Survival of Rhipicephalus appendiculatus (Acarina: Ixodidae) and persistence of Theileria parva (Apicomplexa: Theileriidae) in the field

Newson R. M., Chiera J. W., Young A. S., Dolan T. T., Cunningham M. P. and Radley D. D. 1984: Survival of Rhipicephalus appendiculatus (Acarina: Ixodidae) and persistence of Theileria parva (Apicomplexa: Theileriidae) in the field. International Journal for Parasitology14; 483–489. Two paddocks with populations of the African brown ear tick (Rhipicephalus appendicuiatus), one of which carried virulent Theileria parva (the causative organism of East Coast fever [ECF] of cattle), were left unstocked for periods of 338 and 354 days. Groups of 1–3, ECF-susceptible cattle were then introduced eight times during the following year to assess the tick and disease challenge, The ticks were also monitored continuously on the ground. The test cattle developed fatal ECF from adult ticks which had fasted for up to 554 days. A non-pathogenic, antigenicaliy distinct, Theileria species was also detected which was still transmitted by adult ticks after 600 days. The mean survival time of unfed larvae was 175 days (max. 240 days); unfed nymphs 270 and 450 (max. 540 days); unfed adults 420 days (max. 730 days), with females in a clear majority after 270 days.     

Cryopreservation of infective particles of Theileria parva

Cryopreservation of infective particles of Theileria parva. International Journal for Parasitology3: 583–587. Infective particles of Theileria parva, the causative organism of East Coast fever of cattle, were obtained from infected Rhipicephalus appendiculatus ticks, either by using an in vitro feeding technique or by grinding the ticks in a suitable medium. If foetal calf serum containing 15% glycerol (v/v) was added to the infective material and it was then distributed either to glass capillary tubes (in vitro tick feed) or glass tubes (ground tick supernate) it could be slowly frozen to either ?80°C or ?196°C without loss of viability. Stabilates, tested by rapid thawing and inoculation into ECF-susceptible cattle, remained viable for up to a year at these temperatures.     

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.     

Some factors controlling the stimulation of sporogony of Theileria parva in its tick vector Rhipicephalus appendiculatus

Young A. S., Leitch B. L. and Mutugi J. J. 1984. Some factors controlling the stimulation of sporogony of Theileria parva in its tick vector, Rhipicephalus appendiculatus. International Journal for Parasitology14: 97–102. The effect of various temperature treatments on the sporogony cycle of Theileria parva in the salivary glands of unfed adult Rhipicephalus appendiculatus ticks of various ages was investigated. It was found that ticks incubated at 28 or 37°C would develop sporozoites infective to cattle but never in such large numbers as in ticks fed on rabbits. Heat stimulation of sporogony was possible for isolates of T. parva with minimal laboratory handling. The age of the ticks incubated at 28 or 37°C was important since sporozoites could only be induced at the earliest on day 27 or 28 p.repl. (post-repletion) and at the latest by day 41 p.repl. The age of ticks fed on rabbits was not as important for the production of sporozoites.     

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.     

Growth of <Emphasis Type="Italic">Theileria parva</Emphasis>-infected Bovine Lymphoid Cells in Irradiated Mice

SEVERAL attempts to establish Theileria parva, the causative organism of East Coast fever of cattle, in laboratory animals^1–4 have failed. The recent successful establishment of T. parva macroschizont-infected bovine lymphoid cells in tissue culture⁵ provided a concentrated source of material which could be used in further attempts to infect laboratory animals.     

East coast fever: 60Co-irradiation of Theileria parva in its tick vector, Rhipicephalus appendiculatus

Three experiments were carried out in which Theileria parva was irradiated in its tick vector, Rhipicephalus appendiculatus. In the first experiment, infected unfed adult ticks were irradiated at doubling doses from 4 to 32 krad. Some of the ticks were then fed for 4, 5, 6, 7 and 8 days on rabbits, and the parasites in their salivary glands examined. Five male and 5 female ticks from each irradiation dose were put onto each of a pair of susceptible cattle, whose reactions were recorded. Increasing doses of irradiation resulted in progressive destruction of the parasites. All cattle receiving ticks irradiated at doses up to and including 16 krad died of East Coast fever (ECF), and one of the cattle receiving ticks irradiated at 32 krad died.In the second experiment, recently engorged nymphs were irradiated at 1, 2 or 4 krad, and moulting nymphs at doses of 2, 4, 8, 12, 16 or 32 krad. The salivary glands of the resultant adult ticks were examined after the ticks had fed for 4, 5, 6, 7 or 8 days on rabbits. Engorged nymphs irradiated at 4 krad failed to moult, whilst moulting nymphs irradiated at 32 krad moulted but failed to attach to rabbits. Doses of irradiation survived by the ticks had no apparent morphological effect on the parasites they contained.In the third experiment, infected unfed adult ticks were irradiated at 0, 5, 10, 15, 20 25 30, 35, 40, 45, 50 or 60 krad. The ticks were fed on rabbits for 5, 6 or 7 days. Some of them were then examined morphologically, whilst others were ground in MEM/BPA and aliquots of the supernatant used to inoculate groups of 5 cattle. The reaction of these cattle, together with the morphological examination of the parasites, suggested that increasing doses of irradiation destroyed increasing numbers of parasites.     

