v6.0 with maximum-likelihood algorithm. Bootstrap values were calculated with 1 000 replicates. Number on each branch indicates bootstrap values. Red circle: sequence amplified from pangolin sample in this study. Red triangle: sequences amplified from tick samples in this study.
Analysis of co-infection.
co-infection of ticks was common in this study (Table 2), including single ticks carrying multiple pathogens and different ticks on the same pangolin carrying different pathogens. Multiple infections were found in 33.33% (11/33) of samples. Rickettsia sp. (30/33; 90.91%) was most frequently associated with multiple infection, followed by Babesia sp. (5/33;15.15%). Double infections accounted for 24.24% (8/33) of samples, with Rickettsia sp. + Babesia sp. co-infection showing the highest overall prevalence (12.12%; 4/33), followed by Rickettsia sp. + Colpodella sp. (9.09%; 3/33), and Rickettsia sp. + Anaplasma sp. (3.03%; 1/33). Triple infections also occurred in several ticks, including Rickettsia sp. + Ehrlichia sp. + Babesia sp. (3.03%; 1/33) and Rickettsia sp. + Ehrlichia sp. + Colpodella sp. (6.06%; 2/33). In pangolins, 9 cases (42.86%, 9/21) were infected with Rickettsia sp. alone, 1 case (4.76%, 1/21) was co-infected with Rickettsia sp. and Ehrlichia sp., and 1 case (4.76%, 1/21) was co-infected with Rickettsia sp. and Babesia sp.
Discussion
Among the 21 Malayan pangolins seized by customs, 80.95% (17/21) were parasitized by at least one tick, which is much higher than previous reports on Malayan pangolins [9] and Formosan pangolins [12]. This high infection rate may be related to mutual transmission among pangolins during the smuggling process. When seized, all pangolins were in an unhealthy state. Pangolins require specialized diets and feed only on ants and termites. Furthermore, they also show adverse stress responses when stimulated, resulting in immune, respiratory, and digestive system abnormalities. The poor environments, food deficiencies, and stress factors experienced during smuggling caused physical deterioration, providing an ideal breeding ground for ticks and pathogenic transmission.
As early as the 1950s, Kohls (1957) reported that Sunda pangolins, wild boars, bats, hyenas, bears, sambar, water monitors, long-tailed skinks, and hill turtles could be infected with A. javanense ticks. Subsequent research showed that A. javanense is the most common tick in Asian, Indian, Sunda, Malayan, and Chinese pangolins and can infect humans and transmit zoonotic pathogens [9]. In our research, ticks collected from the confiscated Malayan pangolins were identified as A. javanense. After ruling out viral infection, we tested for bacteria that may be carried by A. javanense, including Rickettsia, Anaplasma, Ehrlichia, Babesia, Theileria, and Hepatozoon.
Rickettsiaceae are a diverse group of obligatory intracellular gram-negative bacteria and include the Rickettsia, Anaplasma, Ehrlichia, Orientia, and Coxiella genera [20], which is a neglected pathogen mainly transmitted by ticks, and its infection status in animals, especially in wild animals, is not yet clear. Moreover, human rickettsial diseases are mostly endemic in nature and under-diagnosed in developing countries such as India [21], and underdeveloped countries in Asian and Africa [22]. Pangolins are wild animals that is difficult to raise on a large scale artificially, which poses a significant obstacle to the study of pathogens that can infect pangolins and cause them to become diseased or even die. Therefore, there are no explicit reported about the severity or even death of pangolin infections caused by pathogens including Rickettsiaceae. The Rickettsia genus is divided into the typhus group (TG) and spotted fever group (SFG), while Orientia tsutsugamushi and Orientia chuto belong to the scrub typhus group (STG) [23]. Common clinical symptoms of spotted fever include anorexia, fever, rash, cutaneous ulcers, coma, convulsions, and other neurological symptoms [24]. The symptoms and lesions of the confiscated pangolins were highly consistent with the above description of spotted fever.
