PERTANIKA JOURNAL OF TROPICAL AGRICULTURAL SCIENCE

Molecular methods coupled with phylogenetic analysis are sensitive tools for detecting and classifying parasites. This study used nuclear and mitochondrial gene markers to investigate the host-vector interaction of the vector-borne haemoparasites. The population genetic structures of important vector-borne haemoparasites in dogs, namely, Anaplasma platys, Ehrlichia canis, Babesia vogeli, and Babesia gibsoni, were determined from the nuclear gene of 16S or 18S rRNA gene, gltA and groESL and mitochondrial gene of COX1 across dogs and vector ticks. A total of 220 blood samples and 140 ticks were collected from shelter dogs in Peninsular Malaysia. Out of the positive samples for the vector-borne haemoparasites, 28 positive blood isolates and six tick isolates were selected and characterised. There was a low diversity in tick sequences, while varying degree of variability was observed in dogs’ sequences. Overlapped haplotypes were observed in sequences of dogs and ticks, revealing the possibility of the same infection origin. No regional separation was detected, but similar haplotypes from different regions were observed. These findings contribute to the epidemiology of vector-borne haemoparasites in dogs in Malaysia.

• Abd-Rani, P. A. M., Irwin, P. J., Coleman, G. T., Gatne, M., & Traub, R. J. (2011). A survey of canine tick-borne diseases in India. Parasites and Vectors, 4, 141. https://doi.org/10.1186/1756-3305-4-141

• Adao, D. E. V., Herrera, C. M. T., Galarion, L. H., Bolo, N. R., Carlos, R. S., Carlos, E. T., Carlos, S. S., & Rivera, W. L. (2017). Detection and molecular characterization of Hepatozoon canis, Babesia vogeli, Ehrlichia canis, and Anaplasma platys in dogs from Metro Manila, Philippines. Korean Journal of Veterinary Research, 57(2), 79-88. https://doi.org/10.14405/kjvr.2017.57.2.79

• Aguiar, D. M., Hagiwara, M. K., & Labruna, M. B. (2008). In vitro isolation and molecular characterization of an Ehrlichia canis strain from São Paulo, Brazil. Brazilian Journal of Microbiology, 39, 489-493. https://doi.org/10.1590/S1517-83822008000300014

• Aguirre, E., Sainz, A., Dunner, S., Amusategui, I., López, L., Rodríguez-Franco, F., Luaces, I., Cortés, O., & Tesouro, M. A. (2004). First isolation and molecular characterization of Ehrlichia canis in Spain. Veterinary Parasitology, 125(3-4), 365-372. https://doi.org/10.1016/j.vetpar.2004.08.007

• Aktas, M., Altay, K., Dumanli, N., & Kalkan, A. (2009). Molecular detection and identification of Ehrlichia and Anaplasma species in ixodid ticks. Parasitology Research, 104, 1243–1248. https://doi.org/10.1007/s00436-009-1377-1

• Alhassan, A., Hove, P., Sharma, B., Matthew-Belmar, V., Karasek, I., Lanza-Perea, M., Werners, A. H., Wilkerson, M. J., & Ganta, R. R. (2021). Molecular detection and characterization of Anaplasma platys and Ehrlichia canis in dogs from the Caribbean. Ticks and Tick-borne Diseases, 12(4), 101727. https://doi.org/10.1016/j.ttbdis.2021.101727

• Andersson, M. O., Tolf, C., Tamba, P., Stefanache, M., Waldenström, J., Dobler, G., & Chitimia-Dobler, L. (2017). Canine tick-borne diseases in pet dogs from Romania. Parasites and Vectors, 10, 155. https://doi.org/10.1186/s13071-017-2092-x

• Araya-Anchetta, A., Busch, J. D., Scoles, G. A., & Wagner, D. M. (2015). Thirty years of tick population genetics: A comprehensive review. Infection, Genetics, and Evolution, 29, 164–179. https://doi.org/10.1016/j.meegid.2014.11.008

