Abstract
Pangolins are mammals of the order Pholidota, with most species listed as endangered or critically endangered. They are distributed in both Asia and Africa, with the Indian pangolin (Manis crassicaudata) found in South Asia, including Pakistan. The tick genus Amblyomma, in the family Ixodidae, is widely distributed in tropical and subtropical regions and infests a variety of terrestrial and semi terrestrial vertebrates. The bacterial genus Borrelia is Gram-negative and consists of three main groups, one of which is mainly linked to reptiles, some birds, and mammals. This study focuses on investigating the Indian pangolin as a potential host for Amblyomma pakhtunensis, a recently described tick species previously reported only on monitor lizards and with no known microbial associations. It also aims to determine whether ticks from pangolins contain Borrelia species and assesses their phylogenetic relationships. Ticks collected from a pangolin were morphologically identified as A. pakhtunensis. DNA from a subset of ticks was extracted to confirm their molecular identity and to detect Borrelia spp. in these ticks using conventional PCR. For the former, a partial fragment of tick 16S rRNA was targeted, while for the latter, partial fragments of the Borrelia 16S rRNA, flaB, and glpQ genes were targeted for amplification. A single live pangolin, conventionally identified as M. crassicaudata, was found. Twenty‑seven ticks were collected (16 males, 11 females), all morphologically identified as A. pakhtunensis. BLAST analysis of A. pakhtunensis 16S rDNA sequence showed 99.75% identity with the same tick species. BLAST analysis of Borrelia 16S rDNA and flaB sequences revealed highest identities of 99.25% and 95.91%, respectively, with Candidatus Borrelia javanense. The overall Borrelia infection rate was 50% (5/10), with infections detected in 4/5 female ticks and 1/5 male tick. In both 16S rDNA and flaB based phylogenetic analyses, the detected Borrelia species clustered with Candidatus B. javanense from China. In addition to expanding the vertebrate host diversity of A. pakhtunensis, this study also expands knowledge of the third group of Borrelia by extending their known vertebrate host range and genetic diversity. This study can enhance our understanding of the regional epidemiology of tick-borne diseases and may specifically aid pangolin conservation strategies by taking into account the control of ticks and their associated pathogens.
Data availability
All DNA sequences generated in this study are publicly available in GenBank under the following accession numbers: PX387109, PX387536, and PX394402.
References
Gaudin, T. J., Emry, R. J. & Wible, J. R. The phylogeny of living and extinct pangolins (Mammalia, Pholidota) and associated taxa: a morphology based analysis. J. Mamm. Evol. 16, 235–305 (2009).
IUCN, MA. "The IUCN red list of threatened species. Version 2019‐2." (2019).
Waseem, M. et al. Occupancy, habitat suitability and habitat preference of endangered Indian pangolin (Manis crassicaudata) in Potohar Plateau and Azad Jammu and Kashmir, Pakistan. Glob. Ecol. Conserv. 23, e01135 (2020).
Nowak, R. M. Walker’s Mammals of the World 6th edn. (Johns Hopkins University Press, 1999).
Chao, J. T., Li, H. F. & Lin, C. C. The role of pangolins in ecosystems. In Pangolins 43–48 (Acad. Press, 2020).
Sun, N. C. M., Lin, J. S. & Pei, K. J. C. Characteristics, location, and usage patterns of resting burrows in Chinese pangolins: insights from radio-telemetry tracking. Glob. Ecol. Conserv. 57, e03406 (2025).
Mohapatra, R. K., Panda, S., Nair, M. V. & Acharjyo, L. N. Check list of parasites and bacteria recorded from pangolins (Manis sp.). J. Parasit. Dis. 40(4), 1109–1115 (2016).
Barker, S.C. and Barker, D. Ticks of Australasia: 125 species of ticks in and around Australia (2023).
