Merogony in in vitro cultures of Theileria parva

In vitro studies were focussed on the duration and cessation of merogony in Theileria parva infected blood lymphocyte cell cultures. The cultures were infected using purified tick stabilates as an alternative to in vitro infections, using sporozoites obtained by labour intensive dissections of salivary glands from infected ticks. After establishment of infection in peripheral blood lymphocytes (PBL), merozoites were temporarily produced for about 2 months after which lymphoblasts only contained schizonts.     

In vitro infection of bovine alloreactive cytotoxic T cell lines with sporozoites of Theileria annulata and T parva

Bovine alloreactive cytotoxic lymphocyte (CTL) lines of known target specificity were infected in vitro with sporozoites of Theileria annulata and T parva and cultured in limiting dilution. The phenotypes of the CTL lines both pre- and post infection were assessed using a panel of monoclonal antibodies specific for defined bovine lymphocyte subpopulations. The effector function of the resultant infected cell lines was determined using a Cr51 release assay and compared to the uninfected control CTL line. The results indicated that T parva sporozoites consistently infected and transformed the CTL lines very efficiently even at the lowest cell doses. In contrast the T annulata sporozoites were largely unable to infect and transform the alloreactive CTL except at the very highest cell and sporozoite doses. A factor which appeared to influence susceptibility to T annulata infection was an increased level of class II expression on the CTL line. None of the cell lines showed cytotoxic effector function after infection with either T annulata or T parva sporozoites.     

The early events of infection with Theileria parva in cattle: infectivity for cattle of leukocytes incubated in vitro with sporozoites

Leukocytes were isolated from bovine blood and, after short periods of incubation in vitro with sporozoites of Theileria parva, were washed thoroughly, and their infectivity tested in autologous and allogeneic hosts. Using a standard inoculum of 10(6) viable cells, it was found that, after incubation in vitro for either 1 or 24 h, the cells initiated lethal infections in autologous cattle, but failed to infect allogeneic animals. Autologous and allogeneic erythrocytes and mouse lymphocytes similarly incubated with sporozoites failed to infect cattle. The supernatant from bovine lymphocyte suspensions incubated with sporozoites for 1 h produced lethal infections whereas after 24 h of incubation the supernatant was non-infective. All cattle which did not develop detectable infection were fully susceptible to subsequent challenge with a stabilate of sporozoites. By inoculating cattle with graded doses of autologous blood leukocytes which had been incubated for 24 h with sporozoites, it was found that as few as 2 X 10(3) cells gave rise to infection. The results indicate that this approach can be used to evaluate different cell populations as targets for infection and transformation by sporozoites of T. parva.     

Ultrastructural characteristics of in vitro parasite-lymphocyte behaviour in invasions with Theileria annulata and Theileria parva

Host-parasite relationships have been studied by electron microscopy using glutaraldehyde-OsO4-fixed pellets of lymphoid cultures infected in vitro by Theileria annulata and T. parva. Intracellular presence of the parasite resulted in a progressive and marked lymphoblastoid transformation. The schizont stage periodically provoked the formation of, and adopted an intimate association with, cytoplasmic annulate lamellae in the interphase cell. Annulate lamellae developed from the outer nuclear membrane of the host cell by a delamination process and were taken into the cytoplasmic matrix of the schizont by phagotrophy through the cytostome. Schizont nuclei themselves were seen to divide at the prometaphase stage of host cell mitosis, the division being characterized by the development of intranuclear spindle microtubules anchored in spindle pole bodies. A hypothesis is propounded that Theileria parasites, consequent on interiorization, provoke the blastoid transformation and the formation of annulate lamellae through the influence of components of their genomic material on host cell deoxyribonucleic acid (DNA) and that the annulate lamellae represent a species of messenger ribonucleic acid (mRNA) and serve as a monitoring device for the schizont, facilitating the accurate timing of the host cell cyclical events.     

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.     

