Serological and molecular surveys of Anaplasma spp. in Egyptian cattle reveal high A. marginale infection prevalence

Document Type : Full paper (Original article)


1 Department of Animal Medicine (Infectious Diseases), Faculty of Veterinary Medicine, Benha University, Toukh 13736, Egypt

2 Department of Animal Wealth Development, Faculty of Veterinary Medicine, Benha University, Toukh 13736, Egypt

3 Department of Parasitology and Animal Diseases, National Research Center, Dokki, Giza, Egypt

4 Higher Institute of Biotechnology of Sidi Thabet, University of Manouba, Manouba, Tunisia, and Laboratory of Microbiology, National School of Veterinary Medicine of Sidi Thabet, University of Manouba, Manouba, Tunisia

5 Department of Pathobiology, Faculty of Veterinary Science, Bu-Ali Sina University, Hamedan, Iran, and Zoonotic Diseases Research Center, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran


Background: Bovine anaplasmosis is an infectious disease with worldwide distribution. It spreads by various routes mainly through tick bites. Aims: This study aimed to investigate bovine related Anaplasma spp. in cattle from three northern governorates of Egypt by serological and molecular assays, to evaluate the associated risk factors and to analyze the phylogeny of revealed A. marginale isolates. Methods: During 2020, a total of 650 blood samples were collected from asymptomatic cattle in the governorates of Kafr El-Sheikh (n=240), Menofia (n=230), and Al-Gharbia (n=180). Sera samples were examined using the Anaplasma antibody test kit, cELISA v2. Blood genomic DNA of seropositive cattle was then examined by PCRs specific to A. marginale, A. centrale, and A. bovis. Selected positive samples were subjected to nucleotide sequencing. Risk factors (i.e. geographical area, breed, type of production, sex, age, herd size, season, husbandry system, tick infestation, and application of acaricides) were evaluated by logistic regression approach. Results: In total, 130 cattle (20%, 95% CI: 17.1–23.3) were recorded seropositive for Anaplasma species. Major risk factors associated with seropositivity were being crossbred, dairy cattle, aged more than 5 years, summer season, herd size of below 300, pasture grazing, tick infestation, and not being subjected to regular treatment with acaricides. By using species-specific PCR, only A. marginale was detected. Nucleotide sequencing showed the occurrence of two different msp4 genotypes. Conclusion: This study shows the high prevalence of A. marginale in cattle of Kafr El-Sheikh, Al-Gharbia, and Menofia. However, the connection between Anaplasma species and their tick vectors remains unknown in Egypt and merits further investigations. Since these infections primarily spread through ixodid tick bites, effective ectoparasite control strategies, regular examination of cattle and successful chemoprophylaxis are recommended.


