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Research (Published online: 20-12-2016)

20. Multiple infections of Anaplasma platys variants in Philippine dogs - Adrian Patalinghug Yba˝ez, Rochelle Haidee Daclan Yba˝ez, Naoaki Yokoyama and Hisashi Inokuma

Veterinary World, 9(12): 1456-1460



   doi: 10.14202/vetworld.2016.1456-1460


Adrian Patalinghug Yba˝ez: Department of Biology and Environmental Studies, College of Sciences, University of the Philippines Cebu, Lahug, Cebu City 6000, Philippines; Department of Research, Gullas College of Medicine, University of the Visayas, Banilad, Mandaue City 6014, Cebu, Philippines; Department of Veterinary Clinical Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Inada Cho, Hokkaido 080-8555 Japan;

Rochelle Haidee Daclan Yba˝ez: Department of Biology and Environmental Studies, College of Sciences, University of the Philippines Cebu, Lahug, Cebu City 6000, Philippines;

Naoaki Yokoyama: Research Unit for Molecular Diagnosis, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Inada Cho, Hokkaido 080-8555, Japan;

Hisashi Inokuma: Department of Veterinary Clinical Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Inada Cho, Hokkaido 080-8555 Japan;


Received: 25-07-2016, Accepted: 23-11-2016, Published online: 20-12-2016


Corresponding author: Adrian Patalinghug Yba˝ez, e-mail:

Citation: Yba˝ez AP, Yba˝ez RHD, Yokoyama N, Inokuma H (2016) Multiple infections of Anaplasma platys variants in Philippine dogs, Veterinary World, 9(12): 1456-1460.

Aim: Anaplasma platys, the causative agent of infectious canine cyclic thrombocytopenia, is a tick-borne pathogen that also has been implicated as potentially zoonotic. To provide molecular evidence on the multiple infections of A. platys variants in Philippine dogs.

Materials and Methods: DNA fragments of A. platys from infected dogs in the Philippines were molecularly characterized. For screening, 25 dogs suspected to have canine anaplasmosis were tested using a 16S rRNA-based nested polymerase chain reaction (PCR). Infection was confirmed by sequencing of positive amplicons. Second round PCR targeting a longer 16S rRNA fragment was subsequently performed on the first round PCR amplicons of the positive samples. Further characterization using the heat-shock operon (groEL) gene was also performed on the A. platys-positive samples.

Results: 10 16S rRNA sequences were obtained and found 99.6-100% identical to each other and 99.6-99.7% identical to the closest registered A. platys sequences. On the other hand, 36 groEL clone sequences were obtained and found to be 85.1-99.8% identical with each other and 85.0-88.9% identical to the closest previously registered A. platys sequences. Four dogs were found coinfected with 2-3 groEL variant sequences. Phylogenetic analyses suggest that the detected A. platys in the Philippines may represent unique variants.

Conclusion: A. platys variants were detected in Philippine dogs. Coinfection of different A. platys variants in dogs was also demonstrated. The present study may indicate the potential genetic diversity of A. platys in the country.

Keywords: Anaplasma platys, dogs, Philippines, variants.

