Development Of Suspension Culture For Theileria Annulata Schizont-infected Cells And Studies On Its Biological Characterization

Tropical theileriosis is a tick-borne disease of bovine caused by Theileria annulata.The typical clinical symptoms of bovine theileriosis are fever,anemia,enlargement of superficial lymph nodes,jaundice and respiratory distress,even leading to death of cattle.It occurs around the world,and results in great economic losses for livestock industry.The prevention and treatment of T.annulata mainly are chemical drugs and vaccination.In Middle East,Africa and China,the control of tropical theileriosis mainly is based on application the vaccine of T.annulata schizont-infected cells.In the 1970s,T.annulata schizonts infected vaccine was successfully developed in China,and the production process of the vaccine was improved from adherent culture to rolling bottle,thereby achieved the large-scale production and commercial supply of the vaccine,which has been extensively applied more than 40years in 13 provinces of north in China.Although the disease occurs sporadic,the administration of this vaccine basically controlled its pandemic.However,the quality of vaccines produced by rolling process was unstable in intra and inter-batch,even incomplete attenuation that leads to obvious morbidity of bovine.The vaccine manufacturers in Ningxia and Xinjiang have been halted the vaccine production since the GMP management implemented by bio-products factories in China.However,the vaccine still has some demand in the market because the tropical theileriosis happens on occasion in cattle and dairy cattle in pastoral and agricultural areas in northern China.In recent years,the progress of large-scale suspension cell culture has been established for vaccine of viral disease,such as foot-and mouth virus,rabies virus and so on.The suspension culture technique improves the productivity and quality of vaccine,which provides the possibility for process improvement and large-scale production in T.annulata schizont-infected cell vaccine.In this study,The T.annulata（Takashi）infected bovine lymphocyte cell line was successfully established based on the diversity characteristics of T.annulata in China,and the cells were adapted to suspension culture in vitro.The safety and immunogenicity of the suspension cell to immunized animals are evaluated,which provides vital supports for the large-scale production of T.annulata vaccine.The main results are as follows:1)The establishment of T.annulata schizont-infected cell line:50 Hyalomma anatolicum anatolicum ticks infected T.annulata（Ta Kashi）were feed on cattle,whose rectal temperature began to rise at 12 d,the peak reached to 41℃,and the schizont was observed when the swell of anterior shoulder and anterior femoral lymph nodes by puncturing.The T.annulata schizont-infected cell line was successfully built based on the method of primary cell culture.The cell line can be stably passaged in culturing RPMI1640 medium plus with 10%FBS at 37℃.And the cell density could reach to2.8～3.0×10⁶cells/m L after in vitro culture 72 h.Moreover,the genetic evolution between T.annulata and other theileria species or strains was analyzed using the target genes-18S RNA and Tams1.The results indicated that T.annulata was mostly related to the strains（Henan and Inner Mongolia）in phylogenetic relationships.2)The establishment of suspension culture method for T.annulata schizont-infected cells:the special optimized medium for suspension culture of T.annulata-transformed cells was 75%GB,25%RPMI1640 plus certain supplements containing amino acids,glucose and 7 kinds of growth factors by basic medium screening,superior amino acids consumption and their by-products detection during suspension culture.The cell density ranged from 0.5×10⁶ cells/m L to 4.3×10⁶ cells/m L,and the cell viability was>90%and the cells could stably passage using the optimized culture medium in suspension culture for 72 h at 37℃and 80 rpm for 5%CO₂ in incubator.3)The preparation for sporozoite suspension and pathogenicity detection of T.annulata infection:the sporozoite suspension was prepared using unfed adult ticks infected T.annulata according to the previous studies and the preparation protocol was confirmed.The optimal dose for animal challenge using the sporozoite suspension was between 8 and 20 ticks,and the preservation method of sporozoite suspension was developed that was in liquid nitrogen.Moreover,the cattle were artificially infected with T.annulata sporozoites and the clinical symptoms and pathology changes were investigated from body temperature detection,blood analysis,infection rate calculation,necropsy and histological characteristics.4)Safety and immunogenicity evaluation of T.annulata schizont-infected cells:the different cell passages（F5,F12,F18,F26）of T.annulata-transformed cells by the suspension culture at the density of5×10⁶ cells challenging cattle were evaluated.The findings of our study demonstrated that the cattle in the group（F5）appeared transient fever and parasitaemia,but the T.annulata in other groups were not detected and no clinical symptoms.The cattle were challenged by sporozoite suspension containing 16ticks after immunization using T.annulata schizont-infected cells at different cell passages（F5,F12,F18 and F26）,the results showed that the cattle of these four groups acquired protective effects,whereas the groups（F5 and F26）performed the typical clinical features,F12 and F26 groups were slightly transient fever and parasitaemia but no clinical characteristics,the cattle of control group were all dead.The above findings of our study demonstrated that T.annulata schizont-infected cells using suspension culture process can induce effective immune response and provides effective protection against T.annulata.