Main Subjects

Abdel-Shafy, S; Allam, N and Mahmoud, M (2016). Molecular description of Anaplasma biodiversity regarding 16SrDNA, msp4, hsp60, and rpoB profiles in ixodid ticks infesting animals from some Egyptian Provinces. Bull. Natl. Res. Cent., 41: 121-136.
Ait Hamou, S; Rahali, T; Sahibi, H; Belghyti, D; Losson, B; Goff, W and Rhalem, A (2012). Molecular and serological prevalence of Anaplasma marginale in cattle of North Central Morocco. Res. Vet. Sci., 93: 1318-1323.
Alanazi, AD; Nguyen, VL; Alyousif, MS; Manoj, RR; Alouffi, AS; Ridolfi, D; Sazmand, A; Mendoza-Roldan, JA; Dantas-Torres, F and Otranto, D (2020). Ticks and associated pathogens in camels (Camelus dromedarius) from Riyadh Province, Saudi Arabia. Parasit. Vectors. 13: 110.
Alderink, FJ and Dietrich, R (1983). Economic and epidemiological implications of anaplasmosis in Texas beef cattle herds. Bulletin of Texas Agricultural Experiment Station, Number 1426. PP: 66-75.
Alekseev, AN; Dubinina, HV; Semenov, AV and Bolshakov, CV (2001). Evidence of ehrlichiosis agents found in ticks (Acari: Ixodidae) collected from migratory birds. J. Med. Entomol., 38: 471-474.
AL-Hosary, A; Răileanu, C; Tauchmann, O; Fischer, S; Nijhof, AM and Silaghi, C (2020). Epidemiology and genotyping of Anaplasma marginale and co-infection with piroplasms and other Anaplasmataceae in cattle and buffaloes from Egypt. Parasit. Vectors. 13: 495.
AL-Hosary, A; Răileanu, C; Tauchmann, O; Fischer, S; Nijhof, AM and Silaghi, C (2021). Tick species identification and molecular detection of tick-borne pathogens in blood and ticks collected from cattle in Egypt. Ticks Tick Borne Dis., 12: 101676.
Altschul, SF; Madden, TL; Schäffer, AA; Zhang, J; Zhang, Z; Miller, W and Lipman, DJ (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res., 25: 3389-3402.
Aubry, P and Geale, DW (2011). A review of bovine anaplasmosis. Transbound. Emerg. Dis., 58: 1-30.
Battilani, M; De Arcangeli, S; Balboni, A and Dondi, F (2017). Genetic diversity and molecular epidemiology of Anaplasma. Infect. Genet. Evol., 49: 195-211.
Belkahia, H; Said, MB; Alberti, A; Abd, K; Issaoui, Z; Hattab, D; Gharbi, M and Messadi, L (2015). First molecular survey and novel genetic variants’ identification of Anaplasma marginale, A. centrale and A. bovis in cattle from Tunisia. Infect. Genet. Evol., 34: 361-371.
Ben Said, M; Asker, AB; Belkahia, H; Ghribi, R; Selmi, R and Messadi, L (2018a). Genetic characterization of Anaplasma marginale strains from Tunisia using single and multiple gene typing reveals novel variants with an extensive genetic diversity. Ticks Tick Borne Dis., 9: 1275-1285.
Ben Said, M; Belkahia, H; Alberti, A; Zobba, R; Bousrih, M; Yahiaoui, M; Daaloul-Jedidi, M; Mamlouk, A; Gharbi, M and Messadi, L (2015). Molecular survey of Anaplasma species in small ruminants reveals the presence of novel strains closely related to A. phagocytophilum in Tunisia. Vector Borne Zoonotic Dis., 15: 580-590.
Ben Said, M; Belkahia, H; El Mabrouk, N; Saidani, M; Alberti, A; Zobba, R; Cherif, A; Mahjoub, T; Bouattour, A and Messadi, L (2017a). Anaplasma platys-like strains in ruminants from Tunisia. Infect. Genet. Evol., 49: 226-233.
Ben Said, M; Belkahia, H; El Mabrouk, N; Saidani, M; Hassen, MB; Alberti, A; Zobba, R; Bouattour, S; Bouattour, A and Messadi, L (2017b). Molecular typing and diagnosis of Anaplasma spp. closely related to Anaplasma phagocytophilum in ruminants from Tunisia. Ticks Tick borne Dis., 8: 412-422.
Ben Said, M; Belkahia, H and Messadi, L (2018b). Anaplasma spp. in North Africa: a review on molecular epidemiology, associated risk factors and genetic characteristics. Ticks Tick Borne Dis., 9: 543-555.
