Preliminary PCR Screening and Optimization for Five Suspected Viruses in Haemaphysalis bispinosa Ticks from Moa Buffalo (Bubalus bubalis)
DOI:
10.29303/jppipa.v12i1.13910Published:
2026-02-15Downloads
Abstract
Tick-borne viruses are becoming a serious threat to livestock health, especially in tropical regions where monitoring systems are still limited. H. bispinosa, the main external parasite of water buffalo in Eastern Indonesia, is suspected of carrying several veterinarians' important viral pathogens. This study aims to detect five viruses consisting of DNA viruses (BuGyKV, BoHV-1, and OvHV-2) and RNA viruses (FMDV and TAMV). FMDV and TAMV, both RNA viruses, need to be converted into complementary DNA (cDNA) through reverse transcription, which is then used as a template for PCR amplification using virus-specific primers. All viruses were detected using specific primers and extensive annealing temperature optimization. The results showed weak and nonspecific amplification bands at one temperature for BuGyKV, BoHV-1, and OvHV-2, but these bands did not match the expected amplicon size. No amplification bands were observed for FMDV and TAMV under any conditions. These findings suggest that this amplification failure is likely due to primer mismatch with local virus variants resulting from geographical differences. Overall, it can be concluded that the five target viruses were not detected in the H. bispinosa samples examined. This preliminary study provides an initial virological overview of H. bispinosa ticks from Moa Island and emphasizes the need for more sensitive detection methods, such as nested PCR, qPCR, or metagenomic approaches, to improve the detection of low-titer viruses in ticks.
Keywords:
BuGyKV Haemaphysalis bispinosa Moa buffalo PCR Tick-borne virusesReferences
Abbasi, F., Abbasi, I. H. R., Nissa, T. F., Bhutto, Z. A., Arain, M. A., Soomro, R. N., Siyal, F. A., & Fazlani, S. A. (2017). Epidemiological study of tick infestation in buffalo of various regions of district Khairpur, Pakistan. Vet World, 10(6), 688–694. https://doi.org/10.14202/vetworld.2017.688-694 DOI: https://doi.org/10.14202/vetworld.2017.688-694
Abdallah, M. O., Niu, Q., Yang, J., Hassan, M. A., Yu, P., Guan, G., Chen, Z., Liu, G., Luo, J., & Yin, H. (2017). Identification of 12 piroplasms infecting ten tick species in China using reverse line blot hybridization. Journal Parasitol, 103(3), 221–227. https://doi.org/10.1645/16-161 DOI: https://doi.org/10.1645/16-161
Ali, A., Khan, M. A., Zahid, H., Yaseen, P. M., Khan, M. Q., Nawab, J., Ur Rehman, Z., Ateeq, M., Khan, S., & Ibrahim, M. (2019). Seasonal dynamics, record of ticks infesting humans, wild and domestic animals, and molecular phylogeny of Rhipicephalus microplus in Khyber Pakhtunkhwa Pakistan. Front Physiol, 10, 793. https://doi.org/10.3389/fphys.2019.00793 DOI: https://doi.org/10.3389/fphys.2019.00793
Byamukama, B., Amin, A., Mwiine, F. N., & Ekiri, A. B. (2025). Epidemiology and control strategies for foot-and-mouth disease in livestock and wildlife in Uganda: systematic review. Vet Res Commun, 49(4). https://doi.org/10.1007/s11259-025-10791-z DOI: https://doi.org/10.1007/s11259-025-10791-z