• Beall, M. J., Chandrashekar, R., Eberts, M. D., Cyr, K. E., Diniz, P. P. V., Mainville, C., Hegarty, B. C., Crawford, J. M., & Breitschwerdt, E. B. (2008). Serological and molecular prevalence of Borrelia burgdorferi, Anaplasma phagocytophilum, and Ehrlichia species in dogs from Minnesota. Vector-Borne and Zoonotic Diseases, 8(4), 455-464. https://doi.org/10.1089/vbz.2007.0236

• Bilgic, H. B., Karagenç, T., Shiels, B., Tait, A., Eren, H., & Weir, W. (2010). Evaluation of cytochrome b as a sensitive target for PCR based detection of T. annulate carrier animals. Veterinary Parasitology, 174(3-4), 341–347. https://doi.org/10.1016/j.vetpar.2010.08.025

• Blouin, M. S. (2002). Molecular prospecting for cryptic species of nematodes: Mitochondrial DNA versus internal transcribed spacer. International Journal for Parasitology, 32(5), 527–531. https://doi.org/10.1016/S0020-7519(01)00357-5

• Cao, J., Yang, Q., Zhang, J., Zhou, Y., Zhang, H., Gong, H., & Zhou, J. (2015). Seroprevalence survey of Babesia gibsoni infection and tick species in dogs in east China. Veterinary Parasitology, 214(1-2), 12-15. https://doi.org/10.1016/j.vetpar.2015.10.002

• Çelik, B. A., Ayan, A., Yilmaz, A. B., Çelik, Ö. Y., Kilinç, Ö. O., & Ayan, Ö. O. (2021). Molecular identification of Ehrlichia canis in Rhipicephalus sanguineus ticks from Siirt province. Acta Veterinaria, 71(3), 247-255. https://doi.org/10.2478/acve-2021-0022

• Chao, L. L., Yeh, S. T., Hsieh, C. K., & Shih, C. M. (2016). First detection and molecular identification of Babesia vogeli from Rhipicephalus sanguineus (Acari: Ixodidae) in Taiwan. Experimental and Applied Acarology, 68, 539-551. https://doi.org/10.1007/s10493-015-0010-5

• Clare, E. L. (2011). Cryptic species? Patterns of maternal and paternal gene flow in eight neotropical bats. PLOS One, 6(7), e21460. https://doi.org/10.1371/journal.pone.0021460

• Conrad, P., Thomford, J., Yamane, I., Whiting, J., Bosma, L., Uno, T., Holshuh, H. J., & Shelly, S. (1991). Hemolytic anemia caused by Babesia gibsoni infection in dogs. Journal of the American Veterinary Medical Association, 199(5), 601-605.

• Criado-Fornelio, A., Gonzalez-del-Rio, M. A., Buling-Sarana, A., & Barba-Carretero, J. C. (2003). Molecular characterization of a Babesia gibsoni isolate from a Spanish dog. Veterinary Parasitology, 117(1-2), 123-129. https://doi.org/10.1016/j.vetpar.2003.08.006

• Dantas-Torres, F. (2010). Biology and ecology of the brown dog tick, Rhipicephalus sanguineus. Parasites and Vectors, 3, 26. https://doi.org/10.1186/1756-3305-3-26

• De la Fuente, J., Torina, A., Caracappa, S., Tumino, G., Furlá, R., Almazán, C., & Kocan, K. M. (2005). Serologic and molecular characterization of Anaplasma species infection in farm animals and ticks from Sicily. Veterinary Parasitology, 133(4), 357-362. https://doi.org/10.1016/j.vetpar.2005.05.063

• De la Fuente, J., Torina, A., Naranjo, V., Nicosia, S., Alongi, A., La Mantia, F., & Kocan, K. M. (2006). Molecular characterization of Anaplasma platys strains from dogs in Sicily, Italy. BMC Veterinary Research, 2, 24. http://www.biomedcentral.com/1746-6148/2/24

• Excoffier, L., & Lischer, H. E. L. (2010). Arlequin suite ver 3.5: A new series of programs to perform population genetics analyses under Linux and Windows. Molecular Ecology Resources, 10(3), 564-567. https://doi.org/10.1111/j.1755-0998.2010.02847.x