Guglielmone, A. A., Nava, S. & Robbins, R. G. Geographic distribution of the hard ticks (Acari: Ixodida: Ixodidae) of the world by countries and territories. Zootaxa 5251(1), 1–274 (2023).
Khan, M. et al. New records of Amblyomma gervaisi from Pakistan, with detection of a reptile-associated Borrelia sp. Tick. Tick. Borne Dis. 13(6), 102047 (2022).
Labruna, M. B. et al. Redescription of the female, description of the male, and several new records of Amblyomma parkeri (Acari: Ixodidae), a South American tick species. Exp. Appl. Acarol. 49(3), 243–260 (2009).
Guglielmone, A. A. et al. The Hard Ticks of the World Vol. Vol. 10, 978–994 (Springer, 2014).
Eisen, L. Tick species infesting humans in the United States. Tick. Tick-borne Dis. 13(6), 102025 (2022).
Guzmán Cornejo, C., Robbins, R. G., Guglielmone, A. A., Montiel Parra, G. & Pérez, T. M. The Amblyomma (Acari: Ixodida: Ixodidae) of Mexico: identification keys, distribution and hosts. (2011).
Alasmari, S. M. N. et al. Impact of climate change on the tick-host-pathogen complex: Distribution patterns, disease incidence, and host infestation. Revista. Bras. de Parasitologia Vet. 34(4), e004725 (2025).
Dupuis, A. P., Lange, R. E. & Ciota, A. T. Emerging tickborne viruses vectored by Amblyomma americanum (Ixodida: Ixodidae): heartland and Bourbon viruses. J. Med. Entomol. 60(6), 1183–1196 (2023).
Molaei, G., Khalil, N., Ramos, C. J. & Paddock, C. D. Establishment of Amblyomma maculatum ticks and Rickettsia parkeri the Northeastern United States. Emerg. Infect. Dis. 30(10), 2208 (2024).
Ali, A. et al. Description of a new tick species, closely related to Amblyomma javanense (Supino, 1897), associated with Varanus bengalensis (Squamata: Varanidae) in Pakistan. Tick. Tick-borne Dis. 15(6), 102361 (2024).
Camicas, J. L., Hervy, J. P., Adam, F. & Morel, P. C. The ticks of the world (Acarida, Ixodida): nomenclature, described stages, hosts, distribution. (1998)
Duan, D. Y., Tang, J. M., Chen, Z., Liu, G. H. & Cheng, T. Y. Mitochondrial genome of Amblyomma javanense: a hard tick parasite of the endangered Malayan pangolin (Manis javanica). Med. Vet. Entomol. 34(2), 229–235 (2020).
Dworkin, M. S., Schwan, T. G., Anderson, D. E. Jr. & Borchardt, S. M. Tick-borne relapsing fever. Infect. Dis. Clin. N. Am. 22(3), 449 (2008).
Enferadi, A., Ownagh, A. & Tavassoli, M. Detection of Borrelia turcica from Hyalomma aegyptium ticks and blood of turtles (Testudo graeca) in northern west of Iran. Eur. J. Wildl. Res. 71(2), 18 (2025).
Gofton, A. W. et al. Exploring Borrelia in Australia: isolation, genomic characterisation, and host and vector associations. Tick. Tick. Borne Dis. 16(4), 102505 (2025).
Rahman, Z. U., Khan, M., Khan, Z., Khan, W., Khan, A., Liaqat, I., Ali, A. & Almutairi, M. M. First molecular record of Argas hermanni and Argas sp. closely related to Argas persicus, with detection of Borrelia anserina in Argas persicus from pigeons in Pakistan. (In press).
Binetruy, F. et al. A novel Borrelia species, intermediate between Lyme disease and relapsing fever groups, in neotropical passerine-associated ticks. Sci. rep. 10(1), 10596 (2020).
Fesler, M. C. et al. Lyme disease: diversity of Borrelia species in California and Mexico detected using a novel immunoblot assay. Healthcare 8(2), 97 (2020)(Multidisciplinary Digital Publishing Institute).
