Titrating Theileria parva: single stocks against combination of stocks

Theileria parva is the causative agent of East Coast fever (ECF), an important cattle disease in East and Central Africa. One of the methods for control of ECF is 'infection and treatment', a procedure in which an animal is infected with the live parasite and at the same time treated with a long-acting oxytetracycline formulation, restraining the infection and allowing a protective cellular immune response to develop. Optimal immunizing doses were estimated using models of trichotomous response: dysimmunization (death or severe reaction during immunization), immunization failure (death or severe reaction during lethal challenge) and successful immunization (neither dysimmunization nor immunization failure). In this paper we present methods of interpreting immunization trials and apply these methods to previously unpublished data from two such trials: one with a mixture of three T. parva stocks and one with a single T. parva stock. We explain why titration trials conducted with a cocktail of antigens could predict a suboptimal immunization dose. Indeed it is possible for a combination of three individually efficient stocks to result in a mixture with which optimal immunization response might be difficult to achieve, because of averaging effects. The corresponding interpretation provides insights into why standard immunization trials for T. parva have not yielded the results that might be expected of them. The results of this work may also have implications for the use of antigen cocktails in cancer, HIV and malaria vaccine trials.     

Purification and characterization of precipitating antigens from Theileria parva.

Precipitating antigens from Theileria parva have been partially purified. Two antigens from each of the schizont and piroplasm stages of the parasite were identified; the major antigens from the two stages shared the same specificity. The antigens showed considerable molecular heterogeneity, almost certainly a result of the preparative method, and they always contained large amounts of DNA. The piroplasm antigens were of parasite nuclear origin and the schizont antigens were probably of the same origin. The antigens were weakly antigenic, and the activity against them of humoral antibody from cattle immune to East Coast fever was low. These antigens do not appear to induce protection against East Coast fever.     

Characterization of the Theileria parva sporozoite proteome

East Coast fever is a lymphoproliferative disease caused by the tick-borne protozoan parasite Theileria parva. The sporozoite stage of this parasite, harboured and released from the salivary glands of the tick Rhipicephalus appendiculatus during feeding, invades and establishes infection in bovine lymphocytes. Blocking this initial stage of invasion presents a promising vaccine strategy for control of East Coast fever and can in part be achieved by targeting the major sporozoite surface protein p67. To support research on the biology of T. parva and the identification of additional candidate vaccine antigens, we report on the sporozoite proteome as defined by LC–MS/MS analysis. In total, 4780 proteins were identified in an enriched preparation of sporozoites. Of these, 2007 were identified as T. parva proteins, representing close to 50% of the total predicted parasite proteome. The remaining 2773 proteins were derived from the tick vector. The identified sporozoite proteins include a set of known T. parva antigens targeted by antibodies and cytotoxic T cells from cattle that are immune to East Coast fever. We also identified proteins predicted to be orthologs of Plasmodium falciparum sporozoite surface molecules and invasion organelle proteins, and proteins that may contribute to the phenomenon of bovine lymphocyte transformation. Overall, these data establish a protein expression profile of T. parva sporozoites as an important starting point for further study of a parasitic species which has considerable agricultural impact.     

TpUB05, a Homologue of the Immunodominant Plasmodium falciparum Protein UB05, Is a Marker of Protective Immune Responses in Cattle Experimentally Vaccinated against East Coast Fever

Introduction

East Coast fever, a devastating disease of cattle, can be controlled partially by vaccination with live T. parva sporozoites. The antigens responsible for conferring immunity are not fully characterized. Recently it was shown that the P. falciparum immunodominant protein UB05 is highly conserved in T. parva, the causative agent of East Coast fever. The aim of the present investigation was to determine the role of the homologue TpUB05 in protective immunity to East Coast fever.

Methods

The cloning, sequencing and expression of TpUB05 were done according to standard protocols. Bioinformatics analysis of TpUB05 gene was carried out using algorithms found in the public domain. Polyclonal antiserum against recombinant TpUB05 were raised in rabbits and used for further analysis by Western blotting, ELISA, immunolocalization and in vitro infection neutralization assay. The ability of recombinant TpUB05 (r-TpUB05) to stimulate bovine PBMCs ex-vivo to produce IFN-γ or to proliferate was tested using ELISpot and [3H]-thymidine incorporation assays, respectively.