• Foongladda, S., Inthawong, D., Kositanont, U., & Gaywee, J. (2011). Rickettsia, Ehrlichia, Anaplasma, and Bartonella in ticks and fleas from dogs and cats in Bangkok. Vector-Borne and Zoonotic Diseases, 11(10), 1335-1341. https://doi.org/10.1089/vbz.2010.0174

• Galay, R. L., Manalo, A. A. L., Dolores, S. L. D., Aguilar, I. P. M., Sandalo, K. A. C., Cruz, K. B., Divina, B. P., Andoh, M., Masatani, T., & Tanaka, T. (2018). Molecular detection of tick-borne pathogens in canine population and Rhipicephalus sanguineus (sensu lato) ticks from southern Metro Manila and Laguna, Philippines. Parasites and Vectors, 11, 643. https://doi.org/10.1186/s13071-018-3192-y

• Haanshuus, C. G., Mohn, S. C., Mørch, K., Langeland, N., Blomberg, B., & Hanevik, K. (2013). A novel, single-amplification PCR targeting mitochondrial genome highly sensitive and specific in diagnosing malaria among returned travelers in Bergen, Norway. Malaria Journal, 12, 26. http://www.malariajournal.com/content/12/1/26

• Halos, L., Jamal, T., Vial, L., Maillard, R., Suau, A., LeMenach, A., Boulouis, H. J., Vayssier-Taussat, M. (2004). Determination of an efficient and reliable method for DNA extraction from ticks. Veterinary Research, 35, 709–713. https://doi.org/10.1051/vetres:2004038

• Harrus, S., & Waner, T. (2011). Diagnosis of canine monocytotropic ehrlichiosis (Ehrlichia canis): An overview. Veterinary Journal, 187(3), 292-296. https://doi.org/10.1016/j.tvjl.2010.02.001

• Hill, C. A., & Gutierrez, J. A. (2003). A method for extraction and analysis of high quality genomic DNA from ixodid ticks. Medical and Veterinary Entomology, 17(2), 224–227. https://doi.org/10.1046/j.1365-2915.2003.00425.x

• Hsieh, Y. C., Lee, C. C., Tsang, C. L., & Chung, Y. T. (2010). Detection and characterization of four novel genotypes of Ehrlichia canis from dogs. Veterinary Microbiology, 146(1-2), 70-75. https://doi.org/10.1016/j.vetmic.2010.04.013

• Hu, M., & Gasser, R. B. (2006). Mitochondrial genomes of parasitic nematodes - Progress and perspectives. Trends in Parasitology, 22(2), 78-84. https://doi.org/10.1016/j.pt.2005.12.003

• Huang, C.-C., Hsieh, Y.-C., Tsang, C.-L., & Chung, Y.-T. (2010). Sequence and phylogenetic analysis of the gp200 protein of Ehrlichia canis from dogs in Taiwan. Journal of Veterinary Science, 11(4), 333-340. https://doi.org/10.4142/jvs.2010.11.4.333

• Huang, H., Unver, A., Perez, M. J., Orellana, N. G., & Rikihisa, Y. (2005). Prevalence and molecular analysis of Anaplasma platys in dogs in Lara, Venezuela. Brazilian Journal of Microbiology, 36, 211-216. https://doi.org/10.1590/S1517-83822005000300002

• Hubbard, M. J., Cann, K. J., & Wright, D. J. (1995). Validation and rapid extraction of nucleic acids from alcohol-preserved ticks. Experimental and Applied Acarology, 19(8), 473–478. https://doi.org/10.1007/bf00048266

• Inokuma, H., Brouqui, P., Drancourt, M., & Raoult, D. (2001). Citrate synthase gene sequence: A new tool for phylogenetic analysis and identification of Ehrlichia. Journal of Clinical Microbiology, 39(9), 3031-3039. https://doi.org/10.1128/JCM.39.9.3031-3039.2001

