Isolation of infectious Theileria parva sporozoites secreted by infected Rhipicephalus appendiculatus ticks into an in vitro tick feeding system.

BACKGROUND: Vector-borne diseases pose an increasing threat to global food security. Vaccines, diagnostic tests, and therapeutics are urgently needed for tick-borne diseases that affect livestock. However, the inability to obtain significant quantities of pathogen stages derived from ticks has hindered research. In vitro methods to isolate pathogens from infected tick vectors are paramount to advance transcriptomic, proteomic, and biochemical characterizations of tick-borne pathogens. METHODS: Nymphs of Rhipicephalus appendiculatus were infected with Theileria parva by feeding on a calf during an acute infection. Isolation of sporozoites was accomplished by feeding infected adult ticks on an in vitro tick feeding system. Sporozoite viability was tested using in vitro bovine lymphocytes. RESULTS: We isolated infectious T. parva sporozoites secreted into an in vitro tick feeding system. Infected adult R. appendiculatus ticks attached to and successfully fed on silicone membranes in the in vitro tick feeding system. Bovine blood in the receptacle was replaced with cell-free medium and the ticks were allowed to feed for 3 h to collect secreted T. parva sporozoites. Secreted sporozoites infected in vitro bovine lymphocytes, demonstrating that isolated sporozoites remained viable and infectious. CONCLUSIONS: This work is the first to report the isolation of mature infectious T. parva sporozoites using an in vitro tick feeding system, which represents a significant step towards the development of a more efficient control strategy for T. parva. Isolation of infectious tick-stage parasites will facilitate the examination of the vector-pathogen interface, thereby accelerating the development of next-generation vaccines and treatment interventions for tick-borne pathogens.

CD4 T Cell Responses to Theileria parva in Immune Cattle Recognize a Diverse Set of Parasite Antigens Presented on the Surface of Infected Lymphoblasts.

Parasite-specific CD8 T cell responses play a key role in mediating immunity against Theileria parva in cattle (Bos taurus), and there is evidence that efficient induction of these responses requires CD4 T cell responses. However, information on the antigenic specificity of the CD4 T cell response is lacking. The current study used a high-throughput system for Ag identification using CD4 T cells from immune animals to screen a library of ∼40,000 synthetic peptides representing 499 T. parva gene products. Use of CD4 T cells from 12 immune cattle, representing 12 MHC class II types, identified 26 Ags. Unlike CD8 T cell responses, which are focused on a few dominant Ags, multiple Ags were recognized by CD4 T cell responses of individual animals. The Ags had diverse properties, but included proteins encoded by two multimember gene families: five haloacid dehalogenases and five subtelomere-encoded variable secreted proteins. Most Ags had predicted signal peptides and/or were encoded by abundantly transcribed genes, but neither parameter on their own was reliable for predicting antigenicity. Mapping of the epitopes confirmed presentation by DR or DQ class II alleles and comparison of available T. parva genome sequences demonstrated that they included both conserved and polymorphic epitopes. Immunization of animals with vaccine vectors expressing two of the Ags demonstrated induction of CD4 T cell responses capable of recognizing parasitized cells. The results of this study provide detailed insight into the CD4 T cell responses induced by T. parva and identify Ags suitable for use in vaccine development.

Synergistic Effect of Two Nanotechnologies Enhances the Protective Capacity of the Theileria parva Sporozoite p67C Antigen in Cattle.

