Immunohistochemical analysis of skin and draining lymph nodes of sheep repeatedly infested with the ixodid tick Hyalomma anatolicum anatolicum were studied for different antigen-presenting cells and lymphocyte subpopulations. Infiltration of neutrophils, macrophages and lymphocytes adjacent to the tick bite site were observed. Skin biopsies showed significant increases in dermal infiltration of CD8+ and γδ+ T cells at 72 h and 8 days after both primary and secondary infestation. Infiltrations of MHC-II DR/DQ decreased at 72 h after tick infestation, whereas significant increases were recorded for 8-day skin biopsies. CD1+ cellular infiltrations were observed during secondary infestations at the dermis. Decreased ratios of CD4[ratio ]CD8 T cells and MHC-II[ratio ]CD1 antigen-presenting cells were observed in both infestations compared to healthy skin biopsies. Ratios of αβ[ratio ]γδ T cells increased gradually during infestation compared to uninfested skin. The regional lymph nodes from tick-infested sheep showed an increased CD8+, γδ+ T and CD1+ cellular infiltration compared to control lymph nodes. CD4+ T cells were decreased. There were no significant changes in CD45R+ cellular infiltration either at skin lesions or regional lymph nodes.

—The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of gut supernate antigen (GS Ag) derived from partly fed Hyalomma anatolicum anatolicum Koch, 1844 (Acari: Ixodidae) adult females revealed 26 discrete potypeptide bands of molecular weight ranging from 25–208 kDa. Out of these, seven bandswere of major polypeptides with molecular weights of between 25.2 and 185.8 kDa. On immunoblotting with antisera raised in rabbits against gut supernate (crude extract), eight immunogenic polypeptides with molecular weights of between 51.7 and 185.8 were identified. The implications of these findings for the potential development of anti-tick vaccine are discussed.

A Polymerase Chain Reaction (PCR) and Southern blot hybridization for the detection of Theileria annulata are described. The PCR used primers amplifying a 785 base-pair fragment of the T. annulata gene which encodes the 30 kDa major merozoite surface antigen, Tams1. The sensitivity of the PCR in bovine blood was 1 piroplasm in 1 μl of blood. T. buffeli, T. parva, Babesia bigemina, B. bovis and B. divergens were not detected. The PCR detected down to 1 infected acinus/tick in resting and partially fed adult Hyalomma anatolicum anatolicum ticks and was negative for T. lestoquardi and T. equi, which are transmitted by this tick but are not infective to cattle. The specificity of the PCR was checked using 30 stocks of T. annulata, all of which were detected. Three stocks of T. lestoquardi, 4 of T. equi and 1 each of T. buffeli, T. parva, B. bigemina, B. bovis and B. divergens were used to ascertain there were no cross-reactions. A nested PCR using separate primers for the first reaction and the same primers for the second reaction detected T. annulata to the same sensitivity and specificity in saponin-extracted DNA samples stored for long periods at −20 °C.

The maturation and quantification of Theileria lestoquardi (T. hirci) parasites in unfed and partially fed adult Hyalomma anatolicum anatolicum ticks was studied using (1) methyl green pyronin (MGP) staining of salivary glands, (2) in vitro infection of peripheral blood mononuclear cells (PBM) with parasites harvested from infected ticks and (3) a semi-quantitative polymerase chain reaction (PCR). With MGP staining the greatest infection rate was seen in unfed ticks. Feeding resulted in a gradual reduction in the number of infected acini with a concomitant increase in the maturity of the parasites. In vitro infection of sheep PBM with titrated ground-up tick supernate (GUTS) demonstrated that infectivity peaked between 2 and 4 days of tick feeding whereas GUTS prepared from unfed ticks was not infective. The polymerase chain reaction (PCR) was both sensitive and specific, detecting T. lestoquardi DNA in unfed and partially fed ticks, with a maximum sensitivity of 0·022 infected acinus/tick in 2-day fed ticks, though it gave no indication of the infectivity of the parasite.

Whole body tick collections, carried out at 6-day intervals for 2 months at Nisheishiba, showed significant differences in tick counts between three breeds of cattle. Crossbred (Bos taurus × B. indicus) cows carried 4.5 times more ticks (mean tick count 70.5 ± 84.8) than the B. indicus Kenana (mean tick count 16.7 ± 24.4) and Butana cows (mean tick count 15.0 ± 18.4). In all cases, the SD's were larger than the means reflecting the usual wide variability in tick numbers between individuals. Significant differences also existed in the degree of engorgement achieved by female Hyalomma anatolicum anatolicum ticks on different breeds of cattle. The weight of detaching fully engorged females feeding on Kenana and crossbred cattle was 374.8 mg and 422.0 mg respectively. This was reflected in the amounts of eggs laid by the females which were 191.3 mg for the Kenana-fed ticks and 261.1 mg for those from the crossbreds. The resulting larval challenge from the crossbred-fed ticks was 36.5% greater than from the ticks fed on Kenana cattle.