• Inokuma, H., Fujii, K., Okuda, M., Onishi, T., Beaufils, J. P., Raoult, D., & Brouqui, P. (2002). Determination of the nucleotide sequences of heat shock operon groESL and the citrate synthase gene (gltA) of Anaplasma (Ehrlichia) platys for phylogenetic and diagnostic studies. Clinical and Vaccine Immunology, 9(5), 1132-1136. https://doi.org/10.1128/CDLI.9.5.1132-1136.2002

• Inokuma, H., Raoult, D., & Brouqui, P. (2000). Detection of Ehrlichia platys DNA in brown dog ticks (Rhipicephalus sanguineus) in Okinawa Island, Japan. Journal of Clinical Microbiology, 38(11), 4219-4221. https://doi.org/10.1128/JCM.38.11.4219-4221.2000

• Ionita, M., Mitrea, I. L., Pfister, K., Hamel, D., Buzatu, C. M., & Silaghi, C. (2012). Canine babesiosis in Romania due to Babesia canis and Babesia vogeli: A molecular approach. Parasitology Research, 110, 1659-1664. https://doi.org/10.1007/s00436-011-2683-y

• Isozumi, R., Fukui, M., Kaneko, A., Chan, C. W., Kawamoto, F., & Kimura, M. (2015). Improved detection of malaria cases in island settings of Vanuatu and Kenya by PCR that targets the Plasmodium mitochondrial cytochrome c oxidase III (cox3) gene. Parasitology International, 64(3), 304–308. https://doi.org/10.1016/j.parint.2014.09.006

• Jabbar, A., Jex, A. R., Mohandas, N., Hall, R. S., Littlewood, D. T. J., & Gasser, R. B. (2013). The mitochondrial genome of Aelurostrongylus abstrusus - Diagnostic, epidemiological and systematic implications. Gene, 516(2), 294–300. https://doi.org/10.1016/j.gene.2012.10.072

• Jennett, A. L., Smith, F. D., & Wall, R. (2013). Tick infestation risk for dogs in a peri-urban park. Parasite and Vector, 6, 358. https://doi.org/10.1186/1756-3305-6-358

• Jex, A. R., Hall, R. S., Littlewood, D. T. J., & Gasser, R. B. (2010). An integrated pipeline for next-generation sequencing and annotation of mitochondrial genomes. Nucleic Acids Research, 38(2), 522–533. https://doi.org/10.1093/nar/gkp883

• Kamani, J., Baneth, G., Mumcuoglu, K. Y., Waziri, N. E., Eyal, O., Guthmann, Y., & Harrus, S. (2013). Molecular detection and characterization of tick-borne pathogens in dogs and ticks from Nigeria. PLOS Neglected Tropical Diseases, 7(3), e2108. https://doi.org/10.1371/journal.pntd.0002108

• Kamani, J., Lee, C. C., Haruna, A. M., Chung, P. J., Weka, P. R., & Chung, Y. T. (2013). First detection and molecular characterization of Ehrlichia canis from dogs in Nigeria. Research in Veterinary Science, 94(1), 27-32. https://doi.org/10.1016/j.rvsc.2012.07.031

• Kjemtrup, A. M., Wainwright, K., Miller, M., Penzhorn, B. L., & Carreno, R. A. (2006). Babesia conradae, sp. Nov., a small canine Babesia identified in California. Veterinary Parasitology, 138(1-2), 103-111. https://doi.org/10.1016/j.vetpar.2006.01.044

• Koh, F. X., Panchadcharam, C., & Tay, S. T. (2016). Vector-borne diseases in stray dogs in peninsular Malaysia and molecular detection of Anaplasma and Ehrlichia spp. from Rhipicephalus sanguineus (Acari: Ixodidae) ticks. Journal of Medical Entomology, 53(1), 183-187. https://doi.org/10.1093/jme/tjv153

• Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution, 35(6), 1547–1549. https://doi.org/10.1093/molbev/msy096

• Livanova, N. N., Fomenko, N. V., Akimov, I. A., Ivanov, M. J., Tikunova, N. V., Armstrong, R., & Konyaev, S. V. (2018). Dog survey in Russian veterinary hospitals: Tick identification and molecular detection of tick-borne pathogens. Parasites and Vectors, 11, 591. https://doi.org/10.1186/s13071-018-3161-5