Gofton, A. W. et al. Characterisation and comparative genomics of three new Varanus-associated Borrelia spp. from Indonesia and Australia. Parasit. Vect. 16(1), 317 (2023).
Gupta, R. S. Distinction between Borrelia and Borreliella is more robustly supported by molecular and phenotypic characteristics than all other neighbouring prokaryotic genera: response to Margos’ et al." the genus Borrelia reloaded"(PLoS ONE 13 (12): e0208432). PLoS ONE 14(8), e0221397 (2019).
Kaenkan, W. et al. Reptile-associated Borrelia spp. in Amblyomma ticks, Thailand. Tick. Tick. Borne Dis. 11(1), 101315 (2020).
Margos, G., Henningsson, A. J., Markowicz, M. & Fingerle, V. Borrelia ecology and evolution: Ticks and hosts and the environment. Microorganisms 10(8), 1513 (2022).
Panetta, J. L. et al. Reptile-associated Borrelia species in the goanna tick (Bothriocroton undatum) from Sydney, Australia. Parasit. Vectors. 10(1), 616 (2017).
Takano, A. et al. Isolation and characterization of a novel Borrelia group of tick-borne borreliae from imported reptiles and their associated ticks. Environ. Microbiol. 12(1), 134–146 (2010).
Adeolu, M. & Gupta, R. S. A phylogenomic and molecular marker based proposal for the division of the genus Borrelia into two genera: the emended genus Borrelia containing only the members of the relapsing fever Borrelia, and the genus Borreliella gen. nov. containing the members of the Lyme disease Borrelia (Borrelia burgdorferi sensu lato complex). Antonie Van Leeuwenhoek 105(6), 1049–1072 (2014).
Barbour, A. G. & Gupta, R. S. The family Borreliaceae (Spirochaetales), a diverse group in two genera of tick-borne spirochetes of mammals, birds, and reptiles. J. med. entomol. 58(4), 1513–1524 (2021).
Estrada-Peña, A. & Cabezas-Cruz, A. Phyloproteomic and functional analyses do not support a split in the genus Borrelia (phylum Spirochaetes). BMC Evol. Boil. 19(1), 54 (2019).
Margos, G. et al. There is inadequate evidence to support the division of the genus Borrelia. Int. J. Syst. Evol. Microbiol. 67(4), 1081–1084 (2017).
Sprong, H. et al. Control of Lyme borreliosis and other Ixodes ricinus-borne diseases. Parasit. Vector. 11(1), 145 (2018).
Jiang, B. G. et al. Molecular detection of novel Borrelia species, Candidatus Borrelia javanense, in Amblyomma javanense ticks from pangolins. Pathogens 10(6), 728 (2021).
Tang, T. et al. The global distribution and the risk prediction of relapsing fever group Borrelia: a data review with modelling analysis. Lancet Microbe 5(5), e442–e451 (2024).
Ali, A, Almutairi, M.M., Robbins, R.G., DOBLER, L.C. and Ullah, S., Updated checklist, morphological descriptions, hosts and vector potential of ticks (Acari: Argasidae, Ixodidae) in Pakistan. Zootaxa, 5725(2), pp. 151–202 (2025).
Sambrook, J., Fritsch, E .F. & Maniatis, T. Molecular cloning: a laboratory manual (No. Ed. 2, pp. xxxviii+-1546) (1989).
Stromdahl, E. Y. et al. Evidence of Borrelia lonestari DNA in Amblyomma americanum (Acari: Ixodidae) removed from humans. J. Clin. Microbiol. 41(12), 5557–5562 (2003).
Halperin, T. et al. Detection of relapsing fever in human blood samples from Israel using PCR targeting the glycerophosphodiester phosphodiesterase (GlpQ) gene. Acta Trop. 98(2), 189–195 (2006).
