UDC 636.2:619:579.881.31:577.21:579.083.1

doi: 10.15389/agrobiology.2015.6.825eng

Anaplasma marginale

S.N. Koval’chuk1, G.Yu. Kosovskii1, A.V. Arkhipov1, T.T. Glazko1,2,
V.I. Glazko1,2

1Center for Experimental Embryology and Reproductive Biotechnology, Federal Agency of Scientific Organizations,
12/4, ul. Kostyakova, Moscow, 127422 Russia,
e-mail s.n.kovalchuk@mail.ru, gkosovsky@mail.ru, batler51@yandex.ru,
tglazko@rambler.ru, vglazko@yahoo.com, info-ceerb@mail.ru 
2K.A. Timiryazev Russian State Agrarian University—Moscow Agrarian Academy,
49, ul. Timiryazevskaya, Moscow, 127550 Russia

Received August 14, 2015

Anaplasma marginale is a rickettsial pathogen responsible for bovine anaplosmosis, the acute disease in cattle herds which is associated with anemia, fever, rapid loss of milk production and weight, abortion, and, in some cases, death of the infected cattle. Anaplasma marginale is transmitted by ticks and biting insects. Diagnosis of bovine anaplasmosis is made by microscopic examination of blood smears stained with Giemsa stain, but this method is not useful to detect presymptomatic animals. Several serological tests have used extensively for epidemiological studies, but they do not discriminate between different Anaplasma species. A real-time polymerase chain reaction (PCR) combines high specificity with accurate measurement of DNA copy number and allows quantification of the targeted pathogen DNA. The goal of this study was to develop a real-time PCR assay for differential detection of A. marginale in the blood of cattle. The single-copy gene msp4 was chosen as a target DNA for PCR. Msp4 is a dominant immune protein of outer membrane of all Anaplasma  knowen to date. The primers for phylogenetic analysis in А. marginale based on msp4 were reported earlier by J. de la Fuente et al. (2001), but they were not species-specific. The analysis of msp4 gene sequence of different A. marginale isolates and closely related species, including A. ovis, revealed species-specific areas, which were used for design of primers and TaqMan probe (MSP4-F 5′-CA-TGAGTCACGAAGTGGCT-3′ and MSP4-R 5′-GGCACACT-CACATCAATC-3′, MSP4-probe 5′-(Cy5)-AAGGGGGAGTAATGGGAGGTAGCT-3′) for amplification and detection of 177 bp fragment of msp4 gene by a real time PCR. In the amplified nucleotide sequences a 99 to 100 % homology to msp4 fragments was found in different isolates of A. marginale. To assess analytical sensitivity of our PCR test, we used pGEM-msp4, a constructed recombinant plasmid with 177 bp fragment of msp4 gene, diluted to obtain samples with 100-107 msp4 copies. It was shown that the assay was able to detect as few as 102 of A. marginale msp4 gene in the analyzed DNA sample. Analytical specificity of the developed primers and the MSP4-probe was proved in tests with DNA of sheep naturally infected by A. ovis,and also DNA isolated from cows with Sanguibacter keddieii, Propionibacterium acnes and Pseudomonas aeruginosa infection pre-detected by sequencing. In these samples no increased fluorescence characteristic of probes from animals infected by A. marginale was observed with no PCR products identified. Thus, the method specificity allowed to differ A. marginale and A. ovis. The developed method of A. marginale identification on the basis on amplification and detection of the msp4 gene fragment using a real time PCR differed from known analogues with high sensitivity, rapidity and opportunity of quantitative evaluation of the bacterial load.  The developed method could be used for rapid differential detection and quantification of А. marginale in blood samples from infected cattle for confirmation of anaplasmosis and epidemiological studies.

Keywords: Anaplasma marginale, msp4 gene, cattle, diagnostics, a real-time PCR.