Comparison of the survival on ice of thawed Theileria parva sporozoites of different stocks cryoprotected by glycerol or sucrose

Stabilates of Theileria parva sporozoites are mostly delivered in liquid nitrogen tanks to the East Coast fever immunization points. Using an in vitro titration model, we assessed the loss of infectivity of several stabilates when they are stored in ice baths for up to 24 h. Comparisons, with respect to rates of loss of infectivity, were made between T. parva stocks (Chitongo and Katete), cryoprotectants (sucrose and glycerol) and method of assessment (in vivo and in vitro techniques). Chitongo and Katete stabilates showed similar loss dynamics. The losses were 1-4% (depending on parasite stock) and 3% per hour of storage for glycerol and sucrose stabilates respectively, and the loss rates were not significantly different. The results suggest that Chitongo stabilates and sucrose cryoprotected suspensions can be delivered on ice as is done for Katete. A graphical relationship of in vitro effective dose at 50% infectivity (ED50) and in vivo protection rate was made. The relationship showed a 35% loss of protection for a relatively low corresponding increase of ED50 from 0.006 to 0.007 tick equivalent.     

Theileria parva: In Vitro Studies on the Effects of Holding Temperature,pH and Medium on Sporozoite Infectivity

The effects of holding temperature, pH and medium on the infectivity of Theileria parva sporozoites were investigated using an in vitro infectivity assay. The sporozoite infectivity lasted for 72 h at a holding temperature of 4 C but for only 24 h at 24 degrees C. Sporozoite infectivity was found to be sensitive to pH variations and sporozoites were most infective between pH 7 and pH 8. There was a significant loss in infectivity at pH 5 and infectivity was almost totally abolished at pH 9. Theileria parva sporozoites are usually held and manipulated in Eagle's minimum essential medium (MEM) with Earles' salts. In this study. Leibovitz-15 supplemented with 15% fetal bovine serum gave a significantly better infectivity than Eagle's MEM (3.8 log units versus 1.0 log units) or any other medium. The importance of proper management of the T. parva sporozoite environment in the laboratory or field is emphasized by the findings in these studies and might also explain some of the failures of vaccination when the pH of the holding medium was allowed to deteriorate.     

Cultivation of Theileria. I. Attempts to complete the cycle of Theileria parva in vitro

Microschizonts and free merozoites developed in bovine lymphoblastoid cell cultures containing macroschizonts of 6 different strains of Theileria parva. Clean bovine red cells were added to the cultures, which were incubated in various ways. No penetration of red cells by merozoites was observed, not even when cultures in diffusion chambers were introduced into the peritoneal cavity of non-infected cattle.     

Infectivity and cross-immunity studies of Theileria lestoquardi and Theileria annulata in sheep and cattle: II. In vitro studies.

In the studies previously reported, the tick-borne protozoan parasites Theileria lestoquardi and Theileria annulata were shown to differ in their capacity to infect sheep and cattle. In the studies presented here, these findings were further supported. In vitro infectivity of T. lestoquardi and T. annulata sporozoites for peripheral blood mononuclear cells of sheep and cattle were determined by analysis of cell cultures for cell proliferation, the detection of parasites in Giemsa-stained cytospin smears and the establishment of continuously growing schizont-infected cell lines. In the same way, the development of schizont-infected cells into continuously growing cell lines was studied with material isolated ex vivo from the sheep and cattle undergoing primary infections described elsewhere. Comparisons were also made between development of ex vivo cell lines from animals undergoing primary infections with those of the animals undergoing challenge infection with the other parasite species. Theileria species specific primers were used in a PCR to determine the identity of the parasites in the cell lines. These in vitro studies confirmed earlier observations that T. lestoquardi was unable to infect cattle, whereas infection of all sheep with T. annulata was proven. Moreover, earlier indications of the development of partial cross-immunity in sheep of T. annulata to T. lestoquardi and vice versa were strengthened. These findings may thus have consequences for the understanding of the epidemiology of T. lestoquardi infections of sheep. On the other hand. since piroplasms were not demonstrated in sheep infected with T. annulata, such sheep will not be infective to ticks and will consequently be unlikely to play a role in the maintenance and transmission of T. annulata to cattle.     

Comparative autoradiographic study of parasite-host-cell cyclical relationship in lymphoblastoid cell lines infected with Theileria annulata and Theileria parva in vitro

Cyclical interactions between intracellular schizonts of the Ankara and Hissar strains of Theileria annulata and the Muguga strain of T. parva and the parasitised host lymphoblasts have been studied autoradiographically by following the incorporation of (3H) thymidine into parasite and host cell nuclei, and also by quantitating the number of schizont nuclei per lymphoblast, at various stages and phases of host cell cycle. The synthesis of DNA by Theileria schizonts and the parasitised host lymphoblasts was found to be asynchronous and to occur at different phases of the host cell interphase stage. While the lymphoblast nuclear DNA incorporates (3H) thymidine during the S phase, schizont nuclei were labelled during the G2 phase of the host lymphoblast interphase stage. The replication of schizont nuclei took place before the metaphase stage of host cell cycle, viz, prometaphase, so that the mean schizont nuclear number at host prometaphase and at anaphase--telophase was consistently more or less double the mean nuclear number at interphase.     