A locus conferring tolerance to Theileria infection in African cattle

East Coast fever, a tick-borne cattle disease caused by the Theileria parva parasite, is among the biggest natural killers of cattle in East Africa, leading to over 1 million deaths annually. Here we report on the genetic analysis of a cohort of Bos indicus (Boran) cattle demonstrating heritable tolerance to infection with T. parva (h² = 0.65, s.e. 0.57). Through a linkage analysis we identify a 6 Mb genomic region on bovine chromosome 15 that is significantly associated with survival outcome following T. parva exposure. Testing this locus in an independent cohort of animals replicates this association with survival following T. parva infection. A stop gained variant in a paralogue of the FAF1 gene in this region was found to be highly associated with survival across both related and unrelated animals, with only one of the 20 homozygote carriers (T/T) of this change succumbing to the disease in contrast to 44 out of 97 animals homozygote for the reference allele (C/C). Consequently, we present a genetic locus linked to tolerance of one of Africa’s most important cattle diseases, raising the promise of marker-assisted selection for cattle that are less susceptible to infection by T. parva.     

Loop-mediated isothermal amplification (LAMP) assays for detection of Theileria parva infections targeting the PIM and p150 genes

We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria parva, the causative agent of East Coast fever (ECF), an economically important cattle disease in eastern, central and southern Africa. These assays target the polymorphic immunodominant molecule (PIM) and p150 LAMP genes. The primer set for each gene target consists of six primers, and each set recognises eight distinct regions on the target gene to give highly specific detection of T. parva. The detection limit of each primer set is 1 fg, which is equivalent to one copy of the PIM and p150 T. parva genes. These PIM and p150 LAMP primer sets amplify DNA of T. parva isolates from cattle and buffalo from different countries including Kenya, South Africa, Tanzania, Rwanda, Uganda and Burundi, indicating their ability to detect T. parva from different countries. With the advantages of simplicity, rapidity and cost effectiveness, these LAMP assays are good candidates for molecular epidemiology studies and for monitoring control programs in ECF-endemic, resource poor countries.     

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.     

East Coast fever: quantitative studies of Theileria parva in cattle

A series of experiments is described in which infective material obtained by grinding adult Rhipicephalus appendiculatus ticks containing mature Theileria parva parasites was titrated in East Coast fever-susceptible cattle. The reactions of the cattle to the various inocula, and the rate of multiplication of macroschizonts in their lymph nodes, were studied. In the final experiment, conclusive evidence was produced to support the observations of previous workers that the prepatent period, time to onset of febrile response, and time to death of the animals was dose-dependent, whereas the production of intraerythrocytic piroplasms was totally time-dependent. Furthermore, the effective rate of multiplication of macroschizonts was shown for the first time to be dose-dependent. It was not possible to detect macroschizonts before the fifth day after inoculation, and an occult phase of the parasite's life cycle, between the infective particle and the uninuclear macroschizont, is postulated. The discrepancies between the results of the present work and those of previous workers are discussed.     

Absolute Quantification of the Host-To-Parasite DNA Ratio in Theileria parva-Infected Lymphocyte Cell Lines

Theileria parva is a tick-transmitted intracellular apicomplexan pathogen of cattle in sub-Saharan Africa that causes East Coast fever (ECF). ECF is an acute fatal disease that kills over one million cattle annually, imposing a tremendous burden on African small-holder cattle farmers. The pathology and level of T. parva infections in its wildlife host, African buffalo (Syncerus caffer), and in cattle are distinct. We have developed an absolute quantification method based on quantitative PCR (qPCR) in which recombinant plasmids containing single copy genes specific to the parasite (apical membrane antigen 1 gene, ama1) or the host (hypoxanthine phosphoribosyltransferase 1, hprt1) are used as the quantification reference standards. Our study shows that T. parva and bovine cells are present in similar numbers in T. parva-infected lymphocyte cell lines and that consequently, due to its much smaller genome size, T. parva DNA comprises between 0.9% and 3% of the total DNA samples extracted from these lines. This absolute quantification assay of parasite and host genome copy number in a sample provides a simple and reliable method of assessing T. parva load in infected bovine lymphocytes, and is accurate over a wide range of host-to-parasite DNA ratios. Knowledge of the proportion of target DNA in a sample, as enabled by this method, is essential for efficient high-throughput genome sequencing applications for a variety of intracellular pathogens. This assay will also be very useful in future studies of interactions of distinct host-T. parva stocks and to fully characterize the dynamics of ECF infection in the field.     