Full article (Rus)

Full text (Eng)



  1. Kocan K.M., de la Fuente J., Blouin E.F., Coetzee J.F., Ewing S.A. The natural history of Anaplasma marginale. Vet. Parasitol., 2010, 167: 95-107 CrossRef
  2. Kocan K.M., de la Fuente J., Blouin E.F., Garcia-Garcia J.C. Anaplasma marginale (Rickettsiales: Anaplasmataceae): recent advances in defining host-pathogen adaptations of a tick-borne rickettsia. Parasitology, 2004, 129: 285-300 CrossRef
  3. Scoles G.A., Broce A.B., Lysyk T.J., Palmer G.H. Relative efficiency of biological transmission of Anaplasma marginale (Rickettsiales: Anaplasmataceae) by Dermacentor andersoni (Acari: Ixodidae) compared with mechanical transmission by Stomoxys calcitrans (Diptera: Muscidae). J. Med. Entomol., 2005, 42: 668-675 CrossRef
  4. Guglielmone A.A. Epidemiology of babesiosis and anaplasmosis in South and Central America. Vet. Parasitol., 1995, 57: 109-119.
  5. de la Fuente J., Lew A., Lutz H., Meli M.L., Hofmann-Lehmann R., Shkap V., Molad T. Genetic diversity of Anaplasma species major surface proteins and implications for anaplasmosis serodiagnosis and vaccine development. Anim. Health Res., 2005, 6: 75-89 CrossRef
  6. Gulyukin M.I., Zablotskii V.T., Belimenko V.V. Rossiiskii veterinarnyi zhurnal SKHZH, 2013, 2: 36-40.
  7. Noaman V., Shayan P. Comparison of Microscopy and PCR-RFLP for detection of Anaplasma marginale in carrier cattle. Iran. J. Microbiol., 2010, 2(2): 89-94.
  8. Potgieter F.T., Stoltsz W.H. Anaplasmosis. In: Infectious diseases of livestock with special reference to Southern Africa /J.A.W. Coetzer, G.R. Thompson, R.C. Tustin (eds.). Oxford University Press, Cape Town, South Africa, 1994.
  9. Dreher U.M., Fuente J., Hofmann-Lehmann R., Meli M.L., Pusterla N., Kocan K.M., Woldehiwet Z., Braun U., Regula G., Staerk K.D., Lutz H. Serologic crossreactivity between Anaplasma marginale and Anaplasma phagocytophilum. Clin. Diagn. Lab. Immunol., 2005, 12: 1177-1183 CrossRef
  10. Al-Adhami B., Scandrett W.B., Lovanov V.A., Gajadhar A.A. Serological cross reactivity between Anaplasma marginale and Ehrlichia species in naturally and experimentally infected cattle. J. Vet. Diagn. Invest., 2011, 23: 1181-1188 CrossRef
  11. Strik N.I., Alleman A.R., Barbet A.F., Sorenson H.L., Wamsley H.L., Gaschen F.P., Luckschander N., Wong S., Chu F., Foley J.E., Bjoersdorff A., Stuen S., Knowles D.P. Characterization of Anaplasma phagocytophilum major surface protein 5 and the extent of its cross-reactivity with A. marginale. Clin. Vaccine Immunol., 2007, 14: 262-268 CrossRef
  12. Torioni de Echaide S., Knowles D.P., McGuire T., Palmer G.H., Suarez C.E., McElwain T.F. Detection of cattle naturally infected with Anaplasma marginale in a region of endemicity by nested PCR and a competitive Enzyme-Linked Immunosorbent Assay using recombinant major surface protein 5. J. Clin. Microbiol., 1998, 36: 777-782.
  13. de la Fuente J., Van Den Bussche R.A., Kocan K.M. Molecular phylogeny and biogeography of North American isolates of Anaplasma marginale (Rickettsiaceae: Ehrlichieae). Vet. Parasitol., 2001, 97: 65-76 CrossRef
