Original Article

Establishment and Validation of Theileria annulata Sporozoite Ak-93 Infection in Laboratory-Reared Hyalomma anatolicum Tick Using In Vivo and In Vitro Assays


Background: Tropical Theileriosis caused by Theileria annulata is a tick-borne disease which transmitted by the ix­odid tick members of the genus Hyalomma. Studies on different aspects of disease require to access infective sporozoite of parasite which produced by tick vector. This study was carried out to establish of T. annulata life cycle to achieve T. annulata infected ticks.Methods: Laboratory rabbit and calf were used for rearing of Hyalomma anatolicum different instars. Unfed nymphs were fed on T. annulata infected calf. Clinical signs, Giemsa stained smears and Polymerase Chain Reaction (PCR) methods were used for detection of infection in blood and tick specimens. Susceptible calf was used for confirmation of sporozoites maturation and infectivity in bioassay test.Results: Hyalomma anatolicum two and three-host strategies of life cycle was lasted 90 and 116 days respectively. The PCR confirmed T. annulata infection in blood and tick samples. Maturation of T. annulata sporozoites was confirmed in bioassy test. First clinical symptom of disease was seen earlier in the case of transmission of disease through feeding of live ticks in comparison with blood injection method.Conclusion: Complete life cycle of T. annulata was done and confirmed by clinical signs, microscopic examination, molecular methods and bioassay test. According to published reports to date, this is the first report of establishment of H. anatolicum tick infection with T. annulata using susceptible calf under controlled conditions in Iran.
1. Hashemi-Fesharki R (1988) Control of Theil¬eria annulata in Iran. Parasitol Today. 4(2): 36–40.
2. Purnell RE (1978) Theileria annulata as a hazard to cattle in countries on the north¬ern Mediterranean littoral. Vet Sci Com¬mun. 2(1): 3–10.
3. Jalovecka M, Hajdusek O, Sojka D, Ko-pacek P, Malandrin L (2018) The com-plexity of piroplasms life cycles. Front Cell Infect Microbiol. 8: 248.
4. Shkap V, de Vos AJ, Zweygarth E, Jongejan F (2007) Attenuated vaccines for trop¬ical theileriosis, babesiosis and heart¬wa¬ter: the continuing necessity. Trends Par¬asitol. 23(9): 420–426.
5. McHardy N, Wekesa LS, Hudson AT, Ran-dall AW (1985) Antitheilerial activity of BW720C (buparvaquone): a comparison with parvaquone. Res Vet Sci. 39(1): 29–33.
6. Nabian S, Rahbari S, Changizi A, Shayan P (2009) The distribution of Hyalomma spp. ticks from domestic ruminants in Iran. Med Vet Entomol. 23(3): 281–283.
7. Ghosh S, Azhahianambi P (2007) Labor-atory rearing of Theileria annulata-free Hyalomma anatolicum anatolicum ticks. Exp Appl Acarol. 43(2): 137–146.
8. Kröber T, Guerin PM (2007) An in vitro feeding assay to test acaricides for con-trol of hard ticks. Pest Manag Sci For-mer Pestic Sci. 63(1): 17–22.
9. Tajeri S, Razmi G, Haghparast A (2016) Establishment of an artificial tick feed-ing system to study Theileria lestoquar-di infection. PLoS One. 11(12): e0169053.
10. Mehlhorn H, Schein E (1985) The piro-plasms: life cycle and sexual stages. In Advances in parasitology. Academic Press. 23: 37–103.
11. Samish M (1977) Infective Theileria annu-lata in the tick without a blood meal stim¬ulus. Nature. 270(5632): 51–52.
12. Walker AR, McKellar SB (1983) The mat-uration of Theileria annulata in Hy¬alom-ma anatolicum anatolicum stimulated by incubation or feeding to produce sporo¬zoites. Vet Parasitol. 13(1): 13–21.
13. OIE Terrestrial Manual (2018) Chapter 3.4.14. Theileriosis. pp. 1185–1209. Available at: https://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/3.04.14_THEILIERIO SIS.pdf
14. Modirrousta H, Habibi G, Shayan P, Af¬shari A, Mirjalili A, Abdigoudarzi M (2019) Determination of the Infectivity of Cryopreserved Theileria annulata Spo¬rozoites in Tick Derived Stabilates Iran Ak-93 Strain, by in Vivo and in Vitro Meth¬ods. Iran J Parasitol. 14(4): 572–583.
15. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Proc Natl Acad Sci. USA, 74: 5463–5467.
16. Abdigoudarzi M, Rivaz S (2011) Tick rearing studies of Hyalomma an¬atoli-cum anatolicum (Acari: Ixodidac) and pre¬par¬ing of its life cycle data at laboratory conditions. Vet J. 24(1): 1–12.
17. Habibi GR, Esmaeil-Nia K, Bozorgi S, Najjar E, Hashemi-Fesharki R, Bordbar N (2007) PCR-based detection of Theil-eria infection and molecular character-iza¬tion of Tams1 T. annulata vaccine strain. Arch Razi Inst. 62(2): 83–89.
18. Aktas M, Bendele KG, Altay K, Dumanli N, Tsuji M, Holman PJ (2007) Se¬quence polymorphism in the ribosomal DNA internal transcribed spacers differs among Theileria species. Vet Parasitol. 147(3–4): 221–230.
19. Schnittger L, Katzer F, Biermann R, Shayan P, Boguslawski K, McKellar S, Doreen Beyer D, Shiels B, Ahmed J (2002) Characterization of a poly¬mor-phic Theileria annulata surface protein (TaSP) closely related to PIM of Theil-eria parva: implications for use in di-agnostic tests and subunit vaccines. Mol Biochem Parasitol. 120(2): 247–256.
20. Xu G, Fang QQ, Sun Y, Keirans JE, Durden LA (2005) Hard tick calreticulin (CRT) gene coding regions have only one intron with conserved positions and variable sizes. J Parasitol. 91(6): 1326–1332.
21. Gill BS, Bhattacharyulu Y, Kaur D (1977) Symptoms and pathology of experi-mental bovine tropical theileriosis (Theileria an¬nulata infection). Ann Parasitol Hum Comp. 52: 597–608.
22. Mazlum Z (1969) Transmission of Theil-eria annulata by the crushed infected unfed Hyalomma dromedarii. Parasit¬ol-ogy. 59(3): 597–600.
23. Hooshmand-Rad P, Hawa NJ (1973) Trans¬mission of Theileria hirci in sheep by Hyalomma anatolicum anatolicum. Trop Anim Health Prod. 5(2): 103–109.
24. Tajeri S, Razmi GR (2011) Hyalomma an-atolicum anatolicum and Hyalomma drom¬edarii (Acari: Ixodidae) imbibe bo-vine blood in vitro by utilizing an ar-tificial feeding system. Vet Parasitol. 180(3–4): 332–335.
IssueVol 14 No 3 (2020) QRcode
SectionOriginal Article
DOI https://doi.org/10.18502/jad.v14i3.4559
Theileria annulata Hyalomma anatolicum Tick rearing PCR Bioassay

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
Afshari A, Habibi G, Abdigoudarzi M, Yazdani F. Establishment and Validation of Theileria annulata Sporozoite Ak-93 Infection in Laboratory-Reared Hyalomma anatolicum Tick Using In Vivo and In Vitro Assays. J Arthropod Borne Dis. 14(3):261–269.