Ben Said, M; Belkahia, H; Selmi, R and Messadi, L (2019). Computational selection of minimum length groESL operon required for Anaplasma species attribution and strain diversity analysis. Mol. Cell. Probes., 48: 101467.
Bezerra-Santos, MA; Sgroi, G; Mendoza-Roldan, JA; Khedri, J; Camarda, A; Iatta, R; Sazmand, A and Otranto, D (2021). Ectoparasites of hedgehogs: From flea mite phoresy to their role as vectors of pathogens. Int. J. Parasitol. Parasites Wildl., 15: 95-104.
Chung, C; Wilson, C; Bandaranayaka-Mudiyanselage, CB; Kang, E; Adams, DS; Kappmeyer, LS; Knowles, DP; McElwain, TF; Evermann, JF and Ueti, MW (2014). Improved diagnostic performance of a commercial Anaplasma antibody competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5–glutathione S-transferase fusion protein as antigen. J. Vet. Diagn. Investig., 26: 61-71.
Dantas-Torres, F and Otranto, D (2017). Anaplasmosis. In: Marcondes, CB (Ed.), Arthropod borne diseases. Springer, Cham, Switzerland. PP: 215-222.
de la Fuente, J; Thomas, EJG; Van Den Bussche, RA; Hamilton, RG; Tanaka, EE; Druhan, SE and Kocan, KM (2003). Characterization of Anaplasma marginale isolated from North American bison. Appl. Environ. Microbiol., 69: 5001-5005.
de la Fuente, J; Vicente, JN; Höfle, U; Ruiz-Fons, F; De Mera, IGF; Van Den Bussche, RA; Kocan, KM and Gortazar, C (2004). Anaplasma infection in free-ranging Iberian red deer in the region of Castilla-La Mancha, Spain. Vet. Microbiol., 100: 163-173.
Dreher, U; de la Fuente, J; Hofmann-Lehmann, R; Meli, ML; Pusterla, N; Kocan, K; Woldehiwet, Z; Braun, U; Regula, G and Staerk, K (2005). Serologic cross-reactivity between Anaplasma marginale and Anaplasma phagocytophilum. Clin. Diagn. Lab. Immunol., 12: 1177-1183.
El-Ashker, M; Hotzel, H; Gwida, M; El-Beskawy, M; Silaghi, C and Tomaso, H (2015). Molecular biological identification of Babesia, Theileria, and Anaplasma species in cattle in Egypt using PCR assays, gene sequence analysis and a novel DNA microarray. Vet. Parasitol., 207: 329-334.
El-Dakhly, KM; Arafa, WM; Soliman, S; Abdel-Fatah, OR; Wahba, AA; Esteve-Gasent, MD and Holman, PJ (2020). Molecular detection, phylogenetic analysis, and genetic diversity of Theileria annulata, Babesia bigemina, and Anaplasma marginale in cattle in three districts of Egypt. Acta Parasitol., 65: 620-627.
Elhariri, MD; Elhelw, RA; Hamza, DA and Soliman, DE (2017). Molecular detection of Anaplasma marginale in the Egyptian water buffaloes (Bubalus bubalis) based on major surface protein 1α. J. Egypt Soc. Parasitol., 47: 247-252.
El-Naga, T and Barghash, S (2016). Blood parasites in camels (Camelus dromedarius) in Northern West Coast of Egypt. J. Bacteriol. Parasitol., 7: 1000258.
Felsenstein, J (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 39: 783-791.
Fereig, RM; Mohamed, SG; Mahmoud, HY; AbouLaila, MR; Guswanto, A; Nguyen, TT; Mohamed, AEA; Inoue, N; Igarashi, I and Nishikawa, Y (2017). Seroprevalence of Babesia bovis, B. bigemina, Trypanosoma evansi, and Anaplasma marginale antibodies in cattle in southern Egypt. Ticks Tick Borne Dis., 8: 125-131.
Hall, TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Sym. Ser., 41: 95-98.
Hildebrandt, A; Franke, J; Meier, F; Sachse, S; Dorn, W and Straube, E (2010). The potential role of migratory birds in transmission cycles of Babesia spp., Anaplasma phagocytophilum, and Rickettsia spp. Ticks Tick Borne Dis., 1: 105-107.
Jongejan, F and Uilenberg, G (2004). The global importance of ticks. Parasitology. 129: S3-S14.