Ci-Xiu, L., Mang, S., Jun-Hua, T., Xian-Dan, L., Yan-Jun, K., Liang-Jun, C., Xin-Cheng, Q., Jianguo, X., Holmes, E. C., & Zhang, Y.-Z. (2015). Unprecedented genomic diversity of RNA viruses in arthropods reveals the ancestry of negative-sense RNA viruses. Microbiology and Infectious Disease. https://doi.org/10.7554/eLife.05378 DOI: https://doi.org/10.7554/eLife.05378
Cui, M., Zhu, H. C., Wang, X., Cao, Y., Liu, D., Carr, M. J., Guan, Y., Zhou, H., & Shi, W. (2025). Tamdy virus pathogenesis in immunocompetent and immunocompromised mouse models. Virulence, 16(1). https://doi.org/10.1080/21505594.2025.2503457 DOI: https://doi.org/10.1080/21505594.2025.2503457
Dias, B. . P., Barbosa, C. C., Ferreira, C. S., Santos, S., Arrieta, O. A. P., Malta, W. C., Gomes, M. L. M. D., Alves, Silva, M., Fonseca, J. . M., Borges, L. P., & Silva, B. . M. (2023). Challenges in Direct Detection of Flaviviruses: A Review. Pathogens, 12(5), 643. https://doi.org/10.3390/pathogens12050643 DOI: https://doi.org/10.3390/pathogens12050643
Durden, L. A., Merker, S., & L, B. (2008). The tick fauna of Sulawesi, Indonesia (Acari: Ixodoidea: Argasidae and Ixodidae. Exp Appl Acarol, 45(1–2), 85–110. https://doi.org/10.1007/s10493-008-9144-z DOI: https://doi.org/10.1007/s10493-008-9144-z
Elnekave, E., Maanen, K., Shilo, H., Gelman, B., Storm, N., Berdenstain, S., Berke, O., & Klement, E. (2016). Prevalence and risk factors for foot and mouth disease infection in small ruminants in Israel. Prev Vet Med, 125, 82–8. https://doi.org/10.1016/j.prevetmed.2015.12.019 DOI: https://doi.org/10.1016/j.prevetmed.2015.12.019
Fuente, J., Antunes, S., Bonnet, S., Cabezas-Cruz, A., Domingos, A. G., Estrada-Peña, A., Johnson, N., Kocan, K. M., Mansfield, K. L., Nijhof, A. M., Papa, A., Rudenko, N., Villar, M., Alberdi, P., Torina, A., Ayllón, N., Vancova, M., Golovchenko, M., Grubhoffer, L., … Rego, R. O. M. (2017). Tick-pathogen interactions and vector competence: Identification of molecular drivers for tick-borne diseases. Front Cell Infect Microbiol, 7, 114. https://doi.org/10.3389/fcimb.2017.00114 DOI: https://doi.org/10.3389/fcimb.2017.00114
Gao, W., Xia, X., Wang, T., Liu, Y., Wang, J., Li, Y., Xia, L., Jia, N., Jiang, J., Sun, Y., & Cui. (2025). Diverse virome and potential pathogens in five tick species from metropolis surroundings of Beijing and Tianjin, China. NPJ Biofilms and Microbiomes, 1–34. https://doi.org/10.1038/s41522-025-00878-5 DOI: https://doi.org/10.1038/s41522-025-00878-5
Karim, S., Budachetri, K., Mukherjee, N., Williams, J., Kausar, A., Hassan, M. J., Adamson, S., Dowd, S. E., Apanskevich, D., Arijo, A., Sindhu, Z. U., Kakar, M. A., Khan, R. M. D., Ullah, S., Sajid, M. S., Ali, A., & Iqbal, Z. (2017). A study of ticks and tick-borne livestock pathogens in Pakistan. PLoS Negl Trop Dis, 11(6), 5681. https://doi.org/10.1371/journal.pntd.0005681 DOI: https://doi.org/10.1371/journal.pntd.0005681
König, M. T., Frölich, K., Jandowsky, A., Knauf-Witzens, T., Langner, C., Dietrich, R., Märtlbauer, E., & Didier, A. (2023). First insights into the occurrence of circular single-stranded DNA genomes in Asian and African cattle. Animals (Basel), 13(9). https://doi.org/10.3390/ani13091492 DOI: https://doi.org/10.3390/ani13091492
