Comparison of cryoprotectants in the preservation of Theileria parva sporozoites using an in vitro infectivity assay.

An in vitro infectivity assay was used to examine five cryoprotectants for their suitability for preserving Theileria parva sporozoites. All five were capable of preserving T. parva sporozoites through freezing, the optimal concentrations being 7.5% for glycerol, 5% for dimethyl sulphoxide (DMSO), poly (vinylpyrrolidone) (PVP) and poly(ethylene glycol) (PEG), and 2.5% for hydroxyethyl starch (HES). When the five cryoprotectants were compared at their optimal concentrations, using a modification of the standard method of stabilate preparation, glycerol was significantly better than the others (p < 0.05). Measurement of the effects of each cryoprotectant on the osmolality of the media revealed that glycerol and DMSO elevated the osmolality significantly (p < 0.05). Resuscitation of glycerol-preserved sporozoites required the presence of glycerol in the diluent to maintain infectivity. Studies on the effects of equilibration time in glycerol on the infectivity of sporozoites showed that those frozen immediately after mixing (2 min) were as infective as those frozen after 60 min of equilibration.

Detection of antibody to Theileria parva schizonts and cell surface membrane antigens of infected lymphoblastoid cells by immunoperoxidase techniques.

Peroxidase-labeled antibody procedures were described for detecting bovine antibodies reactive with intracellular Theileria parva schizonts and cell surface membrane antigens of infected lymphoblastoid cells. Indirect tests were performed where the reacting bovine antibodies were localized with affinity purified rabbit-anti-bovine IgG coupled to horseradish peroxidase. A 4- to 8-fold increase in sensitivity for detecting bovine antibodies was obtained with unlabeled rabbit-anti-bovine IgG which in turn was detected with peroxidase labeled goat-anti-rabbit IgG. The T. parva infected cells used as antigen were attached to poly-l-lysine treated glass slides and all reaction steps were performed on the slides. The intracellular schizonts and cell surface staining reactions were dependent upon the status of the cells; acetone-fixed cells were required for schizont reactions and viable unfixed cells for cell surface membrane reactions. Sera from cattle stimulated in various ways with T. parva were examined by the techniques. Cattle infected by stabilate inoculation or inoculated with infected autologous lymphoblastoid cells developed relatively high levels of antibody to schizonts, but no detectable antibody to cell surface membrane antigens. This would indicate that parasite antigens do not occur on the surface of infected lymphoblasts. Cattle inoculated with infected allogeneic lymphoblasts developed low-levels of antibody to schizonts and readily demonstrable antibody to cell surface antigens. The immunoperoxidase procedures have certain advantages over immunofluorescence in that light microscopy is used; therefore, the reactions do not fade which permits a more detailed examination and provides a relatively permanent record, the preparations can be counterstained, and the reagents may be used for immunoelectron-microscopy. The procedures could provide suitable alternatives to immunofluorescence methods for East Coast fever investigations and other systems having intracellular and/or cell surface membrane antigens.

Infection of mammalian cells with Theileria species.

Experiments were carried out to determine the susceptibility of mammalian cells to infection with different species of Theileria in vitro. Sporozoites of Theileria parva (parva), Theileria parva (lawrencei) and Theileria taurotragi were isolated from Rhipicephalus appendiculatus ticks by grinding infected ticks in medium, filtering the suspension and concentrating by centrifugation. The sporozoites were used in attempts to infect in vitro peripheral blood leucocytes harvested from 16 different mammalian species which included 12 species of Bovidae from 6 different sub-families. The technique was shown to be both sensitive and reproducible. The sporozoites of T. parva (parva) infected and transformed cells from 2 species of the sub-family Bovinae, the two cattle types and African buffalo. Theileria parva (lawrencei) infected and transformed cells from the two cattle types, African buffalo and Defassa waterbuck. Theileria taurotragi sporozoites infected in vitro cells from 11 different species of Bovidae which were members of 6 sub-families; Bovinae, Tragelaphinae, Reduncinae, Alcelaphinae, Antilopinae and Caprinae. Transformed lymphoblastoid cell lines were established from 7 of the species infected. Sporozoite attachment and infection was not observed with non-susceptible bovid host cells, nor were any of the non-bovid leucocytes infected by the parasites. The host range observed in this study corresponded to the known host range in vivo.

A comparative study of the disease in cattle caused by Theileria parva or T. lawrencei: II. Hematology, clinical chemistry, coagulation studies and complement.

Theileria lawrencei tick-derived stabilate infection of 8 cattle resulted in the development of panleukopenia and hypoproteinemia. In addition to these changes, T. parva infection caused mild normocytic, normochromic, non-responsive anemia at either of two dose rates. Disseminated intravascular coagulation, as indicate by positive protamine paracoagulation tests, prolonged prothrombin and partial thromboplastin times, and thrombocytopenia, developed in cattle infected with either of the Theileria spp., and was probably an important intermediary mechanism leading to death. Infection of calves with a high dose of T. parva stabilate resulted in more rapid onset of clinico-pathologic changes than did the low dose infection. Theileria lawrencei infection produced a severe, acute syndrome, the clinico-pathologic alterations of which varied in time of onset and severity between those of the T. parva high dose and low dose groups.

Cell-mediated immunity to Theileria-transformed cell lines.

In East and Central Africa the protozoan parasite Theileria parva causes a disease of cattle called East Coast fever (ECF). In Kenya alone between 60,000 and 85,000 cattle die from ECF every year. Infected animals can recover from ECF either naturally or after treatment with tetracyclines or menoctone and are subsequently able to resist challenge with the homologous strain of parasite. That this acquired resistance is due to cell-mediated rather than humoral immunity has been suspected but never decisively shown. A major difficulty in studying immunity to ECF has been the lack of inbred animals for studying Theileria-specific immunity in the absence of allogeneic histocompatibility barriers. We have avoided this problem by measuring cell-mediated immune responses in a syngeneic system in vitro. Unidirectional mixed lymphocyte cultures (MLC) were set up using bovine peripheral blood lymphocytes (PBL) as responder cells and autologous cell lines transformed in vitro by T. parva as stimulator cells. In these cultures, DNA synthesis was induced in PBL from both normal and Theileria-immune animals. However, cytotoxic lymphocytes were induced only in cultures containing responder lymphocytes from Theileria-immune cattle. The results show that Theileria-transformed cells express antigens which are recognized by effector cells and provide evidence that cell-mediated cytotoxic mechanisms function in immunity to ECF.