In the present study, Rickettsia had the highest positive rate in tick samples and pangolin samples (90.91% and 42.86%, respectively). Phylogenetic analysis was performed using the Rickettsiaceae 17 kDa gene. Results showed that more than half of all Rickettsia spp. found in the tick and all the Rickettsia spp. found in pangolin samples closely resembled spotted fever group (SFG) members, Candidatus Rickettsia and Rickettsia rhipicephali (GenBank MH932038, MH932031, and CP003342). The confiscated pangolins, already suffering from malnutrition and weakened immunity, were highly likely to accelerated in their deaths by spotted fever caused by Rickettsia spp. from A. javanense.
We also found great differences between the Rickettsiaceae 17 kDa genes amplified from different ticks on the same pangolin. Of note, of the six tick samples collected from Pangolin No. 16, the similarity between sequences was 76.30%–100.00%, whereas the similarity between the PT5-1 and PT5-2 tick samples was only 62.10%. These results not only suggest that Rickettsia infection may be the leading cause of death in the pangolins, but also imply that pangolin smuggling may create ideal conditions for the transmission and recombination of Rickettsia spp..
The tick samples positive rates of Anaplasma spp. and Ehrlichia spp. were 6.06%, respectively, and Ehrlichia sp. was only positive in Pangolin12. Phylogenetic analysis of the 16S rRNA genes showed that the two Anaplasma spp. were most closely related to the uncultured Anaplasma sp. (GenBank KU189193) (99.30% and 99.65%) and formed a clade with Anaplasma bovis. The Ehrlichia spp. obtained from pangolin and tick samples exhibited 99.46%, 99.65%, and 99.64% similarity, respectively, with E. ruminantium (GenBank NR_074155.1) (Fig 5), which is considered to be the pathogenic agent of Heartwater [25]. Although the positive rate is very low, tick bites caused Ehrlichia transmission in pangolins cannot be ignored.
We also carried out detection of the Babesia, Theileria, and Hepatozoon genera, which live in mammalian blood cells and can cause potentially fatal diseases in infected animals [26]. Babesia spp. were detected in both tick and pangolin samples, with positive rates of 15.15% and 4.76%, respectively, while Theileria sp. and Hepatozoon sp. were not detected. The clinical symptoms observed in some pangolins partially coincided with symptoms of Babesia infection, such as anorexia, hematuria, and dyspnea [27]. Thus, we hypothesized that Babesia infection was not the primary cause, not rule out it might accelerate death of a few pangolins.
Of note, we also detected Colpodella spp. in the tick samples, which are close relatives of the phylum Apicomplexa, including Babesia and Plasmodium [28]. While earlier research suggested that most Colpodella species are free-living and feed on protists [29], subsequent studies have identified these species in blood, tick, soil, and fecal samples [30–32]. Studies in China indicate that Colpodella may also infect humans and induce neurological symptoms [31, 33]. Among the 33 ticks tested in this study, six (overall prevalence 18.18%) were infected with Colpodella, with two sequences similar to ticks from Qinghai (GenBank MH012046) and Yunnan (GenBank MH208621), and the other four sequences similar to a Colpodella sp. (GenBank KT600661) implicated to cause neurological symptoms upon infection. This study is the first to report on Colpodella sp. infection in A. javanense ticks carried on pangolins. Given the limited research, we cannot currently speculate on the relationship between ticks carrying Colpodella sp. and the clinical symptoms or death in the Malayan pangolins. The route of transmission of Colpodella is also unknown, but it is likely to be through tick bites [31]. While the public health threat posed by Colpodella is not yet known, further research is needed on the role of wild animals that may carry ticks, such as pangolins, in the transmission of this parasite.
Conclusions
This study suggests that spotted fever caused by Rickettsia spp. from A. javanense accelerates the death of most confiscated unhealthy pangolins. Co-infection of Rickettsia spp. with Ehrlichia spp., or Babesia spp. may have accelerated deterioration and eventual death of a few pangolins. Identification of the Colpodella pathogen in A. javanense ticks carried by the pangolins warrants further study to investigate its transmission and pathogenic features. Furthermore, the diagnosis, treatment, and surveillance of TBDs are needed to improve rescue operations for pangolins and other wild animals, which should also shed light on the prevention and control of zoonotic diseases.
Acknowledgments
We thank Jiajie Xiao from Guangdong provincial wildlife rescue center and Jinping Chen from Institute of Zoology, Gangdong Academy of Sciences for their kind help in this work.
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