Remil Linggatong GALAY, Kristina Andrea Claveria SANDALO, Angelito Pangan DELA CRUZ, Tetsuya TANAKA, Flor Marie Immanuelle Rafallo PILAPIL AMANTE

Molecular detection of Anaplasma spp. in blood and milk of dairy cattle in the Philippines

Anaplasmosis has become a major concern in the cattle industry throughout the world due to its great economic impact.The causative agents, Anaplasma species, are primarily transmitted by ticks, occurring intracellularly within blood cells, with somespecies being zoonotic. In this study, the presence of Anaplasma spp. was investigated in the blood and milk of dairy cattle in thePhilippines. Blood and milk samples were collected from 98 dairy cattle from selected farms in five provinces in the southern part ofLuzon Island in the Philippines. After DNA extraction, a conventional PCR for the control gene actin was performed, followed by nestedPCR for Anaplasma spp. Selected amplicons were purified and subjected to sequence analysis. It was found that 97 (98.97%) bloodsamples and 6 (6.12%) milk samples were positive for Anaplasma. Sequence analysis revealed that the positive amplicons from milksamples and their corresponding blood samples shared a high identity (98%–100%) with reported Anaplasma marginale isolates. To theauthors’ knowledge, this study provides the first molecular evidence of the presence of A. marginale in milk from dairy cattle under fieldconditions in the Philippines.

PDF

___

1. National Dairy Authority Central Office. Reports of Accomplishments First Semester 2018. Quezon City, the Philippines: National Dairy Authority; 2018.
2. Aubry P, Geale DW. A review of bovine anaplasmosis. Transboundary and Emerging Diseases 2011; 58: 1-30.
3. Reinbold JB, Coetzee JF, Sirigireddy KR, Ganta RR. Detection of Anaplasma marginale and A. phagocytophilum in bovine peripheral blood samples by duplex real-time reverse transcriptase PCR assay. Journal of Clinical Microbiology 2010; 48: 2424-2432.
4. Kocan KM, de la Fuente J, Blouin EF, Coetzee JF, Ewing SA. The natural history of Anaplasma marginale. Veterinary Parasitology 2010; 167: 95-107.
5. Atif FA. Anaplasma marginale and Anaplasma phagocytophilum: Rickettsiales pathogens of veterinary and public health significance. Parasitology Research 2015; 114: 3941-3957.
6. Correa WM, Correa CN, Gottschalk AF. Bovine abortion associated with Anaplasma marginale. Canadian Journal of Comparative Medicine 1978; 42: 227-228.
7. Ybañez AP, Mingala CN, Ybañez RHD. Historical review and insights on the livestock tick-borne disease research of a developing country: the Philippine scenario. Parasitology International 2018; 67: 262-266.
8. de Echaide ST, Bono MF, Lugaresi C, Aguirre N, Mangold A et al. Detection of antibodies against Anaplasma marginale in milk using a recombinant MSP5 indirect ELISA. Veterinary Microbiology 2005; 106: 287-292.
9. Pusterla N, Huder J, Wolfensberger C, Braun U, Lutz H. Laboratory findings in cows after experimental infection with Ehrlichia phagocytophila. Clinical and Diagnostic Laboratory Immunology 1997; 4: 643-647.
10. Chang YF, Novosel V, Dubovi E, Wong SJ, Chu FK et al. Experimental infection of the human granulocytic ehrlichiosis agent in horses. Veterinary Parasitology 1998; 78: 137-145.
11. Granquist EG, Aleksandersen M, Bergström K, Dumler SJ, Torsteinbø WO et al. A morphological and molecular study of Anaplasma phagocytophilum transmission events at the time of Ixodes ricinus tick bite. Acta Veterinaria Scandinavica 2010; 52: 43.
12. Berzina I, Krudewig C, Silaghi C, Matise I, Ranka R et al. Anaplasma phagocytophilum DNA amplified from lesional skin of seropositive dogs. Ticks and Tick-borne Diseases 2014; 5: 329- 335.
13. Zhang Y, Lu Y, Cui Y, Wang J, Cao S et al. First molecular evidence for the presence of Anaplasma DNA in milk from sheep and goats in China. Parasitology Research 2016; 115: 2789-2795.
14. Haguingan JB, Gordoncillo MN, Bautista JN, Hikiba M, Sarmago IG. Detection and identification of bacterial carriage of subclinical mastitis cases in backyard dairy cows. Philippine Journal of Veterinary Medicine 2010; 47: 21-25.
15. Ybañez AP, Sivakumar T, Ybañez RHD, Ratilla JC, Perez ZO et al. First molecular characterization of Anaplasma marginale in cattle and Rhipicephalus (Boophilus) microplus ticks in Cebu, Philippines. Journal of Veterinary Medical Science 2013; 75: 27-36.
16. Ochirkhuu N, Konnai S, Mingala CN, Okagawa T, Villanueva M et al. Molecular epidemiological survey and genetic analysis of vector-borne infections of cattle in Luzon Island, the Philippines. Veterinary Parasitology 2015; 212: 161-167.
17. Ybañez AP, Ybañez RHD, Claveria FG, Cruz-Flores MJ, Xuenan X et al. High genetic diversity of Anaplasma marginale detected from Philippine cattle. Journal of Veterinary Medical Science 2014; 76: 1009-1014.
18. Aquino AJB, Divina BP, Bombio AM, Pilapil FMIR. Detection of Anaplasma marginale infection in a dairy cattle farm by stained blood smear examination and nested polymerase chain reaction. Philippine Journal of Veterinary and Animal Science 2018; 44: 68-75.
19. Philippine Statistics Authority. Performance of Philippine Agriculture October-December 2018. Quezon City, the Philippines: Philippine Statistics Authority; 2018.
20. Ybañez AP, Sivakumar T, Ybañez RHD, Vincoy MRB, Tingson JA et al. Molecular survey of bovine vector-borne pathogens in Cebu, Philippines. Veterinary Parasitology 2013; 196: 13-20.
21. Paramanandham K, Mohankumar A, Puttahonnappa Suresh K, Susan Jacob S, Roy P. Prevalence of Anaplasma species in India and the World in dairy animals: A systematic review and meta-analysis. Research in Veterinary Science 2019; 123: 159- 170.
22. George LW, Divers TJ, Ducharme N, Welcome FL. Diseases of the teats and udder. In: Divers TJ, Peek SF (editors). Rebhun’s Diseases of Dairy Cattle. 2nd ed. St. Louis, MO, USA: Saunders- Elsevier; 2008. p. 335.
23. Griffiths MW. The microbiological safety of raw milk. In: Griffiths MW (editor). Improving the Safety and Quality of Milk. Cambridge, UK: Woodhead Publishing Limited; 2010. p. 28.
24. Burton JL, Erskine RJ. Immunity and mastitis some new ideas for an old disease. Veterinary Clinics of North America Food Animal Practice 2003; 19: 1-45.
25. Kobayashi K, Oyama S, Numata A, Rahman MM, Kumura H. Lipopolysaccharide disrupts the milk-blood barrier by modulating claudins in mammary alveolar tight junctions. PLoS One 2013; 8: e62187.