Kang, JG; Kim, HC; Choi, CY; Nam, HY; Chae, HY; Chong, ST; Klein, TA; Ko, S and Chae, JS (2013). Molecular detection of Anaplasma, Bartonella, and Borrelia species in ticks collected from migratory birds from Hong-do Island, Republic of Korea. Vector Borne Zoonotic Dis., 13: 215-225.
Kawahara, M; Rikihisa, Y; Lin, Q; Isogai, E; Tahara, K; Itagaki, A; Hiramitsu, Y and Tajima, T (2006). Novel genetic variants of Anaplasma phagocytophilum, Anaplasma bovis, Anaplasma centrale, and a novel Ehrlichia sp. in wild deer and ticks on two major islands in Japan. Appl. Environm. Microbiol., 72: 1102-1109.
Khodadadi, N; Nabavi, R; Sarani, A; Saadati, D; Ganjali, M; Mihalca, AD; Otranto, D and Sazmand, A (2021). Identification of Anaplasma marginale in long-eared hedgehogs (Hemiechinus auritus) and their Rhipicephalus turanicus ticks in Iran. Ticks Tick Borne Dis., 12: 101641.
Kocan, KM; de la Fuente, J; Blouin, EF; Coetzee, JF and Ewing, S (2010). The natural history of Anaplasma marginale. Vet. Parasitol., 167: 95-107.
Kocan, KM; de la Fuente, J and Cabezas-Cruz, A (2015). The genus Anaplasma: new challenges after reclassifica-tion. Rev. Sci. Tech., 34: 577-586.
Lew-Tabor, A and Valle, MR (2016). A review of reverse vaccinology approaches for the development of vaccines against ticks and tick borne diseases. Ticks Tick Borne Dis., 7: 573-585.
Loftis, AD; Reeves, WK; Szumlas, DE; Abbassy, MM; Helmy, IM; Moriarity, JR and Dasch, GA (2006). Rickettsial agents in Egyptian ticks collected from domestic animals. Exp. Appl. Acarol., 40: 67-81.
M’ghirbi, Y; Bèji, M; Oporto, B; Khrouf, F; Hurtado, A and Bouattour, A (2016). Anaplasma marginale and A. phagocytophilum in cattle in Tunisia. Parasit. Vectors. 9: 556.
Nasreldin, N; Ewida, RM; Hamdon, H and Elnaker, YF (2020). Molecular diagnosis and biochemical studies of tick-borne diseases (anaplasmosis and babesiosis) in Aberdeen Angus Cattle in New Valley, Egypt. Vet. World. 13: 1884.
Noaman, V; Nabinejad, A; Shahmoradi, A and Esmaeilkhanian, S (2016). Molecular detection of bovine leukocytic Anaplasma species in Isfahan, Iran. Res. Mol. Med., 4: 47-51.
Noaman, V and Shayan, P (2010). Molecular detection of Anaplasma bovis in cattle from central part of Iran. Vet. Res. Forum., 1: 117-122.
Ogden, NH; Lindsay, LR; Hanincová, K; Barker, IK; Bigras-Poulin, M; Charron, DF; Heagy, A; Francis, CM; O’Callaghan, CJ and Schwartz, I (2008). Role of migratory birds in introduction and range expansion of Ixodes scapularis ticks and of Borrelia burgdorferi and Anaplasma phagocytophilum in Canada. Appl. Environ. Microbiol., 74: 1780-1790.
OIE, World Organization for Animal Health (2018). Bovine anaplasmosis manual of diagnostic tests and vaccines for terrestrial animals. OIE, Paris, France. fileadmin/Home/eng/Health_standards/tahm/3.04.01_BOVINE_ANAPLASMOSIS.pdf (accessed 28 December 2021).
Parvizi, O; El-Adawy, H; Melzer, F; Roesler, U; Neubauer, H and Mertens-Scholz, K (2020). Seroprevalence and molecular detection of bovine anaplasmosis in Egypt. Pathogens. 9: 64.
Radwan, M; Abdel Fatah, A and El Hamied, O (2013). Epidemiological studies and molecular diagnosis of Anaplasma marginale in cattle and biochemical changes associated with it in Kaliobia Governorate. Am. J. Infec. Dis. Microbiol., 1: 46-49.
Rjeibi, MR; Ayadi, O; Rekik, M and Gharbi, M (2018). Molecular survey and genetic characterization of Anaplasma centrale, A. marginale and A. bovis in cattle from Algeria. Transbound. Emerg. Dis., 65: 456-464.
Saitou, N and Nei, M (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol., 4: 406-425.
Salih, DA; Abdel Rahman, MB; Mohammed, AS; Ahmed, R; Kamal, S and El Hussein, AM (2009). Seroprevalence of tick-borne diseases among cattle in the Sudan. Parasitol. Res., 104: 845-850.