High-resolution predictive mapping for Rhipicephalus appendiculatus (Acari: Ixodidae) in the Horn of Africa

The brown ear tick Rhipicephalus appendiculatus, vector of East Coast fever (ECF) and related cattle diseases caused by Theileria parva has never been reported from the Horn of Africa. Habitat suitability for this tick species was predicted using Maxent modelling technique based on R. appendiculatus records in Sub-Saharan Africa. Two models were developed: the first is based on the tropical R. appendiculatus distribution and the one is based on the distribution records in the temperate region of Sub-Saharan Africa. The tropical model shows favourable habitat in much of the Ethiopian highlands. The whole Djibouti, the south eastern Ethiopian lowlands, majority of Somalia and Eritrea were found to be not suitable for the survival and development of this tick species. Highly suitable areas occur in areas which have moderate temperature and high precipitation. Introductions of R. appendiculatus into the Horn of Africa probably have been prevented by the natural barrier between the known R. appendiculatus distribution range in East Africa and the Horn of Africa. The effect of an introduction of R. appendiculatus and thereby ECF into the Horn of Africa could be catastrophic since the cattle in this area have no immunity against ECF, and mortality might be considerable in all age groups of cattle.     

Construction of a genetic map for Theileria parva: identification of hotspots of recombination

The tick-borne protozoan parasite Theileria parva is the causal agent of East Coast Fever (ECF), a severe lymphoproliferative disease of cattle in eastern, central and southern Africa. The life cycle of T. parva is predominantly haploid, with a brief diploid stage occurring in the tick vector that involves meiotic recombination. Resolved genetic studies of T. parva are currently constrained by the lack of a genome-wide high-definition genetic map of the parasite. We undertook a genetic cross of two cloned isolates of T. parva to construct such a map from 35 recombinant progeny, using a genome-wide panel of 79 variable number of tandem repeat markers. Progeny were established by in vitro cloning of cattle lymphocytes after infection with sporozoites prepared from Rhipicephalus appendiculatus ticks fed on a calf undergoing a dual infection with the two clonal parental stocks. The genetic map was determined by assigning individual markers to the four chromosome genome, whose physical length is approximately 8309 kilobasepairs (Kb). Segregation analysis of the markers among the progeny revealed a total genetic size of 1683.8 centiMorgans (cM), covering a physical distance of 7737.62 Kb (∼93% of the genome). The average genome-wide recombination rate observed for T. parva was relatively high, at 0.22 cM Kb⁻¹ per meiotic generation. Recombination hot-spots and cold-spots were identified for each of the chromosomes. A panel of 27 loci encoding determinants previously identified as immunorelevant or likely to be under selection were positioned on the linkage map. We believe this to be the first genetic linkage map for T. parva. This resource, with the availability of the genome sequence of T. parva, will promote improved understanding of the pathogen by facilitating the use of genetic analysis for identification of loci responsible for variable phenotypic traits exhibited by individual parasite stocks.     

Dose dependent responses of cattle to Theileria parva stabilate

Dolan T. T., Young A.S., Losos G.J., McMillan I., Minder Ch.E. and Soulsby K. 1984. Dose dependent responses of Theileria parva stabilate. International Journal for Parasitology14: 89–95. A tick derived stabilate of Theileria parva (Maguga) was titrated in a large group of Boran (Bos indicus) cattle of the same age, sex and origin. The infectivity data was analysed using the independent action model. The cattle were identified as heterogeneous in their response to infection with 75% showing one ID₅₀ (0.0014) and 25% showing another (0.01). The disease responses of the cattle given different dose levels were compared for a variety of parameters. The results obtained showed these parameters to be dose dependent including the time to onset of piroplasm parasitaemia. The stabilate is of large volume and can be used for controlled challenge in immunity studies and for comparison of susceptibility between cattle of different breeds and from different epidemiological backgrounds.     