Metabolic energy pathways in Theileria annulata sporozoites and their significance in sporozoite-bovine lymphocyte interactions in vitro

The existence of metabolic energy pathways has been studied in extracellular T. annulata sporozoites using chemicals known to inhibit specific energy-generating pathways, and their role during invasion of bovine peripheral blood lymphocytes (PBL) by the sporozoites determined in an in vitro system. An inverse relationship was depicted between the dose of various chemicals and the number of T. annulata sporozoites invading PBL: as the concentrations of the inhibitor drugs increased, the number of T. annulata sporozoites within the lymphocytes decreased. An ultracytochemical study demonstrated the presence of the respective pathway marker enzymes, i.e., lactic dehydrogenase (LDH) in the cytosol and within mitochondria, succinic dehydrogenase (SDH) on the mitochondrial membranes and in the contiguous matrix, and cytochrome oxidase (CO) between the inner and outer mitochondrial membranes, in infective T. annulata sporozoites fixed in situ within whole salivary glands of 3-day fed Hyalomma anatolicum anatolicum ticks.     

Immunisation of cattle against theileriosis in Coast Province, Kenya: laboratory evaluation of a Theileria parva parva stabilate for use in 'infection and treatment' immunisation in the field

Theileria parva parva Marikebuni stock, previously shown to give good protection to immunised cattle in Kilifi District, Coast Province of Kenya, was chosen for large scale immunisation in the district. A large sporozoite stabilate was prepared and evaluated for efficacy and safety in the 'infection and treatment' method, using a long or short acting formulation of oxytetracycline. Susceptible cattle were infected with selected doses of stabilate (10(0), 10(-1), 10(-1.7) and left either as untreated controls, or treated with one of the two oxytetracycline formulations. It was concluded that stabilate dilution at 10(-0.7) or 10(-1) in combination with either formulation of oxytetracycline would effect satisfactory immunisation. The short acting oxytetracycline treatment was judged to be the most efficacious in protecting cattle against homologous challenge. On heterologous challenge it was found that T p parva Marikebuni immune cattle were protected against seven T p parva stocks from Kilifi District and also against four stocks of T p parva from other areas of Kenya. In addition, the Marikebuni stock provided partial protection against challenge by T p lawrencei stocks. Furthermore, cattle immune to T p parva and T p lawrencei were protected against lethal challenge of T p parva Marikebuni stock. Thus, it appears that large scale immunisation of cattle against theileriosis in Kilifi District could be undertaken using the Marikebuni stock. With continued assessment, this stock could provide a master theilerial stock for immunisation against cattle theileriosis in areas free of buffaloes elsewhere in Kenya.     

Validation of an in vitro method to determine infectivity of cryopreserved sporozoites in stabilates of Theileria spp.

The infectivity of 15 cryopreserved Theileria spp. sporozoite stabilates was assessed semi-quantitatively by titration using naive peripheral blood mononuclear cells (PBM) in vitro in multi-well plates. Using the method described, the effective dilution, which would result in 50% of replicate wells infected (ED50), was calculated. The ED50 for 11 Theileria annulata stabilates in bovine PBM ranged from 10(-2.6) to 10(-4.2) dilutions of 1 tick equivalent (t.e.) ml(-1), one stabilate of Theileria parva 10(-2.2)t.e.ml(-1); and three Theileria lestoquardi stabilates in ovine PBM, from 10(-1.5) to 10(-1.8)t.e.ml(-1). Two of the T. annulata stabilates had been used individually to infect groups of calves: stabilate 52 produced more severe disease responses than stabilate 67, as measured by prepatent period, parasitosis, parasitaemia and death or recovery. This corresponded with the sixfold difference found in vitro between the ED50's of these two stabilates. This method is useful not only to measure the infection potential of the sporozoite stabilates but also as an in vitro model for chemotherapeutic and immunological studies of the early stages of theileriosis.     