Salih, DA; Hassan, S; Julla, I; Kyule, M; Zessin, KH and El Hussein, A (2008). Distribution and application of ELISA for the seroprevalence of tick-borne diseases in Central Equatoria State, Sudan. Transbound. Emerg Dis., 55: 257-262.
Salm, F; Younis, E; Hegazy, N and El-Sawalhy, A (2011). Epidemiological studies on bovine anaplasmosis. Bull. Anim. Health Prod. Afr., 59: 179-189.
Sazmand, A; Alipoor, G; Zafari, S; Zolhavarieh, SM; Alanazi, AD and Sargison, ND (2020). Assessment of knowledge, attitudes and practices relating to parasitic diseases and anthelmintic resistance among livestock farmers in Hamedan, Iran. Front. Vet. Sci., 7: 584323.
Sazmand, A; Eigner, B; Mirzaei, M; Hekmatimoghaddam, S; Harl, J; Duscher, GG; Fuehrer, HP and Joachim, A (2016). Molecular identification of hemoprotozoan parasites in camels (Camelus dromedarius) of Iran. Iran J. Parasitol., 11: 568-573.
Sazmand, A; Harl, J; Eigner, B; Hodžić, A; Beck, R; Hekmatimoghaddam, S; Mirzaei, M; Fuehrer, HP and Joachim, A (2019). Vector-borne bacteria in blood of camels in Iran: New data and literature review. Comp. Immunol. Microbiol. Infect. Dis., 65: 48-53.
Selim, A; Alanazi, AD; Sazmand, A and Otranto, D (2021). Seroprevalence and associated risk factors for vector-borne pathogens in dogs from Egypt. Parasit. Vectors. 14: 175.
Selmi, R; Ben Said, M; Dhibi, M; Ben Yahia, H; Abdelaali, H and Messadi, L (2020). Genetic diversity of groEL and msp4 sequences of Anaplasma ovis infecting camels from Tunisia. Parasitol. Int., 74: 101980.
Selmi, R; Said, MB; Dhibi, M; Yahia, HB and Messadi, L (2019). Improving specific detection and updating phylogenetic data related to Anaplasma platys-like strains infecting camels (Camelus dromedarius) and their ticks. Ticks Tick Borne Dis., 10: 101260.
Sharifiyazdi, H; Jafari, S; Ghane, M; Nazifi, S and Sanati, A (2017). Molecular investigation of Anaplasma and Ehrlichia natural infections in the dromedary camel (Camelus dromedarius) in Iran. Comp. Clin. Pathol., 26: 99-103.
Tamura, K and Nei, M (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol., 10: 512-526.
Thrusfield, M; Christley, R; Brown, H; Diggle, PJ; French, N; Howe, K; et al. (2018). Surveys. In: Thrusfield, M; Christley, R; Brown, H; Diggle, PJ; French, N; Howe, K; et al. (Eds.), Veterinary epidemiology. (4th Edn.), West Sussex: John Wiley & Sons Ltd., PP: 270–295.
Torina, A; Agnone, A; Blanda, V; Alongi, A; D’Agostino, R; Caracappa, S; Marino, AM; Di Marco, V and de la
Fuente, J
(2012). Development and validation of two PCR tests for the detection of and differentiation between Anaplasma ovis and Anaplasma marginale. Ticks Tick Borne Dis., 3: 283-287.
Tumwebaze, MA; Lee, SH; Moumouni, PFA; Mohammed-Geba, K; Sheir, SK; Galal-Khallaf, A; Abd El Latif, HM; Morsi, DS; Bishr, NM and Galon, EM (2020). First detection of Anaplasma ovis in sheep and Anaplasma platys-like variants from cattle in Menoufia governorate, Egypt. Parasitol. Int., 78: 102150.
Uilenberg, G (1995). International collaborative research: significance of tick-borne hemoparasitic diseases to world animal health. Vet. Parasitol., 57: 19-41.
Younis, E; Hegazy, N; El-Deeb, W and El-Khatib, R (2009). Epidemiological and biochemical studies on bovine anaplasmosis in Dakahlia and Demiatta governorates in Egypt. Bull. Anim. Health Prod. Afri., 57, doi: 10.4314/bahpa.v57i4.51668.
Ziam, H and Benaouf, H (2004). Prevalence of blood parasites in cattle from wilayates of Annaba and El Tarf east Algeria. Arch. Inst. Pasteur Tunis., 81: 27-30.
Zobba, R; Anfossi, AG; Pinna Parpaglia, ML; Dore, GM; 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.