Theileria parva epidemics: a case study in eastern Zambia

This paper presents the results of the follow-up of three sentinel herds between 1994 and 2000 during an East Coast fever (ECF) epidemic in eastern Zambia. The animals of the sentinel herds were closely monitored clinically and serologically together with detailed Rhipicephalus appendiculatus counts. Peaks of disease incidence occurred in the rainy season (December-February) and the dry months of May-July with nymph-to-adult tick transmission dominating the infection dynamics. A second wave of adult R. appendiculatus at the start of the dry season is essential for the occurrence of a full-blown epidemic while the size of the susceptible cattle population acts as a most important limiting factor. The majority of adult cattle of the sentinel herds became infected less than 2 years after the introduction of the disease. The median age at first contact for calves born towards the end of the study (1999) was about 6 months. The case-fatality ratio (including sub-clinical cases) is estimated at 60%. It is argued that part of the so-called 'natural mortality' is actually due to ECF and that ECF occurrence and mortality are systematically underestimated. The direct financial cost of the epidemic, based on loss of animals and cost of treatment only and calculated over 4 years running, is estimated at about 6 US dollars per year per animal at risk. The value of the traditional seroprevalence survey as a tool for monitoring ECF epidemiology is put in question and the prevalence of maternal antibodies in new-born calves, reflecting the immune status of the dam population, is introduced as an alternative. It is demonstrated that an efficient immunisation campaign should concentrate its efforts in the period of low adult R. appendiculatus abundance (July-October).     

Cellular immunity against Theileria parva and its influence on parasite diversity

Theileria parva, a tick-borne parasite of African cattle, causes a fatal disease known as East Coast fever. Cattle that recover from the disease develop strong parasite-specific MHC-class I-restricted cytotoxic T-lymphocyte responses. Protection can be transferred between immune and na?ve calves in the CD8+ T cell fraction emanating from a responding lymph node. In vitro studies suggest that this response requires input from activated CD4+ T cells. The T parva life cycle involves developmental stages in mammalian and tick hosts and can lead to a number of different endemic scenarios for the disease. These range from a stable situation with high prevalence of herd infection, but low fatality rates, to a low prevalence/high fatality scenario. The impact on endemic stability is an important consideration for the design of vaccine implementation strategies. For subunit vaccines targeted at T parva schizonts, the principal issue in this regard is whether development of the piroplasm stage is blocked by immunity.     

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.     

Theileria parva: the immune status of calves born of dams immunised against East Coast fever

Eight Friesian cross cows three months pregnant to a single Friesian bull were immunised against East Coast fever by infection with Theileria parva (Muguga) sporozoite stabilate and treatment with pyrrolidino-methyl tetracycline. They were challenged with the homologous stock four times before calving and a fifth time after calving, and resisted all five challenges which killed all of the five groups of five susceptible controls. Calves born to these hyperimmunised dams were fully susceptible on challenge with the same stabilate, as were susceptible cows from the same farm and their calves. In both instances the calves died three to seven days earlier than the cows which were approximately 10 times heavier. These results show that one- to two-month-old taurine calves from artificially immunised dams are not protected from experimental T parva sporozoite challenge and that there is no inherent calfhood resistance to East Coast fever.     

Immunization of cattle against Theileria parva bovis and their exposure to natural challenge

Theileria parva bovis isolates were tested for their immunizing capacity under natural field challenge on Willsbridge Farm in the highveld of Zimbabwe. Fifteen susceptible Sussex yearlings were immunized with the Boleni stock and 15 with a mixture of three isolates from the farm, using tick-derived sporozoite stabilates. No chemoprophylaxis was used. A dose of 0.1 ml of stabilate appeared to be safe in preliminary laboratory experiments, but the reactions were severe in the Sussex cattle and one died despite treatment. Twenty-nine immunized animals and 10 controls first experienced a mild infection, starting about 15 days after their arrival at the farm. Ten of the immunized animals and four controls had schizonts in peripheral lymph nodes for variable periods; one third of those had pyrexia. Nymphal Rhipicephalus appendiculatus ticks applied to three of the reacting immunized calves transmitted Theileria taurotragi to two animals and T. parva to a third. A second Theileria infection, due to T. parva bovis, was detected shortly after the first one. Schizonts were detected in seven out of 10 controls. Pyrexia was more severe and prolonged. Two of the controls died of theileriosis. At the same time schizonts were seen in three immune animals and eight of them had short periods of pyrexia. Intercurrent infections with Babesia bigemina, Borrelia theileri and Eperythrozoon were detected and may have contributed to the fever. Tick infestations were low during the exposure. In the second year of exposure, four out of eight new control animals had severe reactions, and one died. None of the immunized animals became ill, but one animal from the first year control group, which had not reacted previously, had clinical theileriosis. It is concluded that immunization provided an effective protection against field challenge.     

The biological and practical significance of antigenic variability in protective T cell responses against Theileria parva

The evolution of antigenically distinct pathogen strains that fail to cross-protect is well documented for pathogens controlled primarily by humoral immune responses. Unlike antibodies, which recognise native proteins, protective T cells can potentially recognise epitopes in a variety of proteins that are not necessarily displayed on the pathogen surface. Moreover, individual hosts of different MHC genotypes generally respond to different sets of epitopes. It is therefore less easy to envisage how strain restricted immunity can arise for pathogens controlled by T cell responses, particularly in antigenically complex parasites. Nevertheless, strain restricted immunity is clearly a feature of a number of parasitic infections, where immunity is known to be mediated by T cell responses. One such parasite is Theileria parva which induces potent CD8 T cell responses that play an important role in immunity. CD8 T cells specific for parasitized lymphoblasts exhibit strain specificity, which appears to correlate with the ability of parasite strains to cross-protect. Studies using recently identified T. parva antigens recognised by CD8 T cells have shown that the strain restricted nature of immunity is a consequence of the CD8 T cell response in individual animals being focused on a limited number of dominant polymorphic antigenic determinants. Responses in animals of different MHC genotypes are often directed to different parasite antigens, indicating that, at the host population level, a larger number of parasite proteins can serve as targets for the protective T cell response. Nevertheless, the finding that parasite strains show overlapping antigenic profiles, probably as a consequence of sexual recombination, suggests that induction of responses to an extended but limited set of antigens in individual animals may overcome the strain restricted nature of immunity.     