1. Dumler, J.S., Barbet, A.F., Bekker, C.P.J., Dasch, G.A., Palmer, G.H., Ray, S.C., Rikihisa, Y. and Rurangirwa, F.R. (2001) Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: Unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila. Int. J. Syst. Evol. Microbiol., 51: 2145-2165.
2. Maggi, R.G., Mascarelli, P.E., Havenga, L.N., Naidoo, V. and Breitschwerdt, E.B. (2013) Co-infection with Anaplasma platys, Bartonella henselae and Candidatus Mycoplasma haematoparvum in a veterinarian. Parasit. Vectors, 6: 103.
PMid:23587235 PMCid:PMC3637287
3. Breitschwerdt, E.B., Hegarty, B.C., Qurollo, B.A., Saito, T.B., Maggi, R.G., Blanton, L.S. and Bouyer, D.H. (2014) Intravascular persistence of Anaplasma platys, Ehrlichia chaffeensis, and Ehrlichia ewingii DNA in the blood of a dog and two family members. Parasit. Vectors, 1: 298.
PMid:24984562 PMCid:PMC4089936
4. Arraga-Alvarado, C.M., Qurollo, B.A., Parra, O.C., Berrueta, M.A., Hegarty, B.C. and Breitschwerdt, E.B. (2014) Molecular evidence of Anaplasma platys infection in two women from Venezuela. Am. J. Trop. Med. Hyg., 91: 1161-1165.
PMid:25266347 PMCid:PMC4257640
5. Yba-ez, A.P., Perez, Z.O., Gabotero, S.R., Yandug, R.T., Matsumoto, K. and Inokuma, H. (2012) First molecular detection of Ehrlichia canis and Anaplasma platys in ticks from Dogs in Cebu, Philippines. Ticks Tick Borne Dis., 3: 288-293.
6. Ybanez, A.P. (2014) First molecular evidence of Ehrlichia canis infection in dogs with probable disease relapse in the Philippines. J. Adv. Vet. Res., 4: 184-188.
7. Suksawat, J., Xuejie, Y., Hancock, S.I., Hegarty, B.C., Nilkumhang, P. and Breitschwerdt, E.B. (2001) Serologic and molecular evidence of coinfection with multiple vector-borne pathogens in dogs from Thailand. J. Vet. Intern. Med., 15: 453-462.
8. Wen, B., Cao, W. and Pan, H. (2003) Ehrlichiae and Ehrlichial diseases in china. Ann. N. Y. Acad. Sci., 990: 45-53.
9. Kim, C.M., Yi, Y.H., Yu, D.H., Lee, M.J., Cho, M.R., Desai, A.R., Shringi, S., Klein, T.A., Kim, H.C., Song, J.W., Baek, L.J., Chong, S.T., O`Guinn, M.L., Lee, J.S., Lee, I.Y., Park, J.H., Foley, J. and Chae, J.S. (2006) Tick-borne Rickettsial pathogens in ticks and small mammals in Korea. Appl. Environ. Microbiol., 72: 5766-5776.
PMid:16957192 PMCid:PMC1563606
10. Inokuma, H., Beppu, T., Okuda, M., Shimada, Y. and Sakata, Y. (2003) Epidemiological survey of Anaplasma platys and Ehrlichia canis using ticks collected from Dogs in Japan. Vet. Parasitol., 115: 343-348.
11. Chang, A.C., Chang, W.L., Lin, C.T., Pan, M.J. and Lee, S.C. (1996) Canine infectious cyclic thrombocytopenia found in Taiwan. J. Vet. Med. Sci., 58: 473-476.
12. Haschke-Becher, E., Bernauer, R., Walleczek, A.M., Apfalter, P., Afazel-Saeedi, S., Kraus, J. and Strasser, P. (2010) First detection of the Anaplasma phagocytophilum groEL-A genotype in man. J. Infect., 60: 300-305.
13. Ybanez, A. (2013) First report on Anaplasma platys infection in a dog in the Philippines. Iran. J. Vet. Med., 7: 227-231.
14. Harrus, S., Alleman, A.R., Bark, H., Mahan, S.M. and Waner, T. (2002) Comparison of three enzyme-linked immunosorbent assays with the indirect immunofluorescent antibody test for the diagnosis of canine infection with Ehrlichia canis. Vet. Microbiol., 86: 361-368.
15. Yba-ez, A.P., Sivakumar, T., Yba-ez, R.H., Vincoy, M.R., Tingson, J.A., Perez, Z.O., Gabotero, S.R., Buchorno, LP., Inoue, N., Matsumoto, K., Inokuma, H. and Yokoyama, N. (2013) Molecular survey of bovine vector-borne pathogens in Cebu, Philippines. Vet. Parasitol., 196: 13-20.
16. Ybanez, A.P., Sashika, M. and Inokuma, H. (2014) The phylogenetic position of Anaplasma bovis and inferences on the phylogeny of the Genus Anaplasma. J. Vet. Med. Sci., 76(2): 307.