  14. Carelli G., Decaro N., Lorusso A., Elia G., Lorusso E., Mari V., Ceci L., Buonavoglia C. Detection and quantification of Anaplasma marginale DNA in blood samples of cattle by real-time PCR. Vet. Microbiol., 2007, 124: 107-114 CrossRef
  15. Fyumagwa R.D., Simmler P., Meli M.L., Hoare R., Hofmann-Lehmann R., Lutz H. Prevalence of Anaplasma marginale in different tick species from Ngorongoro Crater, Tanzania. Vet. Parasitol., 2009, 161(1-2): 154-157 CrossRef
  16. Picoloto G., Lima R.F., Olegário L.A.O., Carvalho C.M.E., Lacerda A.C.R., Tomás W.M., Borges P.A.L., Pellegrin A.O., Madruga C.R. Real-time polymerase chain reaction to diagnose Anaplasma marginale in cattle and deer (Ozotoceros bezoarticus leucogaster) of the Brazilian Pantanal. Brazilian Journal of Veterinary Parasitology, 2010, 19(3): 186-188 CrossRef
  17. Torina A., Agnone A., Blanda V., Alongi A., D’Agostino R., Caracappa S., Marino A.M., Di Marco V., de la Fuente J. Development and validation of two PCR tests for the detection of and differentiation between Anaplasma ovis and Anaplasma marginale. Ticks and Tick-borne Diseases, 2012, 3(5-6): 283-287 CrossRef
  18. Reinbold J.B., Coetzee J.F., Sirigireddy K.R., Ganta R.R. Detection of Anaplasma marginale and A. phagocytophilum in bovine peripheral blood samples by duplex real-time reverse transcriptase PCR assay. J. Clin. Microbiol., 2010, 48(7): 2424-2432 CrossRef
  19. Molad T., Mazuz M.L., Fleiderovitz L., Fish L., Savitsky I., Krigel Y., Leibovitz B., Molloy J., Jongejan F., Shkap V. Molecular and serological detection of A. centrale- and A. marginale-infected cattle grazing within an endemic area. Vet. Microbiol., 2006, 113: 55-62 CrossRef
  20. Bilgiç H.B., Karagenç T., Simuunza M., Shiels B., Tait A., Eren H., Weir W. Development of multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale incattle. Exp. Parasitol., 2013, 133(2): 222-229 CrossRef
  21. Lew A.E., Bock R.E., Minchin C.M., Masaka S. Amsp1alpha polymerase chain reaction assay for specific detection and differentiation of Anaplasma marginale isolates. Vet. Microbiol., 2002, 86: 325-333 CrossRef
  22. Ybañez A.P., Sivakumar T., Ybañez R.H.D., Ratilla J.C., Perez Z.O., Gabotero S.R., Hakimi H., Kawazu S., Matsumoto K., Yokoyama N., Ino-kuma H. First molecular characterization of Anaplasma marginale in cattle and Rhipicephalus (Boophilus) microplus ticks in Cebu, Philippines. J. Vet. Med. Sci., 2013, 75: 27-36 (doi: 10.1292/jvms.12-0268).
  23. Vidotto M.C., McGuire T.C., McElwain T.F., Palmer G.H., Knowles D.P. Jr. Intermolecular relationships of major surface proteins of Anaplasma marginale. Infect. Immunol., 1994, 62(7): 2940-2946.
  24. Brayton K.A., Kappmeyer L.S., Herndon D.R., Dark M.J., Tibbals D.L., Palmer G.H., McGuire T.C., Knowles D.P. Jr. Complete genome sequencing of Anaplasma marginale reveals that the surface is skewed to two superfamilies of outer membrane proteins. PNAS USA, 2005, 102(3): 844-849 CrossRef
  25. Glazko V.I., Kosovskii G.Yu., Koval'chuk S.N., Arkhipov A.V., Petrova I.O., Dedovich G.O., Glazko T.T. Innovatsionnye tekhnologii v meditsine, 2014, 2(03): 63-79.