East Coast fever (ECF), caused by Theileria parva, is the most important tick-borne disease of cattle in sub-Saharan Africa. Practical disadvantages associated with the currently used live-parasite vaccine could be overcome by subunit vaccines. An 80-aa polypeptide derived from the C-terminal portion of p67, a sporozoite surface Ag and target of neutralizing Abs, was the focus of the efforts on subunit vaccines against ECF and subjected to several vaccine trials with very promising results. However, the vaccination regimen was far from optimized, involving three inoculations of 450 µg of soluble p67C (s-p67C) Ag formulated in the Seppic adjuvant Montanide ISA 206 VG. Hence, an improved formulation of this polypeptide Ag is needed. In this study, we report on two nanotechnologies that enhance the bovine immune responses to p67C. Individually, HBcAg-p67C (chimeric hepatitis B core Ag virus-like particles displaying p67C) and silica vesicle (SV)-p67C (s-p67C adsorbed to SV-140-C18, octadecyl-modified SVs) adjuvanted with ISA 206 VG primed strong Ab and T cell responses to p67C in cattle, respectively. Coimmunization of cattle (Bos taurus) with HBcAg-p67C and SV-p67C resulted in stimulation of both high Ab titers and CD4 T cell response to p67C, leading to the highest subunit vaccine efficacy we have achieved to date with the p67C immunogen. These results offer the much-needed research depth on the innovative platforms for developing effective novel protein-based bovine vaccines to further the advancement.

The Rhipicephalus appendiculatus tick vector of Theileria parva is absent from cape buffalo (Syncerus caffer) populations and associated ecosystems in northern Uganda.

Rhipicephalus appendiculatus is the major tick vector of Theileria parva, an apicomplexan protozoan parasite that causes the most economically important and lethal disease of cattle in East and central Africa. The African cape buffalo (Syncerus caffer) is the major wildlife host of T. parva from southern Uganda and Kenya to southern Africa. We show herein that R. appendiculatus appears to be absent from the two largest national parks in northern Uganda. Syncerus caffer is common in both of these national parks, specifically Murchison falls (MFNP) and Kidepo Valley (KVNP). We re-confirmed the previously reported absence of T. parva in buffalo sampled in the two northern parks based on RLB data using a nested PCR based on the T. parva p104 gene. By contrast, T. parva-infected R. appendiculatus ticks and parasite-infected buffalo were present in Lake Mburo (LMNP) in South central Uganda. This suggests that the distribution of R. appendiculatus, which is predicted to include the higher rainfall regions of northern Uganda, may be limited by additional, as yet unknown factors.

Effect of chemical tick control practices on tick infestation and Theileria parva infection in an intensive dairy production region of Uganda.

Chemical tick control is a major means of preventing East Coast fever (ECF), especially in sub-Saharan Africa. However, in southwestern Uganda, improper tick control practices have led to severe acaricide resistance. The objectives of this study were to determine the risk factors associated with tick infestation in dairy cattle and Theileria parva infection, and to generate evidence for the prioritization of effective countermeasures for tick control. A cross-sectional study was conducted in 30 farms in Mbarara District, and information on tick control practices and tick infestation were collected. Tick samples were collected from 13 farms to test tick acaricide efficacy. A total of 420 blood samples from calves to adults of exotic- and cross-breed dairy cattle were collected, and T. parva diagnosis via polymerase chain reaction was performed. All the 13 tick populations tested were resistant to deltamethrin (synthetic pyrethroid). Resistance to single-formulation organophosphate-chlorfenvinphos was 39 % (5/13); co-formulations (chlorpyrifos + cypermethrin), 69 % (9/13); and amitraz (amidine), 85 % (11/13). The overall prevalence of T. parva infection at the individual-level was 45.2 % (190/420, 95 % confidence interval (CI): 40.4-50.1), and that at the farm-level was 83 % (25/30, 95 %CI: 65-94). A good quality cattle crush was a preventive factor for tick infestation (odds ratio (OR): 0.32, 95 %CI: 0.15-0.63, p =  0.001). Well-managed acaricide storage (OR: 0.36, 95 %CI: 0.17-0.76, p =  0.008), and a good quality measuring cylinder for acaricide were preventive factors (OR: 0.32, 95 %CI: 0.11-0.93, p = 0.036) for T. parva infection. The risk factors for T. parva infection were a longer period of acaricide use of the same brand (OR: 1.06, 95 %CI: 1.01-1.10, p = 0.012), and a higher frequency (twice a week) of acaricide use rather than once a week (OR: 11.70, 95 %CI: 1.95-70.13, p = 0.007). These risk factors should be given high intervention priority in order to effectively control ticks and prevent T. parva infections in dairy farms. Teaching proper practices to dairy farmers and to technical staff should be used to overcome the severe challenge of acaricide resistance in Mbarara District.