Enzymes of glucose and glycerol catabolism in in vitro-propagated Theileria parva schizonts

Theileria parva schizonts propagated in vitro in peripheral blood lymphocytes were purified and assayed for key enzymes of glucose and glycerol catabolism and the citric acid cycle. The activities of glycolytic enzymes were in the range of 21-100 nmol/min/mg protein. Glycerol kinase and alpha -glycerophosphate dehydrogenase activities were more than 16 times lower than the activities of other enzymes catalysing the oxidation of the triose phosphates to lactate. It was suggested that the catabolism of glycerol is negligible and that glucose is catabolized to lactate via the Embden-Meyerhof pathway. The activities of the enzymes catalysing the section of the citric acid cycle that involves the formation of citrate to succinyl-CoA were consistently very low (less than 2.0 nmol/min/mg protein), indicating that this part of the cycle plays a minor role in this parasite. Enzyme activities of the cycle catalysing the formation of succinate from oxaloacetate were relatively higher than those catalysing other sections of the citric acid cycle, suggesting that this section of the cycle could be important to the parasite. Pyruvate carboxylase activity was more than 10 times that of phosphoenolpyruvate carboxykinase. It was suggested that pyruvate could be carboxylated to oxaloacetate. Taken together, these results suggest that the catabolism of glucose in Theileria parva schizonts is mainly via the Embden-Meyerhof pathway and that the citric acid cycle plays a minor role in energy production.     

Infectivity and cross-immunity studies of Theileria lestoquardi and Theileria annulata in sheep and cattle: I. In vivo responses.

In a series of experiments, sporozoite stabilates of a Theileria lestoquardi (Lahr) and a T. annulata (Ankara) stock prepared from Hyalomma anatolicum anatolicum ticks, were used to examine the infectivity of both parasite species for sheep and cattle and to study the development of cross-immunity between these parasite species. In the first experiment sheep and cattle were inoculated with T. lestoquardi sporozoites. Surviving animals and naive sheep and cattle were, in the second experiment, inoculated with T. annulata. In the third experiment, naive sheep and sheep previously infected with T. annulata, were inoculated with T. lestoquardi. The following responses to inoculations were monitored: clinical and haematological signs of infection, appearance of parasitic stages of the parasites in lymph node biopsies and in peripheral blood and serological response to T. lestoquardi and T. annulata schizont antigens. While T. lestoquardi readily infected sheep and caused severe disease, it did not infect cattle. On the other hand, T. annulata infected both cattle and sheep. However, whereas cattle became severely affected, infected sheep showed mild clinical symptoms only and piroplasms did not develop. Despite their different behaviour in the host species examined, cross-immunity studies suggested that the parasite species are very closely related. Experiments in sheep indicated that T. lestoquardi infection protected against subsequent T. annulata infection. On the other hand, recovery from T. annulata infection did not prevent infection by sporozoites of T. lestoquardi, resulting in the establishment of schizonts and their subsequent development into piroplasms, although it protected against the major clinical effects of T. lestoquardi infection.     

Epidemiological studies on Theileriosis and the dynamics of Theileria parva infections in Rwanda

An epidemiological analysis based on three country wide surveys was carried out to determine the prevalence of infections with Theileria spp. in Rwanda. In the 1998 dry season, a total of 264 blood samples were submitted to Theileria spp. characterisation using the 18S species-specific PCR-RFLP assay. The same samples together with 634 samples (317 samples/season) collected during the 2002 dry season and the 2003 wet season were further analysed using the p104 Theileria parva specific PCR. The results from the 18S characterisation showed the presence of four Theileria spp., namely T. parva, T. mutans, T. taurotragi and T. velifera in the field. Half of the animals had multiple Theileria spp. infections. T. parva was the most prevalent and a high correlation (94%) was found between the prevalence results using the 18S and the p104 PCR assays. The prevalence of T. parva infections was stable over time and over season but decreased significantly from the high land to the low land areas. This unexpected trend cannot be explained alone by ecology or the dynamics of the tick population in the different zones, many other components such as breed type, tick control practices and grazing system are likely to play a role. Another important finding was the fact that young animals are infected early in life in all regions except in the high land zone indicating the existence of a particular epidemiological situation in this part of the country.