Isolation of T. annulata infected cells:
Heparinized blood and lymph node aspirates were collected from a three-year-old cow naturally infected with T. annulata. This cow was admitted to the Teaching Veterinary Hospital, Faculty of Veterinary Medicine, Assiut University from EL-Ghanim district, Assiut governorate. The animal showed typical clinical signs of tropical theileriosis. Her body temperature was 41.5°C, with congested conjunctival mucous membranes and enlargement of the superficial lymph nodes. An aspirate was collected from the prescapular lymph nodes. Following sampling and diagnosis, the cow was treated with Buparvaquone (MSD Animal health, New Jersey, USA) at 2.5 mg/kg body weight deeply intramuscular, followed by a second dose after 72 hours. The animal was also treated with non-steroidal anti-inflammatory drugs (Butafenil, Vetoquinol, France) and antibiotics (Marbocyl 10%, Vetoquinol, France) to control potential secondary bacterial infections.
Preparation and attenuation of T. annulata infected cell line:
Preparation of peripheral blood mononuclear cells (PBMC) from heparinized blood was performed by gradually transferring three mL of blood to a Falcon tube containing an equal amount of Ficoll® 400 (Sigma Aldrich, Germany) followed by centrifugation at 1,800 rpm for 40 min. The PBMC layer was subsequently collected and transferred to a new Falcon tube in which it was washed three times with RPMI 1640 (Cat. No, BE12-115F, Lonza, Switzerland) with a centrifugation step at 2,000 rpm for 10 min between washes. The pellet was subsequently resuspended in 2 mL complete RPMI 1640 media with 10% inactivated fetal calf serum (LSP, UK), 1% Amphotericin B (Lonza, Switzerland), 1% Gentamicin (Lonza, Switzerland), 2% Streptomycin and Penicillin (Lonza, Switzerland). Lymph node aspirates were washed three times in RPMI 1640 and resuspended in 2 mL complete media. Both samples were then transferred to two separate filter-cap tissue culture flasks 25T containing 5 mL complete RPMI 1640 and were incubated with 5% CO2 at 37°C. The media was changed every 72 hours. Theileria annulata schizont-infected leukocytes grew continuously and were passaged under sterile laboratory conditions up to 114 serial passages. The obtained cell line was tested to confirm that it was free from any bacterial, mycoplasma or fungal contamination by the regular inoculation of 100 µL from the cell line on enriched 4% sheep blood agar (BioLab ’BAN20500’) and EcoBio, Sabouraud-Dextrose-Agar 4% PH Euro-USP, (BioLab ’ESDA20500’) and incubate the plates at 37°C for 48 hours and 25°C for two week, respectively before inspection 44.
DNA was also extracted and tested by the Reverse Line Blot (RLB) assay to confirm that the cell line was free from any other protozoan or rickettsia pathogen 3. The viability of the cell line was evaluated using Trypan blue (Lonza, Switzerland) exclusion counting before storage of each passage in liquid nitrogen and prior to preparation of the vaccine doses Cell viability ranged between 97–99% before storage and vaccination22–24.
Experimental animals
Twenty-four exotic breed (Friesian) six-month old male calves were purchased from Assiut’s official governmental farm, which is located approx. 50 km north of Assiut city. Twenty-four crossbred male calves of the same age were purchased from a private farm in Arab EL-Awamer village, which is located approx. 75 km north to Assiut city.
Preparation of animals for vaccination trail
All calves were kept in tick-free pens for 6–8 weeks prior to immunization. During this time, they were regularly observed and examined to confirm that they were free from any disease or parasitic infection. Blood samples from each animal were regularly collected and examined before and during the field challenge. Complete blood counts (CBC) and Giemsa-stained blood smears were made to assess the animal’s health status. PCR assays targeting the merozoite-piroplasm surface antigen 1 (Tams-1) of T. annulata and the 18S rRNA gene of Babesia and Theileria species were performed to test the animals for the presence of Babesia and Theileria infections. In addition to this, all animals were screened by an indirect Theileria annulata surface protein (TaSp) ELISA to check for previous T. annulata exposure prior to the start of the vaccination trial 8,26.
Immunization
Both crossbred and exotic groups were divided randomly into two equally sized subgroups (vaccination and control groups). Each animal in the vaccination group was inoculated subcutaneously with 4ml (1 x 106 cells/mL) of the attenuated cell line at passage 85 in the middle third of the neck. Passage 85 has been shown to be the highly immunogenic passage that stimulates a strong immune response in the vaccinated animals during our pretrials to find the most effective passage that will be able to stimulate the immune system to produce protective antibodies against the parasite without any adverse effects.
Animals of the control group were injected with RPMI 1640 culture media only. Both groups were kept in tick-free pens for 3–4 weeks under continuous clinical, hematological, and parasitological monitoring including both molecular and serological examinations that were performed as described above. Both groups were subsequently transported to EL-Wady EL-Geded governorate, a region considered to be highly endemic for T. annulata, where they were kept for three months under natural conditions and exposed to field challenge 1.
Field monitoring
Calves were observed daily to assess the tick infestation rate and to monitor the development of clinical signs. The rectal temperature was recorded three times per day: before sunrise, at noon and after sunset. The mean of the three values was considered as the daily body temperature. EDTA blood was collected three times per week and examined for the presence of T. annulata development stages. Lymph node aspirates from animals that showed enlarged lymph nodes were collected and used for the preparation of Giemsa-stained lymph smears, which were subsequently examined microscopically. The parasitemia’s percentage in each animal was calculated using the following equation (Parasitemia % = (Number of the parasitized RBCs/12500 RBCs x100 which represent the total number of RBCs in 25:50 Microscopic Fields). Serum samples were collected every month for screening using the TaSp ELISA 9,25. PCR was used for detection of T. annulata infection in the blood samples collected from animals before and after immunization and challenge in both groups 45,46. Positive PCR products were purified using QIAGEN PCR purification Kit (Cat. No. 28104, Qiagen, UK) according to manufacturer’s instructions and sequenced in both directions using an ABI 310 Sequencer at the Molecular Biology Research & Studies Institute of Assiut University 47. Sequences were subjected to BLAST similarity searches. A RLB assay for the simultaneous detection of Theileria/Babesia and Anaplasma/Ehrlichia was employed to determine if tick-borne pathogens other than T. annulata were present in the blood samples 2,3.
Necropsy finding
Postmortem examinations were performed in the Department of Pathology and Clinical Pathology, Faculty of Veterinary Medicine, Assiut University on calves that died during the experiment 14.
Tick collection and identification
Ticks were manually collected from animals using a forceps and identified morphologically according to standard morphological keys 48.
Statistical analysis
General linear model analysis was conducted in R. Pairwise analyses were attached by least square means analyses for multiple comparisons under the lsmean package with Tukey adjustment. Significance levels were interpreted as: P-Value ≤ 0.00*** (Highly significant),0.001** (Moderate significant),0.01* (Mild significant), 0.05 (Non-significant) 49–51.
Ethical approval
The study is reported in accordance with ARRIVE guidelines. the working protocol, procedures, and all the experiments were ethically reviewed and approved by the Scientific Research Committee and Ethics Board of Assiut University, Egypt, approval number is IRB no: 04-2022-300018. The working protocol, procedures, and all the experiments conformed to recognized standards of Animal research applied by Assiut University and the animal welfare code in Egypt. Additional ethical approval was obtained from both Assiut governorate (Assiut University), 71526, (616, 24.02.2019) Egypt and New Valley Governorate (686, 2.3.2019) and the Veterinary authorities in the New Valley Governorate (485, 3.3.2019).