Molecular epidemiology of Babesia species, Theileria parva, and Anaplasma marginale infecting cattle and the tick control malpractices in Central and Eastern Uganda.

East Coast fever, babesiosis, and anaplasmosis are the major tick-borne diseases affecting cattle productivity in Uganda. The emergence of acaricide-resistant ticks is suspected to have caused a rise in hemoparasites. This study sought to detect and characterize hemoparasites among farms in acaricide-failure hotspots of central as compared to the acaricide-failure naïve areas in Eastern Uganda. Nested PCR assays were performed to determine the prevalences of Babesia bovis, Babesia bigemina, Theileria parva, and Anaplasma marginale in cattle blood samples sourced from randomly selected farms. Randomly selected isolates were sequenced to determine the genetic diversity of the parasites using the following marker genes: B. bovis spherical body protein 4, B. bigemina rhoptry-associated protein 1a, T. parva 104 kDa microneme-rhoptry antigen, and A. marginale major surface protein 5. Furthermore, partially and fully engorged adult ticks were collected for taxonomy, and tick-control practices were assessed using a semi-structured questionnaire. The prevalences of B. bigemina, T. parva, and A. marginale in cattle were 17.2, 65.1, and 22.0%, and 10.0, 26.5, and 3% in the central and eastern region, respectively. Whilst, B. bovis was not detected in the farms involved. The sequences for B. bigemina, T. parva, and A. marginale from the central region showed 99% identity with those from the eastern region. Of the 548 ticks collected, 319, 147, 76, and 6 were Rhipicephalus (Boophilus) decoloratus, Rhipicephalus appendiculatus, Amblyomma variegatum, and Rhipicephalus evertsi evertsi, respectively. The Rhipicephalus ticks were more abundant in the central region, whereas A. variegatum ticks were more abundant in the eastern region. Tick control malpractices were found in both Central and Eastern Uganda, and 42 of the 56 surveyed farms lacked appropriate restraining facilities and so they utilized either ropes or a 'boma' (enclosure). In summary, B. bigemina, T. parva, A. marginale and their co-infections were more prevalent in the central than eastern region; even though, tick control malpractices were observed in both regions. Therefore, an urgent tick and TBD control strategy is needed.

Mitochondrial phylogeography and population structure of the cattle tick Rhipicephalus appendiculatus in the African Great Lakes region.

BACKGROUND: The ixodid tick Rhipicephalus appendiculatus is the main vector of Theileria parva, wich causes the highly fatal cattle disease East Coast fever (ECF) in sub-Saharan Africa. Rhipicephalus appendiculatus populations differ in their ecology, diapause behaviour and vector competence. Thus, their expansion in new areas may change the genetic structure and consequently affect the vector-pathogen system and disease outcomes. In this study we investigated the genetic distribution of R. appendiculatus across agro-ecological zones (AEZs) in the African Great Lakes region to better understand the epidemiology of ECF and elucidate R. appendiculatus evolutionary history and biogeographical colonization in Africa. METHODS: Sequencing was performed on two mitochondrial genes (cox1 and 12S rRNA) of 218 ticks collected from cattle across six AEZs along an altitudinal gradient in the Democratic Republic of Congo, Rwanda, Burundi and Tanzania. Phylogenetic relationships between tick populations were determined and evolutionary population dynamics models were assessed by mismach distribution. RESULTS: Population genetic analysis yielded 22 cox1 and 9 12S haplotypes in a total of 209 and 126 nucleotide sequences, respectively. Phylogenetic algorithms grouped these haplotypes for both genes into two major clades (lineages A and B). We observed significant genetic variation segregating the two lineages and low structure among populations with high degree of migration. The observed high gene flow indicates population admixture between AEZs. However, reduced number of migrants was observed between lowlands and highlands. Mismatch analysis detected a signature of rapid demographic and range expansion of lineage A. The star-like pattern of isolated and published haplotypes indicates that the two lineages evolve independently and have been subjected to expansion across Africa. CONCLUSIONS: Two sympatric R. appendiculatus lineages occur in the Great Lakes region. Lineage A, the most diverse and ubiquitous, has experienced rapid population growth and range expansion in all AEZs probably through cattle movement, whereas lineage B, the less abundant, has probably established a founder population from recent colonization events and its occurrence decreases with altitude. These two lineages are sympatric in central and eastern Africa and allopatric in southern Africa. The observed colonization pattern may strongly affect the transmission system and may explain ECF endemic instability in the tick distribution fringes.