Results

All the 20 cattle immunised by the infection and treatment method (ITM) developed significantly higher levels of TpUB05 specific antibodies (p<0.0001) compared to the non-vaccinated ones. Similarly, r-TpUB05 highly stimulated bovine PMBCs from 8/12 (67%) of ITM-immunized cattle tested to produce IFN-γ and proliferate (p< 0.029) as compared to the 04 naїve cattle included as controls. Polyclonal TpUB05 antiserum raised against r-TpUB05 also marginally inhibited infection (p < 0.046) of bovine PBMCs by T. parva sporozoites. In further experiments RT-PCR showed that the TpUB05 gene is expressed by the parasite. This was confirmed by immunolocalization studies which revealed TpUB05 expression by schizonts and piroplasms. Bioinformatics analysis also revealed that this antigen possesses two transmembrane domains, a N-glycosylation site and several O-glycosylation sites.

Conclusion

It was concluded that TpUB05 is a potential marker of protective immunity in ECF worth investigating further.     

Methods currently used for the control of East Coast fever: their validity and proposals for future control strategies

Restriction of cattle movements, vector control, treatment and immunization are identified as the main control methods against East Coast fever (ECF). The effectiveness of these methods is very much influenced by cultural practices, economic and political pressures and development of resistance by ticks to acaricides. The proposed strategies for the future include continued restriction of cattle movements, less intensive vector control in enzootic areas so as to maintain enzootic stability, chemotherapy and immunization of the improved cattle. In addition, research efforts to evaluate strategic vector control, develop cheaper drugs, improve the quality and delivery of the stabilate vaccine, and identify and mass produce appropriate immunogenic subunit vaccines should be intensified.     

Control of Theileria annulata in Iran

Tropical theileriosis or Mediterranean Coast Fever - caused by Theileria annulata - is a disease of cattle widely distributed across southern Europe, north Africa and central Asia. Its distribution broadly corresponds with that of its main ixodid tick vectors Hyalomma excavatum and H. detritum (Fig. 1). 'Exotic' cattle (Bos taurus) are particularly susceptible with mortalities up to 40-80% in some areas, whereas indigenous cattle (B. indicus) generally suffer much lower mortalities (about 10%) confined mainly to calves. But because imported non-immune cattle are so susceptible, T. annulata represents a major constraint to livestock improvement programmes in many parts of the middle east and Asia. Cattle that recover from T. annulata infection generally show a solid, long-lasting immunity. For many years there have been programmes to protect cattle by inoculation with blood from sick animals, and more recently using live attenuated T. annulata vaccines prepared from cultured schizont-infected lymphoid cells. This article reviews a 14 year immunization programme against T. annulata in Iran.     

Effect of different East Coast Fever control strategies on fertility, milk production and weight gain of Sanga cattle in the Central Province of Zambia

Five different East Coast Fever (ECF) (Theileria parva infection) control strategies, based on tick control and/or ECF immunization, were tested in groups of traditionally managed Sanga cattle in the Central Province of Zambia over a period of 2.5 years. Two groups were kept under intensive tick control (sprayed weekly), one group immunized and one non-immunized. Two further groups were under no tick control, one group immunized and one non-immunized, while a fifth group was immunized against ECF and maintained under strategic tick control (18 sprays per year). Tick control increased milk production and weight gain but not fertility. Immunization had neither marked detrimental nor beneficial effects on the cattle productivity. The combination of strategic tick control and immunization resulted in the highest level of production and at the same time reduced the potential risk from other tick-borne diseases.     

Theileria parva: significance of leukocytes for infecting cattle

Using an artificial feeding technique, infective particles of Theileria parva were harvested in bovine blood in capillary tubes from prefed female Rhipicephalus appendiculatus over a 2-hr period. Inoculations of this blood feed pool invariably resulted in the establishment of patent East Coast fever in autogeneic or syngeneic cattle, i.e., the blood donors or their monozygotic twins, but not in unrelated animals. Mechanical passage of 4 × 10⁶ macroschizont-infected lymphoid cells, harvested from a heifer with East Coast fever, successfully induced patent theileriosis in the donor's monozygotic twin but not in a susceptible allogeneic bovid. The significance of parasite-cell association and histocompatibility of infected cells is discussed in relation to natural and mechanical transmission of Theileria parva.     

East Coast fever: the infectivity for cattle of infective particles of Theileria parva harvested in various substrates

Cunningham M.P., Brown C.G.D., Burridge M.J., Joyner L.P. and Purnell R.E. 1973. East Coast fever : the infectivity for cattle of infective particles of theileria parva harvested in various substrates. International Journal for Parasitology3: 335–338. Female Rhipicephalus appendiculatus ticks infected with Theileria parva were pre-fed on rabbits for 4 days before being removed and restrained on ‘plasticine’. They were then fed for 2-h periods on capillary tubes containing various substrates, the contents of which were subsequently inoculated into East Coast fever-susceptible cattle. Using this technique, precolostral calf serum and Eagle's Minimum Essential Medium, with the addition of Bovine Albumin Powder, were selected as substrates suitable for further laboratory investigation on East Coast fever as they were acceptable to the ticks and supported the viability of the parasites.     