Radulović, Ž, Milutinović, M., Tomanović, S. & Mulenga, A. Detection of Borrelia-specific 16S rRNA sequence in total RNA extracted from Ixodes ricinus ticks. Arq. Bras. Med. Vet. Zootec. 62, 862–867 (2010).
Khan, M. et al. Molecular evidence of Borrelia theileri and closely related Borrelia spp. in hard ticks infesting domestic animals. Front. Vet. Sci. 10, 1297928 (2023).
Zahid, H. et al. Argas persicus and Carios vespertilionis ticks infesting ducks, domestic fowls and bats in Pakistan: first report on molecular survey and phylogenetic position of Borrelia anserina. Vet. Sci. 10(10), 628 (2023).
Mangold, A. J., Bargues, M. D. & Mas-Coma, S. Mitochondrial 16S rDNA sequences and phylogenetic relationships of species of Rhipicephalus and other tick genera among Metastriata (Acari: Ixodidae). Parasitol. Res. 84(6), 478–484 (1998).
Ullah, Z. et al. Investigation of Hyalomma turanicum and Hyalomma asiaticum in Pakistan, with notes on the detection of tickborne Rickettsiales. Front. Vet. Sci. 11, 1500930 (2024).
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. mole. biol. 215(3), 403–410 (1990).
Hall, T., Biosciences, I. & Carlsbad, C. J. G. B. B. BioEdit: an important software for molecular biology. GERF Bull. Biosci. 2(1), 60–61 (2011).
Thompson, J. D., Higgins, D. G. & Gibson, T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acid. Res. 22(22), 4673–4680 (1994).
Kumar, S. et al. MEGA12: Molecular evolutionary genetic analysis version 12 for adaptive and green computing. Mol. Biol. Evol. 41(12), msae263 (2024).
Guner, E. S., Hashimoto, N., Kadosaka, T., Imai, Y. & Masuzawa, T. A novel, fast-growing Borrelia sp. isolated from the hard tick Hyalomma aegyptium in Turkey. Microbiology 149(9), 2539–2544 (2003).
Kalmar, Z. et al. Transstadial transmission of Borrelia turcica in Hyalomma aegyptium ticks. PLoS ONE 10(2), e0115520 (2015).
Hepner, S. et al. Population structure of Borrelia turcica from Greece and Turkey. Infect. Genet. Evol. 77, 104050 (2020).
Benyahia, H. et al. Molecular and MALDI-TOF MS characterisation of Hyalomma aegyptium ticks collected from turtles and their associated microorganisms in Algeria. Tick. Tick-borne Dis. 13(1), 101858 (2022).
Wodecka, B. & Kolomiiets, V. Genetic diversity of Borreliaceae species detected in natural populations of Ixodes ricinus ticks in northern Poland. Life 13(4), 972 (2023).
Norte, A. C. et al. Molecular investigation of tick-borne pathogens from different regions of Morocco. Tick. Tick Borne Dis. 15(6), 102418 (2024).
Kautman, M. et al. The importance of the tortoise tick Hyalomma aegyptium as a carrier of bacterial agents on a wide range. Tick. Tick Borne Dis. 16(2), 102456 (2025).
Loh, S. M., Gillett, A., Ryan, U., Irwin, P. & Oskam, C. Molecular characterization of ‘Candidatus Borrelia tachyglossi’ (family Spirochaetaceae) in echidna ticks, Bothriocroton concolor. Int. J. Syst. Evol. Microbiol. 67(4), 1075–1080 (2017).
Gofton, A. W. et al. Inhibition of the endosymbiont “Candidatus Midichloria mitochondrii” during 16S rRNA gene profiling reveals potential pathogens in Ixodes ticks from Australia. Parasit. Vect. 8(1), 345 (2015).
Colunga-Salas, P., Sánchez-Montes, S., Ochoa-Ochoa, L. M., Grostieta, E. & Becker, I. Molecular detection of the reptile-associated Borrelia group in Amblyomma dissimile. Mexico. Med. Vet. Entomol. 35(2), 202–206 (2021).