Investigations into the host specificity of Theileria taurotragi.

All Theileria parasites have definitive natural hosts that act as carriers. Incidental infections of uncommon hosts do occur raising questions regarding host specificity and its drivers. Reported hosts for Theileria taurotragi include bushbuck, cattle and eland. More recently T. taurotragi was detected in African buffalo, which may have implications for accurate diagnostics of T. parva. The current study therefore investigated the host specificity of T. taurotragi by developing a specific and sensitive real-time Taqman PCR assay. Animals were screened from areas where Rhipicephalus appendiculatus, the tick vector for both T. parva and T. taurotragi was present. While T. taurotragi was detected in cattle, eland, kudu and nyala, African buffalo (n = 352) was negative. Conversely, these same buffalo showed a prevalence of 72-100% for T. parva. While transmission of T. taurotragi to cattle was successful using the same infected tick batch, transmission to African buffalo did not occur. The results suggest that African buffalo is not a natural host of T. taurotragi and would probably not harbor anti-schizont antibodies against T. taurotragi. This would preclude T. taurotragi as possible source of cross-reactivity in the T. parva immunofluorescent antibody test. Host specificity of T. taurotragi for members of the Tragelaphini, but not buffalo also suggests that host specificity may have been an important driver in the speciation of the T. taurotragi clade. Different scenarios for co-evolution of host and parasite are discussed with implications for time of divergence for this Theileria clade.

Rhipicephalus appendiculatus ticks transmit Theileria parva from persistently infected cattle in the absence of detectable parasitemia: implications for East Coast fever epidemiology.

BACKGROUND: East Coast fever (ECF) is a devastating disease of cattle and a significant constraint to improvement of livestock production in sub-Saharan Africa. The protozoan parasite causing ECF, Theileria parva, undergoes obligate sexual stage development in its tick vector Rhipicephalus appendiculatus. Tick-borne acquisition and transmission occurs transstadially; larval and nymphal ticks acquire infection while feeding and transmit to cattle when they feed after molting to the next stage. Much of the current knowledge relating to tick-borne acquisition and transmission of T. parva has been derived from studies performed during acute infections where parasitemia is high. In contrast, tick-borne transmission during the low-level persistent infections characteristic of endemic transmission cycles is rarely studied. METHODS: Cattle were infected with one of two stocks of T. parva (Muguga or Marikebuni). Four months post-infection when parasites were no longer detectable in peripheral blood by PCR, 500 R. appendiculatus nymphs were fed to repletion on each of the cattle. After they molted to the adult stage, 20 or 200 ticks, respectively, were fed on two naïve cattle for each of the parasite stocks. After adult ticks fed to repletion, cattle were tested for T. parva infection by nested PCR and dot blot hybridization. RESULTS: Once they had molted to adults the ticks that had fed as nymphs on Muguga and Marikebuni infected cattle successfully transmitted Theileria parva to all naïve cattle, even though T. parva infection was not detectable by nested PCR on salivary gland genomic DNA of a sample of individual ticks. However, a salivary gland homogenate from a single Marikebuni infected tick was able to infect primary bovine lymphocytes. Infection was detected by nested p104 PCR in 3 of 4 calves and detected in all 4 calves by T. parva 18S nested PCR/dot blot hybridization. CONCLUSION: We show that R. appendiculatus ticks are able to acquire T. parva parasites from infected cattle even in the absence of detectable parasitemia. Although infection was undetectable in a sample of individual ticks, cumulatively as few as 20 ticks were able to transmit T. parva to naïve cattle. These results have important implications for our understanding of T. parva transmission by R. appendiculatus in ECF endemic regions.