Theileria parva seroprevalence in traditionally kept cattle in southern Zambia and El Nino

Sero-epidemiological surveys involving 27,526 cattle over a period of 8 years show that Theileria parva, the parasite causing East Coast fever (ECF) is found throughout southern Zambia. Higher values of T. parva sero-prevalence were observed in the plateau districts of Monze, Choma and Mazabuka than in the valley districts of Siavonga and Sinazongwe. Our results reveal a strong association between high T. parva sero-prevalence and the presence of the periodic climatic phenomenon known as the El Nino Southern Oscillation. More T. parva sero-positive samples were recorded during El Nino years (1997/98) (P<0.001) than other years in the study period. From this association, we conclude that Multiple El Nino Southern Oscillation Indices can be used to predict years with high or low ECF infection prevalence thereby contributing to the improved control of ECF in the area.     

Protective immunity against the protozoan Leishmania chagasi is induced by subclinical cutaneous infection with virulent but not avirulent organisms

Protective immunity against Leishmania major is provided by s.c. immunization with a low dose of L. major promastigotes or with dihydrofolate-thymidylate synthase gene locus (DHFR-TS) gene knockout L. major organisms. Whether these vaccine strategies will protect against infection with other Leishmania species that elicit distinct immune responses and clinical syndromes is not known. Therefore, we investigated protective immunity to Leishmania chagasi, a cause of visceral leishmaniasis. In contrast to L. major, a high dose s.c. inoculum of L. chagasi promastigotes was required to elicit protective immunity. Splenocytes from mice immunized with a high dose produced significantly greater amounts of IFN-gamma and lower TGF-beta than mice immunized with a low dose of promastigotes. The development of protective immunity did not require the presence of NK cells. Protection was not afforded by s.c. immunization with either attenuated L. chagasi or with L. major promastigotes, and s.c. L. chagasi did not protect against infection with L. major. Subcutaneous immunization with DHFR-TS gene knockouts derived from L. chagasi, L. donovani, or L. major did not protect against L. chagasi infection. We conclude that s.c. inoculation of high doses of live L. chagasi causes a subclinical infection that elicits protective immune responses in susceptible mice. However, L. chagasi that have been attenuated either by long-term passage or during the raising of recombinant gene knockout organisms do not elicit protective immunity, either because they fail to establish a subclinical infection or because they no longer express critical antigenic epitopes.     

Measles virus-specific CD4 T-cell activity does not correlate with protection against lung infection or viral clearance

Acute measles in children can be prevented by immunization with the live attenuated measles vaccine virus. Although immunization is able to induce CD4 and CD8 T cells as well as neutralizing antibodies, only the latter have been correlated with protective immunity. CD8 T cells, however, have been documented to be important in viral clearance in the respiratory tract, whereas CD4 T cells have been shown to be protective in a mouse encephalitis model. In order to investigate the CD4 T-cell response in infection of the respiratory tract, we have defined a T-cell epitope in the hemagglutinin (H) protein for immunization and developed a monoclonal antibody for depletion of CD4 T cells in the cotton rat model. Although the kinetics of CD4 T-cell development correlated with clearance of virus, the depletion of CD4 T cells during the primary infection did not influence viral titers in lung tissue. Immunization with the H epitope induced a CD4 T-cell response but did not protect against infection. Immunization in the presence of maternal antibodies resulted in the development of a CD4 T-cell response which (in the absence of neutralizing antibodies) did not protect against infection. In summary, CD4 T cells do not seem to protect against infection after immunization and do not participate in clearance of virus infection from lung tissue during measles virus infection. We speculate that the major role of CD4 T cells is to control and clear virus infection from other affected organs like the brain.     

A detergent-soluble extract of virus-infected cells free from infectious virus protects mice from lethal herpes simplex virus infection

Lethal infection with herpes simplex virus types 1 (HSV-1) and 2 was effectively prevented by previous immunization with a detergent-soluble extract (DSE) of virus-infected cells free from infectious virus without any adjuvant. This protective immunity seemed to last for at least one month. Neutralizing antibodies were elicited in mice immunized with DSE, but at a lower level than in animals immunized with live or killed virus. DSE did not protect athymic nude mice from death by HSV-1 infection, suggesting that a T cell-mediated immune response plays a major role in the protection.     