Kuleš, J., Potocnakova, L., & Bhide, K. (2017). The challenges and advances in diagnosis of vector-borne diseases: where do we stand? Vector-Borne and Zoonotic Diseases, 17(5), 285–296. https://doi.org/10.1089/vbz.2016.2074 DOI: https://doi.org/10.1089/vbz.2016.2074
Kumar, S., Stecher, G., Suleski, M., Sanderford, M., Sharma, S., & Tamura, K. (2024). MEGA12: Molecular evolutionary genetic analysis version 12 for adaptive and green computing. Mol Biol Evol, 41(12), 263. https://doi.org/10.1093/molbev/msae263 DOI: https://doi.org/10.1093/molbev/msae263
Kwak, M. L., & Ng, A. (2022). The detection of three new Haemaphysalis ticks (Acari: Ixodidae) in Singapore and their potential threat to public health, companion animals, and wildlife. Acarologia, 62(4), 927–940. https://doi.org/10.24349/fz2l-kg9r DOI: https://doi.org/10.24349/fz2l-kg9r
Lannutti, L., Gonzales, F. N., Dus Santos, M. J., Florin-Christensen, M., & Schnittger, L. (2022). Molecular detection and differentiation of arthropod, fungal, protozoan, bacterial and viral pathogens of honeybees. Veterinary Sciences, 9(5), 221. https://doi.org/10.3390/vetsci9050221 DOI: https://doi.org/10.3390/vetsci9050221
Lechmann, J., Ackermann, M., Kaiser, V., & Bachofen, C. (2021). Viral infections shared between water buffaloes and small ruminants in Switzerland. J Vet Diagn Invest, 33(5), 894–905. https://doi.org/10.1177/10406387211027131 DOI: https://doi.org/10.1177/10406387211027131
Liu, Z., Li, L., Xu, W., Yuan, Y., & Liang. (2022). Extensive diversity of RNA viruses in ticks revealed by metagenomics in northeastern China. PLOS Neglected Tropical Diseases, 16(12), 11017. https://doi.org/10.1371/journal.pntd.0011017 DOI: https://doi.org/10.1371/journal.pntd.0011017
Ma, R., Zhao, M., Wang, H., Hou, R., Qin, K., Qian, Y., Zhang, H., Zhou, Y., Wu, W., Gu, J., Wang, X., Shen, Q., Liu, S., Liu, J., Bi, W., Yu, Y., Chong, H., Hong, Y., Ji, L., … W, Z. (2022). Virus of giant Panda-infesting ticks teveals novel Bunyaviruses and other viruses that are genetically close to those from giant Pandas. Microbiol Spectr, 10:e02034-. https://doi.org/10.1128/spectrum.02034-22 DOI: https://doi.org/10.1128/spectrum.02034-22
Ma, X., Gu, W., Li, X., Ding, X., & Zhu, L. (2025). Fatty acid synthase may facilitate the trafficking of bovine alpha herpesvirus 1 out of the Golgi apparatus, potentially promoting viral infection. Microbiol Spectr, 13:e01388-. https://doi.org/10.1128/spectrum.01388-25 DOI: https://doi.org/10.1128/spectrum.01388-25
Moming, A., Bai, Y., Wang, J., Zhang, Y., Tang, S., Fan, Z., Deng, F., & Shen, S. (2024). The known and unknown of global tick-borne viruses. Viruses, 16(12), 1807. https://doi.org/10.3390/v16121807 DOI: https://doi.org/10.3390/v16121807
Moming, A., Shen, S., Fang, Y., Zhang, J., Zhang, Y., Tang, S., Li, T., Hu, Z., Wang, H., Zhang, Y., Sun, S., Wang, L. F., & Deng, F. (2021). Evidence of human exposure to Tamdy Virus, Northwest China. Emerg Infect Dis, 27(12), 3166–3170. https://doi.org/10.3201/eid2712.203532 DOI: https://doi.org/10.3201/eid2712.203532
Nithikathkul, C., Polseela, P., Changsap, B., & Leemingsawat, S. (2002). Ixodid ticks on domestic animals in Samut Prakan Province, Thailand. Southeast Asian J Trop Med Public Health, 33(S3), 41–44. https://europepmc.org/article/med/12971472