• Low, V. L., Prakash, B. K., Lim, Y. A. L., Tan, T. K., Vinnie-Siow, W. Y., Sofian-Azirun, M., & AbuBakar, S. (2018). Detection of Anaplasmataceae agents and co-infection with other tick-borne protozoa in dogs and Rhipicephalus sanguineus sensu lato ticks. Experimental and Applied Acarology, 75, 429-453. https://doi.org/10.1007/s10493-018-0280-9

• Low, V. L., Prakash, B. K., Tan, T. K., Sofian-Azirun, M., Anwar, F. H. K., Vinnie-Siow, W. Y., & AbuBakar, S. (2017). Pathogens in ectoparasites from free-ranging animals: Infection with Rickettsia asembonensis in ticks, and a potentially new species of Dipylidium in fleas and lice. Veterinary Parasitology, 245, 102–105. https://doi.org/10.1016/j.vetpar.2017.08.015

• Lv, S., Zhang, Y., Zhang, L. Liu, Q., Liu, H. X., Hu, L., Wei, F. R., Steinmann, P., Graeff-Teixeira, C., Zhou, X. N., & Utzinger, J. (2012). The complete mitochondrial genome of the rodent intra-arterial nematodes Angiostrongylus cantonensis and Angiostrongylus costaricensis. Parasitology Research, 111, 115–123. https://doi.org/10.1007/s00436-011-2807-4

• Malik M. I., Qamar M., Ain Q., Hussain M. F., Dahmani M., Ayaz M., Mahmood A. K., Davoust B., Shaikh R. S., & Iqbal F. (2018). Molecular detection of Ehrlichia canis in dogs from three districts in Punjab (Pakistan). Veterinary Medicine and Science, 4(2), 126-132. https://doi.org/10.1002/vms3.94

• Martin, A. R., Brown, G. K., Dunstan, R. H., & Roberts, T. K. (2005). Anaplasma platys: An improved PCR for its detection in dogs. Experimental Parasitology, 109(3), 176-180. https://doi.org/10.1016/j.exppara.2004.11.007

• Martin, A. R., Dunstan, R. H., Roberts, T. K., & Brown, G. K. (2006). Babesia canis vogeli: A novel PCR for its detection in dogs in Australia. Experimental Parasitology, 112(1), 63–65. https://doi.org/10.1016/j.exppara.2005.09.001

• Martins, F. M., Templeton, A. R., Pavan, A. C. O., Kohlback, B. C., & Morgante, J. S. (2009). Phylogeography of the common vampire bat (Desmodus rotundus): Marked population structure, Neotropical Pleistocene vicariance and incongruence between nuclear and mtDNA markers. BMC Evolutionary Biology, 9, 294. http://www.biomedcentral.com/1471-2148/9/294

• Mokhtar, A. S., Lim, S. F., & Tay, S. T. (2013). Molecular detection of Anaplasma platys and Babesia gibsoni in dogs in Malaysia. Tropical Biomedicine, 30(2), 345-348.

• Moraes, P. H. G., Rufino, C. P., Baraúna, A. R. F., Reis, T., Agnol, L. T. D., Meneses, A. M. C., Aguiar, D. C. F., Nunes, M. R. T., & Gonçalves, E. C. (2015). Molecular characterization of Babesia vogeli in dogs from Belém, northern Brazil. Genetics and Molecular Research, 14(4), 16364-16371. https://doi.org/10.4238/2015.December.9.4

• Morgan, E. R., & Van-Dijk, J. (2012). Climate and the epidemiology of gastrointestinal nematode infections of sheep in Europe. Veterinary Parasitology, 189(1), 8-14. https://doi.org/10.1016/j.vetpar.2012.03.028

• Nazari, M., Lim, S. Y., Watanabe, M., Sharma, R. S. K., Cheng, N. A. B. Y., & Watanabe, M. (2013). Molecular detection of Ehrlichia canis in dogs in Malaysia. PLOS Neglected Tropical Diseases, 7(1), e1982. https://doi.org/10.1371/journal.pntd.0001982