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.     

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.     

The influence of age and maternal antibodies on the postvaccinal response against swine influenza viruses in pigs

The influence of age and maternal immunity on the development and duration of postvaccinal humoral response against swine influenza viruses (SIV) were investigated under experimental conditions. Piglets born to immune and non-immune sows were vaccinated twice with bivalent inactivated vaccine. Vaccination was done according to 5 different schedules: 1+4, 1+8, 4+8, 8+10 or 8+12 weeks of age. Antibodies to the haemagglutinin type 1 and 3 were determined using the haemagglutination inhibition (HI) test. Maternally derived antibodies (MDA) against H1N1 and H3N2 in the serum of unvaccinated piglets born to immune sows were above the positive level until about 13-14 and 9-10 weeks of life, respectively. No serological responses were seen in any of the groups after the first vaccination. After the second dose of vaccine production of antibodies was observed even before the complete disappearance of maternal antibodies. MDA, however, were associated with reduced antibody response. In MDA-negative piglets, an active humoral postvaccinal response was developed in all vaccinated pigs. The age at which the vaccine was given was associated with the differences in the magnitude of antibody response to SIV. In general those pigs that were vaccinated for the first time at the age of 1 week, developed lower maximum titres after the second vaccination, and become seronegative earlier than pigs that were vaccinated for the first time at 4 or 8 weeks of age.     

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.     

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.     

Laboratory and field investigations into the Theileria parva carrier-state in cattle in Zimbabwe.

The Theileria parva carrier-state in cattle on commercial farms on Zimbabwe was investigated using parasitological and serological methods. The proportion of cattle showing Theileria piroplasms on two farms, which had recent histories of disease outbreaks, were 64% (n = 106, total of heifers and weaned calves examined) and 71.5% (n = 60) while the proportion of T. parva antibodies for the same animals were 59% and 98.5%, respectively. On four farms where no cases of the disease occurred for over 10 years, the average proportion of animals showing piroplasms and antibodies were 55.4% (range 32-82, n = 223) and 73% (range 47-91, n = 223), respectively. However, on another three farms which had no history of theileriosis outbreaks these proportions were very low, being 11.4% (0-24, n = 157) for piroplasms and 12.2% (5-23, n = 157) for antibodies. The mean infection rate in unfed Rhipicephalus appendiculatus adults collected from farms with a high prevalence of cattle which were carriers of Theileria piroplasms during the tick activity season was 29% (range 12-60%) with 9.3 (range 2-18.7) mean infected acini per infected tick. The infectivity of different tick batches to susceptible cattle produced a wide spectrum of theileriosis reactions. Laboratory controlled experiments were carried out to study the persistence of T. parva (Boleni) piroplasms in cattle immunized with this strain as well as its infectivity for ticks and its subsequent transmissibility to cattle. Examination of the salivary glands of 15 batches of ticks collected from six immunized cattle on three different occasions over 18 months showed that none were infected with Theileria parasites. However, the infectivity of other ticks in the same batches to susceptible animals was demonstrated 6, 10 and 18 months after cattle had been immunized with Boleni stabilate.     

Immunization against East Coast fever: the use of selected stocks of Theileria parva for immunization of cattle exposed to field challenge

Two antigenically different stocks of Theileria parva parva (Kilifi and Marikebuni), previously characterized as belonging to groups A and C respectively on monoclonal antibody (MAb) profiles, were selected for immunization of different breeds of cattle against East Coast fever (ECF) by the infection and treatment method. A total of 52 immunized cattle and 33 susceptible controls of different group sizes were exposed to field challenge by ticks for periods of 42-90 days at three field sites where ECF is endemic on the Kenyan coast. All immunized cattle survived ECF challenge, but 87% of the controls died of the disease. The cattle exposed at one site had been immunized 1 year earlier and maintained tick-free in the intervening period. The level of immunity in these cattle was similar to that of cattle which had been immunized 1 or 2 months prior to exposure. Thus, immunity had not waned over the 1-year period. A study at another site showed that acaricidal treatment of immunized cattle could be safely extended from twice a week to once every three weeks, whereas in susceptible cattle even twice weekly spraying did not control ECF. The isolates made from infected controls during the trials indicated the presence of three T. p. parva stocks as defined by MAb profiles. Of the two stocks used for immunization, T. p. parva Marikebuni induced broader protection. In view of the apparent limited antigenic diversity of T. p. parva strains within the Coast Province it is suggested that the Marikebuni stock might represent a key stock for vaccination in this area.     

Isolation and transmission of Theileria mutans (Chisamba) in Zambia

Theileria mutans (Chisamba) was isolated from a steer at Chisamba, Central Province, Zambia by inoculation of blood into a susceptible unsplenectomised calf. The parasite was then transmitted on three occasions by nymphs and once by adult Amblyomma variegatum ticks. Macroschizonts, typical for T mutans, were detected in two calves for short periods. The parasite caused varying degrees of anaemia in all experimental calves, whose sera showed high antibody titres to T mutans in the indirect fluorescent antibody test.     

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.