Detection of Theileria parva in tissues of cattle undergoing severe East Coast fever disease show significant parasite DNA accumulation in the spleen.

Infiltration and proliferation of Theileria parva infected lymphocytes in bovine host lymphoid organs is one of the hallmarks of T. parva infection. The relative abundance of parasites within infected host tissues, both lymphoid and non-lymphoid is however unknown. Using quantitative PCR, we have shown that significantly higher concentrations of T. parva DNA are detected in the spleens of cattle undergoing severe disease compared to other organs.

Induction of humoral immune response to multiple recombinant Rhipicephalus appendiculatus antigens and their effect on tick feeding success and pathogen transmission.

BACKGROUND: Rhipicephalus appendiculatus is the primary vector of Theileria parva, the etiological agent of East Coast fever (ECF), a devastating disease of cattle in sub-Saharan Africa. We hypothesized that a vaccine targeting tick proteins that are involved in attachment and feeding might affect feeding success and possibly reduce tick-borne transmission of T. parva. Here we report the evaluation of a multivalent vaccine cocktail of tick antigens for their ability to reduce R. appendiculatus feeding success and possibly reduce tick-transmission of T. parva in a natural host-tick-parasite challenge model. METHODS: Cattle were inoculated with a multivalent antigen cocktail containing recombinant tick protective antigen subolesin as well as two additional R. appendiculatus saliva antigens: the cement protein TRP64, and three different histamine binding proteins. The cocktail also contained the T. parva sporozoite antigen p67C. The effect of vaccination on the feeding success of nymphal and adult R. appendiculatus ticks was evaluated together with the effect on transmission of T. parva using a tick challenge model. RESULTS: To our knowledge, this is the first evaluation of the anti-tick effects of these antigens in the natural host-tick-parasite combination. In spite of evidence of strong immune responses to all of the antigens in the cocktail, vaccination with this combination of tick and parasite antigens did not appear to effect tick feeding success or reduce transmission of T. parva. CONCLUSION: The results of this study highlight the importance of early evaluation of anti-tick vaccine candidates in biologically relevant challenge systems using the natural tick-host-parasite combination.

Transfection of live, tick derived sporozoites of the protozoan Apicomplexan parasite Theileria parva.

Theileria parva is an important veterinary protozoan causing the tick-borne disease East Coast fever. Transfection of Theileria parasites will facilitate the investigation of many aspects of this apicomplexan infection and its unique host-parasite interaction. The pathogen has the extraordinary capacity of transforming B and T cells into clonally dividing cancerous cell lines in a reversible way. Sequence data of the entire T. parva genome are available and in vitro infected cell lines can easily be generated, thereby eliminating the use of animals in the evaluation of the evolution of the transfected sporozoites. Here we report, for the first time, on experiments towards successful transient transfection of T. parva sporozoites, making use of a new generation transfection device. Plasmid DNA containing the strong EF-1α promoter and an Azami Green reporter gene were integrated by nucleofection into freshly purified T. parva sporozoites. Expression of Azami Green was detected with a fluorescence microscope and confirmed by counter staining with a monoclonal directed against a sporozoite protein. Despite the fact that transfection efficiencies are still low, this is the first step towards a stable infection method of T. parva parasites. In the long run, transfected parasites might become an alternative way to induce immunity without clinical signs.

Prevalence and spatial distribution of Theileria parva in cattle under crop-livestock farming systems in Tororo District, Eastern Uganda.