Theileria parva: Early events in the development of bovine lymphoblastoid cell lines persistently infected with macroschizonts

The cellular origin and development of bovine lymphoblastoid cell lines persistently infected with macroschizonts of Theileria parva was studied. Cultures of lymphoblastoid cells isolated from cattle with patent East Coast fever were compared with those obtained by infecting normal lymphocytes in vitro with sporozoites. The young lines were contrasted with a continuous line which had been isolated earlier. The mononuclear cells were separated from the blood and the inoculum enriched for lymphoblastoid cells and/or lymphocytes by removing the monocytes. The lines arose directly from lymphoblastoid cells transplanted into culture or from lymphocytes infected by sporozoites. In primary cultures of lymphoblastoid cells from the peripheral blood, there was an increase in the proportion of infected cells without the eclipse of the parasite, the macroschizonts were larger than those observed in the inoculum or the continuous line, and there was concurrent microschizont differentiation. In lymphocyte cultures challenged with sporozoites, small mononucleated trophozoites were observed after 2 days which differentiated into typical macroschizonts but microschizonts were rare. In all cultures, the infected cells had mitotic indices of 4 to 5%. As the young lines were passaged, the parasites came to resemble those of the continuous line. The macroschizont size in the continuous line was stable and most had six to eight nuclei but when cultured at high cell concentrations the number of parasite nuclei increased. Minicultures of lymphocytes were used to quantitate the infectivity of sporozoites obtained from organ cultures of Rhipicephalus appendiculatus savliary glands. Sporozoites from ticks fed on rabbits for 5 days were approximately six times more infective than those from glands of ticks fed for 2 days and then cultured at 32 °C for 3 days. Glands from unfed ticks cultured for 5 days failed to yield infective sporozoites.     

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.     

The incorporation of radio-labelled nucleic acid precursors by Theileria parva in bovine blood and salivary glands of Rhipicephalus appendiculatus ticks

Irvin A.D., Boarer C.D.H., Kurtti T.J. and Ocama J.G.R. 1981. The incorporation of radio-labelled nucleic acid precursors by Theileria parva in bovine blood and salivary glands of Rhipicephalus appendiculatus ticks. International Journal for Parasitology11:451–456. The uptake of radio-labelled nucleic acid precursors by blood and tick salivary gland forms of Theileria pana was studied. Piroplasms took up tritiated purines, particularly hypoxanthine, but not pyrimidines. Similar uptake was recorded by T. parva, both in tick saliva and in salivary glands maintained in vitro. Intermediate parasite stages were those most readily labelled in glands; this reflected active nucleic acid synthesis associated with rapid parasite division. Radio-labelling of T. parva in tick salivary glands could be of value in procedures used for concentrating and purifying theilerial sporozoites.     

Whole-Genome Sequencing of Theileria parva Strains Provides Insight into Parasite Migration and Diversification in the African Continent

The disease caused by the apicomplexan protozoan parasite Theileria parva, known as East Coast fever or Corridor disease, is one of the most serious cattle diseases in Eastern, Central, and Southern Africa. We performed whole-genome sequencing of nine T. parva strains, including one of the vaccine strains (Kiambu 5), field isolates from Zambia, Uganda, Tanzania, or Rwanda, and two buffalo-derived strains. Comparison with the reference Muguga genome sequence revealed 34 814–121 545 single nucleotide polymorphisms (SNPs) that were more abundant in buffalo-derived strains. High-resolution phylogenetic trees were constructed with selected informative SNPs that allowed the investigation of possible complex recombination events among ancestors of the extant strains. We further analysed the dN/dS ratio (non-synonymous substitutions per non-synonymous site divided by synonymous substitutions per synonymous site) for 4011 coding genes to estimate potential selective pressure. Genes under possible positive selection were identified that may, in turn, assist in the identification of immunogenic proteins or vaccine candidates. This study elucidated the phylogeny of T. parva strains based on genome-wide SNPs analysis with prediction of possible past recombination events, providing insight into the migration, diversification, and evolution of this parasite species in the African continent.     

Feeding electrograms and fluid uptake measurements of cattle tick Boophilus microplus attached on artificial membranes

Waladde S. M., Kemp D. H. and Rice M. J. 1979. Feeding electrograms and fluid uptake measurements of cattle tick Boophilus microplus attached on artificial membranes. International Journal for Parasitology9: 89–95. Newly moulted adult females of the cattle tick Boophilus microplus readily attach on a modified Baudruche membrane. Apparatus design permits the media offered below the membrane to be kept at 37°C and to be changed easily. Patterns of feeding activity were recorded within 2 h of placing the ticks on the membrane by monitoring the changes in electrical resistance between a tick and the medium with a high input impedance electric circuit. Differences in patterns of sucking and salivation were related to the chemical composition of media presented below the membrane. These observations suggest that the newly discovered cheliceral taste receptors of B. microplus are able to mediate changes in feeding patterns in response to stimulation by different chemical solutions in the feeding lesion. Incorporation of phosphorus-32 into the medium allows the volume ingested by ticks to be measured. The techniques described here facilitate the study of host factors that influence attachment, engorgement and detachment of the cattle tick.