Sophia, H. F., Supriyono, S., Soviana, S., Novianto, D. & Hadi, U.K. Molecular detection of Borrelia spp. (Spirochaetales: Borreliaceae) in ticks (Acari: Ixodidae) collected from tortoises in Java, Indonesia. Biodivers. J. Biol. Divers. 24(12) (2023).
Supriyono, S., Sophia, H. F., Hadi, U. K. & Soviana, S. Tick-borne pathogens detection from ticks infesting Malayopython reticulatus (Reptilia: Pythonidae) snakes in Indonesia. Biotropia 32(1), 111–117 (2025).
Ahmad, T., Ali, A., Poudel, T. R., Rahim, A. & Li, B. Ecological drivers and conservation challenges of Indian pangolins (Manis crassicaudata) in Western Pakistan. Glob. Ecol. Conserv. 61, e03653 (2025).
Mahmood, T., Hussain, R., Irshad, N., Akrim, F. & Nadeem, M. S. Illegal mass killing of Indian pangolin (Manis crassicaudata) in Potohar region, Pakistan. Pak. J. Zool. 44(5), 1457–1461 (2012).
Wang, X. M. et al. To save pangolins: a nutritional perspective. Animal 12(22), 3137 (2022).
Apanaskevich, D. A., Oliver, J. H., Sonenshine, D. E. & Roe, R. M. Life cycles and natural history of ticks. Biol. Tick. 1, 59–73 (2014).
Fingerle, V. et al. “Candidatus Borrelia kalaharica” detected from a febrile traveller returning to Germany from vacation in Southern Africa. PLoS Negl. Trop. Dis. 10(3), e0004559 (2016).
Ali, A. et al. Molecular survey and spatial distribution of Rickettsia spp. in ticks infesting free-ranging wild animals in Pakistan (2017–2021). Pathogens 11(2), 162 (2022).
Green, S. J., Venkatramanan, R. & Naqib, A. Deconstructing the polymerase chain reaction: understanding and correcting bias associated with primer degeneracies and primer-template mismatches. PLoS ONE 10(5), e0128122 (2015).
Khan, S. M. et al. Phylogenetic position of Haemaphysalis kashmirensis and Haemaphysalis cornupunctata, with notes on Rickettsia spp. Genes 14(2), 360 (2023).
Muñoz‐Leal, S. et al. Novel Borrelia genotypes in bats from the Macaregua Cave, Colombia. Zoonoses Pub. Health 68(1), 12–18 (2021).
Li, B. et al. Molecular identification of tick-borne Rickettsia, Anaplasma, Ehrlichia, Babesia, and Colpodella in confiscated Malayan pangolins. PLoS Negl. Trop. Dis. 18(11), e0012667 (2024).
Chong, S. Q. et al. Detection of a novel Babesia sp. in Amblyomma javanense, an ectoparasite of Sunda pangolins. Parasit. Vect. 16(1), 432 (2023).
Dao, T. T. H. et al. Detection of tick-borne pathogens in the pangolin tick, Amblyomma javanense, from Vietnam and Laos, including a novel species of Trypanosoma. Acta Trop. 260, 107384 (2024).
Shi, Y. N. et al. Detection of a novel Pestivirus strain in Java ticks (Amblyomma javanense) and the hosts Malayan pangolin (Manis javanica) and Chinese pangolin (Manis pentadactyla). Front. Microbiol. 13, 988730 (2022).
Parragué-Migone, C., Santodomingo, A., Thomas, R., Muñoz-Leal, S. & Acosta-Jamett, G. Multilocus sequence typing unveils two novel genospecies of Borrelia burgdorferi sensu lato in ticks infesting cricetid rodents of northern Chile. Transbound. Emerg. Dis. 2025(1), 8841276 (2025).
https://www.wwfpak.org/?390818%2FWWF-Pakistan-urges-immediate-action-to-save-pangolins.