PMid:24189581 PMCid:PMC3982816
17. Chae, J.S., Foley, J.E., Dumler, J.S. and Madigan, J.E. (2000) Comparison of the nucleotide sequence of 16S rRNA, 444 Ep-ank, and groESL heat shock operon genes in naturally occurring Ehrlichia equi and human granulocytic ehrlichiosis agent isolates from northern California. J. Clin. Microbiol., 38: 1364-1369.
PMid:10747108 PMCid:PMC86446
18. Yba-ez, A.P., Matsumoto, K., Kishimoto, T. and Inokuma, H. (2012) Molecular analyses of a potentially novel Anaplasma species closely related to Anaplasma phagocytophilum detected in sika deer (Cervus nippon yesoensis) in Japan. Vet. Microbiol., 157: 232-236.
19. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011) MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol., 28: 2731-2739.
20. Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., H÷hna, S., Larget, B., Liu, L., Suchard, M.A. and Huelsenbeck, J.P. (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol., 61: 539-542.
21. Gaunt, S., Beall, M., Stillman, B., Lorentzen, L., Diniz, P., Chandrashekar, R. and Breitschwerdt, E. (2010) Experimental infection and co-infection of dogs with Anaplasma platys and Ehrlichia canis: Hematologic, serologic and molecular findings. Parasit. Vectors, 3. DOI: 10.1186/1756-3305-3-33.
22. Zobba, R., Anfossi, A.G., Parpaglia, M.L.P., Dore, G.M., Chessa, B., Spezzigu, A., Rocca, S., Visco, S., Pittau, M. and Alberti, A. (2014) Molecular investigation and phylogeny of Anaplasma spp. in Mediterranean ruminants reveal the presence of neutrophil-tropic strains closely related to A. platys. Appl. Environ. Microbiol., 80: 271-280.
PMid:24162569 PMCid:PMC3911010
23. Zobba, R., Anfossi, A.G., Visco, S., Sotgiu, F., Dedola, C., Parpaglia, M.L.P., Battilani, M., Pittau, M. and Alberti, A. (2015) Cell tropism and molecular epidemiology of Anaplasma platys-like strains in cats. Ticks Tick Borne Dis., 6: 272-280.
24. Yushan, H., Lei, L., Weijia, L. and Xiaoguang, C. (2010) Sequence analysis of the groEL gene and its potential application in identification of pathogenic bacteria. Afr. J. Microbiol. Res., 4: 1733-1741.
25. Bown, K.J., Lambin, X., Ogden, N.H., Petrovec, M., Shaw, S.E., Woldehiwet, Z. and Birtles, R.J. (2007) High-resolution genetic fingerprinting of European strains of Anaplasma phagocytophilum by use of multilocus variable-number tandem-repeat analysis. J. Clin. Microbiol., 45: 1771-1776.
PMid:17442796 PMCid:PMC1933113
26. Rar, V.A., Epikhina, T.I., Livanova, N.N., Panov, V.V., Doroschenko, E.K., Pukhovskaya, N.M. and Ivanov, L.I. (2011) Genetic variability of Anaplasma phagocytophilum in Ixodes persulcatus ticks and small mammals in the Asian part of Russia. Vector Borne Zoonotic Dis., 11: 1013-1021.
27. Yabsley, M.J., Murphy, S.M., Luttrell, M.P., Wilcox, B.R., Howerth, E.W. and Munderloh, U.G. (2008) Characterization of 'Candidatus Neoehrlichia lotoris' (family Anaplasmataceae) from raccoons (Procyon lotor). Int. J. Syst. Evol. Microbiol., 58: 2794-2798.
PMid:19060060 PMCid:PMC4278589
28. Inokuma, H., Fujii, K., Okuda, M., Onishi, T., Beaufils, J.P., Raoult, D. and 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. Clin. Diagn. Lab. Immunol., 9: 1132-1136.
29. Yba-ez, A.P., Yba-ez, R.H.D., Yokoyama, N. and Inokuma, H. (2015) RNA polymerase sub-unit β (rpoB) characterization of Ehrlichia canis detected from dogs and Rhipicephalus sanguineus ticks in Cebu, Philippines. Philipp. Vet. Arch., 85: 601-608.
30. Yba-ez, A.P., Yba-ez, R.H.D., Villavelez, R.R., Malingin, H.P.F., Barrameda, D.N.M., Naquila, S.V. and Olimpos, S.M.B. (2016) Retrospective analyses of dogs found serologically positive for Ehrlichia canis in Cebu, Philippines from 2003 to 2014. Vet. World, 9: 43.
PMid:27051183 PMCid:PMC4819348