Chemical Restraint of Exotic Animals in a Disease Regulatory Program

The African red tick (Rhipicephalus evertsi Neuman) is widely distributed in Africa but had never been identified in North America until September 1960 when it was found in a wild animal compound in Florida. The red tick is a vector of cattle fever (piroplasmosis) and several other exotic diseases including the highly fatal East Coast fever. The 130 acre compound contained more than 300 African and Asian animals, including giraffes, camels, zebras, elands, nilghais, blackbuck, aoudads, ostriches and Abyssinian asses. To determine the extent of the infestation, representative numbers of animals were immobilized with succinylcholine chloride for visual and manual examination. The grounds were treated with 2 lbs. of DDT per acre at three week intervals for six weeks and another group of animals were immobilized to determine the efficacy of the program. In all more than 50 wild animals were successfully immobilized with succinylcholine.     

An update on the ecological distribution of Ixodid ticks infesting cattle in Rwanda: countrywide cross-sectional survey in the wet and the dry season

As part of the epidemiological studies aimed at developing an East Coast fever (ECF) immunisation control strategy, which combines an infection and treatment method with strategic tick control, a countrywide tick survey was carried out in both the dry and the wet season to determine the abundance and the dynamics of the tick populations infesting cattle in Rwanda. Six Ixodid tick species where identified from a total of 12,814 tick specimens collected. Rhipicephalus appendiculatus, the main vector of ECF was the most abundant (91.8%) followed by Boophilus decoloratus (6.1%) and Ambyomma variegatum (1.2%). Few ticks from the three other less economically important Ixodid species (Rhipicephalus compositus, R. evertsi evertsi and Ixodes cavipalpus) were recovered. Both adult and immature stages of the most dominant tick species were found to be widespread with a year round presence. The numbers of ticks were high in low land and medium zones and declined markedly in the higher regions of Rwanda. The geographical distribution of various tick species throughout the country and their epidemiological implications are discussed.     

Nordosten der USA

Field trip: The US‐East‐Coast The US‐East‐Coast is a flyway for millions of birds. The Chesapeake Bay with the Delmarva‐Peninsula and her National Wildlife Refuges (NWR) are most attractive destinations to study nature and her history in her habitats for understanding evolution and ecology.     

Assessment of Theileria infections in Rhipicephalus appendiculatus ticks collected from the field

Collections of adult Rhipicephalus appendiculatus ticks were made from bait cattle and vegetation at two field sites in areas of Kenya in which East Coast fever caused by Theileria parva is endemic. These ticks, together with two experimentally infected batches of ticks, were examined for infection with Theileria by four methods. Whole salivary glands were stained with methyl green pyronin or Feulgen's stain. Whole ticks were ground in medium, the suspensions were filtered and centrifuged and the treated material was examined microscopically and tested for infectivity by inoculation into cattle. All field collections and experimental batches of ticks were infected with Theileria and all four methods detected the infections. Approximately 1.5% of the ticks in the field collections were found to be infected with Theileria and the treated material from these ticks transmitted T. parva to cattle. It is considered that it will be feasible to survey field infection rates quantitatively by collecting ticks from bait cattle and vegetation for examination by a combination of salivary gland staining and preparation of tick suspensions for microscopy and infectivity tests.     

Salivary gland of the tick vector (R. appendiculatus) of East Coast fever. I. Ultrastructure of the type III acinus

The brown ear tick Rhiplcephalus appendiculatus is the vector for East Coast fever, a disease that seriously limits livestock production in East Africa. The sporozoites of the infectious agent Theileria parva develop in the tick salivary gland. This paper describes the organization of the type III acinus of the gland and establishes unambiguous ultrastructural criteria for identification of the three secretory cell types: the d-cell, e-cell and f-cell. These observations are basic to exploration of possible cell-type specificity of the invading theileria and other aspects of host-parasite relations.     

Protective immune mechanisms against Theileria parva: evolution of vaccine development strategies.

Theileria parva is an intracellular sporozoan parasite that infects and transforms bovine lymphocytes, causing a severe lymphoproliferative disease known as East Coast fever in eastern, central and southern Africa. In this article, Declan McKeever and colleagues summarize the current understanding of immune mechanisms provoked by the parasite with regard to their role in both pathogenesis and protection. In particular, the influence of genomic polymorphism in parasite and host on the development of immunity is discussed, along with the evolution of current vaccine development strategies as a result of immunological research on the disease.