Uribe, J.E., Kelava, S., Nava, S., Cotes-Perdomo, A.P., Castro, L.R., Rivera-Paéz, F.A., Perea, S., Mans, B.J., Gofton, A., Teo, E.J. and Zardoya, R. New insights into the molecular phylogeny, biogeographical history, and diversification of Amblyomma ticks (Acari: Ixodidae) based on mitogenomes and nuclear sequences. Parasites & Vectors, 17(1), 139 (2024).
Takano, A., Fujita, H., Kadosaka, T., Konnai, S., Tajima, T., Watanabe, H., Ohnishi, M. and Kawabata, H. Characterization of reptile‐associated Borrelia sp. in the vector tick, Amblyomma geoemydae, and its association with Lyme disease and relapsing fever Borrelia spp. Environmental microbiology reports, 3(5), 632–637 (2011).
Hassan, M., Sulaiman, M.H. and Lian, C.J. The prevalence and intensity of Amblyomma javanense infestation on Malayan pangolins (Manis javanica Desmarest) from Peninsular Malaysia. Acta tropica, 126(2), 142–145 (2013).
Barbour, A.G. Phylogeny of a relapsing fever Borrelia species transmitted by the hard tick Ixodes scapularis. Infection, Genetics and Evolution, 27, 551–558 (2014).
Güner, E.S., Watanabe, M., Hashimoto, N., Kadosaka, T., Kawamura, Y., Ezaki, T., Kawabata, H., Imai, Y., Kaneda, K. and Masuzawa, T. Borrelia turcica sp. nov., isolated from the hard tick Hyalomma aegyptium in Turkey. International journal of systematic and evolutionary microbiology, 54(5), 1649–1652 (2004).
Khatri-Chhetri, R., Wang, H.C., Chen, C.C., Shih, H.C., Liao, H.C., Sun, C.M., Khatri-Chhetri, N., Wu, H.Y. and Pei, K.J.C. Surveillance of ticks and associated pathogens in free-ranging Formosan pangolins (Manis pentadactyla pentadactyla). Ticks and tick-borne diseases, 7(6), 1238–1244 (2016).
Acknowledgment
The authors are grateful and acknowledge Higher Education Commission (HEC) and the Pakistan Science Foundation (PSF), for providing the necessary funding for this research.
Funding
The authors thank the Ongoing Research Funding Program, (ORF-2026-1440), King Saud University, Riyadh, Saudi Arabia.
Author information
Authors and Affiliations
Contributions
A.A: Conceptualization, Project administration, Supervision, Data curation, Methodology, writing original draft, Writing—review and editing, Visualization; M.E.K.: Data curation, Software, Methodology, Writing original draft, Writing—review and editing, Visualization, Morphology; M.U.K: Data curation, Software, Methodology, Writing original draft, Writing—review and editing, visualization, Morphology; A.M: Data curation, Methodology, Writing original draft, Writing—review and editing, Visualization; B.F: Data curation, Methodology, Writing original draft, Writing—review and editing, Visualization; M.A: Conceptualization, Supervision, Data curation, Methodology, Writing original draft, Writing—review and editing, Visualization; M.M.A: Data curation, Methodology, Writing original draft, Writing—review and editing, Visualization; A.F.A: Data curation, Methodology, Writing original draft, Writing—review and editing, Visualization; A.T.A: Data curation, Methodology, Writing original draft, Writing—review and editing, Visualization.
Corresponding authors
Ethics declarations
Competing of interest
The authors declare no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Khan, M., Adnan, M., Majid, A. et al. New host record of Amblyomma pakhtunensis on the Indian Pangolin (Manis crassicaudata) with detection of a distinct Borrelia lineage. Sci Rep (2026). https://doi.org/10.1038/s41598-026-45336-0
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-026-45336-0