Riaz, A., Dry, I., Dalziel, R., Rehman, S. U., Shah, M. A., Akhtar, H. M., Yousaf, A., & Baig, R. (2021). Molecular detection and characterization of ovine herpesvirus-2 using heminested PCR in Pakistan. J Vet Sci, 4(51). https://doi.org/10.4142/jvs.2021.22.e51 DOI: https://doi.org/10.4142/jvs.2021.22.e51
Tesfaye, Y., Khan, F., & Gelaye, E. (2020). Molecular characterization of foot-and-mouth disease viruses collected from Northern and Central Ethiopia during the 2018 outbreak. Vet World, 13(3), 542–548. https://doi.org/10.14202/vetworld.2020.542-548 DOI: https://doi.org/10.14202/vetworld.2020.542-548
Tokarz, R., Williams, S. H., Sameroff, S., Sanchez Leon, M., Jain, K., & Lipkin, W. I. (2014). Virome analysis of Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis ticks reveals novel highly divergent vertebrate and invertebrate viruses. J Virol, 88(19), 11480–92. https://doi.org/10.1128/JVI.01858-14 DOI: https://doi.org/10.1128/JVI.01858-14
Utami, P., & Kunda, R. M. (2023). Surface ultrastructure of ticks (Acari: Ixodidae) on Moa buffalo from Southwest Maluku District, Indonesia. Biodiversitas, 24(6), 3230–3235. https://doi.org/10.13057/biodiv/d240617 DOI: https://doi.org/10.13057/biodiv/d240617
Utami, P., Kunda, R. M., & Anaktototy, Y. (2024). First metagenome report of Haemaphysalis bispinosa ticks of Moa buffalo from Southwest Maluku District, Indonesia. Nusantara Bioscience, 16(1), 130–138. https://doi.org/10.13057/nusbiosci/n160116 DOI: https://doi.org/10.13057/nusbiosci/n160116
Wu, Y., Zhou, Q., Mao, M., Chen, H., & Qi, R. (2024). Diversity of species and geographic distribution of tick-borne viruses in China. Front Microbiol, 15(1309698). https://doi.org/10.3389/fmicb.2024.1309698 DOI: https://doi.org/10.3389/fmicb.2024.1309698
Yan, T., Wang, Z., Li, R., Zhang, D., Song, Y., & Cheng, Z. (2024). Gyroviruses: current status and challenges. Front Microbiol, 15(1449814). https://doi.org/10.3389/fmicb.2024.1449814 DOI: https://doi.org/10.3389/fmicb.2024.1449814
Yoo, H. S. (2011). Foot and mouth disease: etiology, epidemiology and control measures. Infect Chemother, 43(2), 178–185. https://doi.org/10.3947/ic.2011.43.2.178 DOI: https://doi.org/10.3947/ic.2011.43.2.178
Zeng, W., Yang, L., Cui, L., Liang, C., Zhu, D., Fang, Y., Zhang, Y., & Liu, H. (2025). Virome analysis and detection of ticks and tick-borne viruses in Shanghai, China. Front Microbiol, 16(1699705). https://doi.org/10.3389/fmicb.2025.1699705 DOI: https://doi.org/10.3389/fmicb.2025.1699705
License
Copyright (c) 2026 Prasetyarti Utami, Rony Marsyal Kunda
This work is licensed under a Creative Commons Attribution 4.0 International License.
Authors who publish with Jurnal Penelitian Pendidikan IPA, agree to the following terms:
- Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution 4.0 International License (CC-BY License). This license allows authors to use all articles, data sets, graphics, and appendices in data mining applications, search engines, web sites, blogs, and other platforms by providing an appropriate reference. The journal allows the author(s) to hold the copyright without restrictions and will retain publishing rights without restrictions.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgment of its initial publication in Jurnal Penelitian Pendidikan IPA.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).