• Nguyen, V. L., Colella, V., Iatta, R., Bui, K. L., Dantas-Torres, F., & Otranto, D. (2019). Ticks and associated pathogens from dogs in northern Vietnam. Parasitology Research, 118, 139-142. https://link.springer.com/article/10.1007/s00436-018-6138-6

• Otranto, D., & Wall, R. (2008). New strategies for the control of arthropod vectors of disease in dogs and cats. Medical and Veterinary Entomology, 22(4), 291-302. https://doi.org/10.1111/j.1365-2915.2008.00741

• Otranto, D., Dantas-Torres, F., & Breitschwerdt, E. B. (2009). Managing canine vector-borne diseases of zoonotic concern: Part one. Trends in Parasitology, 25(4), 157-163. https://doi.org/10.1016/j.pt.2009.01.003

• Pinyoowong, D., Jittapalapong, S., Suksawat, F., Stich, R. W., & Thamchaipenet, A. (2008). Molecular characterization of Thai Ehrlichia canis and Anaplasma platys strains detected in dogs. Infection, Genetics and Evolution, 8(4), 433-438. https://doi.org/10.1016/j.meegid.2007.06.002

• Prakash, B. K., Low, V. L., Tan, T. K., Vinnie-Siow, W. Y., Lim, Y. A. L., Morvarid, A. R., Azman, A. S., Yeong, Y. S., AbuBakar, S., & Sofian-Azirun, M. (2018). Detection of Hepatozoon canis in the brown dog tick and domestic dogs in Peninsular Malaysia. Journal of Medical Entomology, 55(5), 1346-1348. https://doi.org/10.1093/jme/tjy081

• Prakash, B. K., Low, V. L., Vinnie-Siow, W. Y., Tan, T. K., Lim, Y. A. L., Movarid, A. R., Abu-Bakar, S., & Sofian-Azirun, M. (2018). Detection of Babesia spp. in dogs and their ticks from Peninsular Malaysia: Emphasis on Babesia gibsoni and Babesia vogeli infections in Rhipicephalus sanguineus sensu lato (Acari: Ixodidae). Journal of Medical Entomology, 55(5), 1337-1340. https://doi.org/10.1093/jme/tjy072

• Qurollo, B. A., Archer, N. R., Schreeg, M. E., Marr, H. S., Birkenheuer, A. J., Haney, K. N., Thomas, B. S., & Breitschwerdt, E. B. (2017). Improved molecular detection of Babesia infections in animals using a novel quantitative real-time PCR diagnostic assay targeting mitochondrial DNA. Parasites and Vectors, 10, 128. https://doi.org/10.1186/s13071-017-2064-1

• Selim A., Abdelhady A., & Alahadeb J. (2021). Prevalence and first molecular characterization of Ehrlichia canis in Egyptian dogs. Pakistan Veterinary Journal, 41(1), 117-121. https://doi.org/10.29261/pakvetj/2020.061

• Sipin, Q., Kamal, F. M., Watanabe, M., Rani, P. A. M. A., Low, V. L., & Aziz, N. A. A. (2020). Molecular detection of tick-borne haemopathogens in shelter dogs and Rhipicephalus sanguineus (sensu lato) ticks from Peninsular Malaysia. Comparative Immunology, Microbiology and Infectious Diseases, 73, 101563. https://doi.org/10.1016/j.cimid.2020.101563

• Šlapeta, J., Chandra, S., & Halliday, B. (2021). The “tropical lineage” of the brown dog tick Rhipicephalus sanguineus sensu lato identified as Rhipicephalus linnaei (Audouin, 1826). International Journal for Parasitology, 51(6), 431-436. https://doi.org/10.1016/j.ijpara.2021.02.001

• Teacher, A. G. F., & Griffiths, D. J. (2011). HapStar: Automated haplotype network layout and visualisation. Molecular Ecology Resources, 11(1), 151-153. https://doi.org/10.1111/j.1755-0998.2010.02890.x

• Tian, Z., Luo, J., Zheng, J., Xie, J., Shen, H., Yin, H., Luo, J., Tian, M., Yuan, X., Wang, F., & Liu, G. (2013). Phylogenetic analysis of Babesia species in China based on cytochrome b (COB) gene. Infection, Genetic and Evolution, 13, 36-40. https://doi.org/10.1016/j.meegid.2012.09.001

• Unver, A., Rikihisa, Y., Kawahara, M., & Yamamoto, S. (2003). Analysis of 16S rRNA gene sequences of Ehrlichia canis, Anaplasma platys, and Wolbachia species from canine blood in Japan. Annals of the New York Academy of Sciences, 990(1), 692-698. https://doi.org/10.1111/j.1749-6632.2003.tb07445.x

• Vargas-Hernandez, G., Andre, M. R., Faria, J. L. M., Munhoz, T. D., Hernandez-Rodriguez, M., Machado, R. Z., & Tinucci-Costa, M. (2012). Molecular and serological detection of Ehrlichia canis and Babesia vogeli in dogs in Colombia. Veterinary Parasitology, 186(3), 254-260. https://doi.org/10.1016/j.vetpar.2011.11.011

• Wen, B., Rikihisa, Y., Mott, J. M., Greene, R., Kim, H. Y., Zhi, N., Couto, G. C., Unver, A., & Bartsch, R. (1997). Comparison of nested PCR with immunofluorescent antibody assay for detection of Ehrlichia canis infection in dogs treated with doxycycline. Journal of Clinical Microbiology, 35(7), 1852-1855. https://doi.org/10.1128/jcm.35.7.1852-1855.1997

• Wiegarg, G., & Remington, S. J. (1986). Citrate synthase: Structure, control, and mechanism. Annual Review of Biophysics and Biophysical Chemistry, 15, 97-117. https://doi.org/10.1146/annurev.bb.15.060186.000525

• Xia, X. (2018). DAMBE7: New and improved tools for data analysis in molecular biology and evolution. Molecular Biology and Evolution, 35(6), 1550–1552. https://doi.org/10.1093/molbev/msy073

• Yabsley, M. J., McKibben, J., Macpherson, C. N., Cattan, P. F., Cherry, N. A., Hegarty, B. C., Breitschwerdt, E. B., O’Connor, T., Chandrashekar, R., Paterson, T., Perea, M. L., Ball, G., Friesen, S., Goedde, J., Henderson, B., & Sylvester, W. (2008). Prevalence of Ehrlichia canis, Anaplasma platys, Babesia canis vogeli, Hepatozoon canis, Bartonella vinsonii berkhoffii, and Rickettsia spp. in dogs from Grenada. Veterinary Parasitology, 151(2-4), 279-285. https://doi.org/10.1016/j.vetpar.2007.11.008

• Ybañez, A. P., Perez, Z. O., Gabotero, S. R., Yandug, R. T., Kotaro, M., & Inokuma, H. (2012). First molecular detection of Ehrlichia canis and Anaplasma platys in ticks from dogs in Cebu, Philippines. Ticks and Tick-borne Diseases, 3(5-6), 288-293. https://doi.org/10.1016/j.ttbdis.2012.10.032

• Yin, F., Gasser, R. B., Li, F., Ba0, M., Huang, W., Zou, F., Zhao, G., Wang, C., Yang, X., Zhou, Y., Zhao, J., Fang, R., & Hu, M. (2013). Genetic variability within and among Haemonchus contortus isolates from goats and sheep in China. Parasites and Vectors, 6, 279. https://doi.org/10.1186/1756-3305-6-279

• Yu, X. J., McBride, J. W., & Walker, D. H. (2007). Restriction and expansion of Ehrlichia strain diversity. Veterinary Parasitology, 143(3-4), 337–346. https://doi.org/10.1016/j.vetpar.2006.08.027

• Zhang, X., Luo, T., Keysary, A., Baneth, G., Miyashiro, S., Strenger, C., Waner, T., & McBride, J. W. (2008). Genetic and antigenic diversities of major immunoreactive proteins in globally distributed Ehrlichia canis strains. Clinical and Vaccine Immunology, 15(7), 1080-1088. https://doi.org/10.1128/CVI.00482-07