BACKGROUND: Tick-borne diseases (TBDs) present a major economic burden to communities across East Africa. Farmers in East Africa must use acaracides to target ticks and prevent transmission of tick-borne diseases such as anaplasmosis, babesiosis, cowdriosis and theileriosis; the major causes of cattle mortality and morbidity. The costs of controlling East Coast Fever (ECF), caused by Theileria parva, in Uganda are significant and measures taken to control ticks, to be cost-effective, should take into account the burden of disease. The aim of the present work was to estimate the burden presented by T. parva and its spatial distribution in a crop-livestock production system in Eastern Uganda. METHODS: A cross sectional study was carried out to determine the prevalence and spatial distribution of T. parva in Tororo District, Uganda. Blood samples were taken from all cattle (n: 2,658) in 22 randomly selected villages across Tororo District from September to December 2011. Samples were analysed by PCR and T. parva prevalence and spatial distribution determined. RESULTS: The overall prevalence of T. parva was found to be 5.3%. Herd level prevalence ranged from 0% to 21% with majority of the infections located in the North, North-Eastern and South-Eastern parts of Tororo District. No statistically significant differences in risk of infection were found between age classes, sex and cattle breed. CONCLUSIONS: T. parva infection is widely distributed in Tororo District, Uganda. The prevalence and distribution of T. parva is most likely determined by spatial distribution of R. appendiculatus, restricted grazing of calves and preferential tick control targeting draft animals.

Role of the polymorphic immunodominant molecule in entry of Theileria parva sporozoites into bovine lymphocytes.

Theileria parva is a tick-transmitted apicomplexan parasite that infects cattle and African buffalo. In cattle, it causes a fatal lymphoproliferative disease called East Coast fever. The polymorphic immunodominant molecule (PIM) is expressed by two stages of the parasite: the sporozoite, which is inoculated by the tick to infect mammalian lymphocytes, and the schizont, the established intralymphocytic stage. Here, we demonstrate that monoclonal antibodies (MAb) to PIM can reduce the ability of sporozoites to infect bovine lymphocytes in vitro. This reduction appears to be due to blocking of sporozoite attachment by binding of the MAb to several regions of PIM. Interestingly, one MAb, which recognizes an epitope in the central variable region of PIM, did not inhibit sporozoite infectivity. We also demonstrate that PIM antigen, as a recombinant molecule, can also reduce sporozoite infectivity in vitro by blocking both attachment and internalization of sporozoites. Electron microscopic studies showed that PIM is present in microspheres below the sporozoite surface and is transported to the parasite surface soon after contact with bovine lymphocytes. The results suggest that at least two sporozoite molecules, PIM and the previously described p67, are involved in the entry of T. parva into mammalian lymphocytes.

Geographic distribution of Theileria sp. (buffalo) and Theileria sp. (bougasvlei) in Cape buffalo (Syncerus caffer) in southern Africa: implications for speciation.

Strict control measures apply to movement of buffalo in South Africa including testing for Theileria parva, the causative agent of Corridor disease in cattle. The official test is a real-time hybridization PCR assay that amplifies the 18S rRNA V4 hyper-variable region of T. parva, T. sp. (buffalo) and T. sp. (bougasvlei). Mixed infections with the latter organisms affect diagnostic sensitivity due to PCR suppression. While the incidence of mixed infections in the Corridor disease endemic region of South Africa is significant, little information is available on the specific distribution and prevalence of T. sp. (buffalo) and T. sp. (bougasvlei). Specific real-time PCR assays were developed and a total of 1211 samples known to harbour these parasites were screened. Both parasites are widely distributed in southern Africa and the incidence of mixed infections with T. parva within the endemic region is similar (∼25-50%). However, a significant discrepancy exists in regard to mixed infections of T. sp. (buffalo) and T. sp. (bougasvlei) (∼10%). Evidence for speciation between T. sp. (buffalo) and T. sp. (bougasvlei) is supported by phylogenetic analysis of the COI gene, and their designation as different species. This suggests mutual exclusion of parasites and the possibility of hybrid sterility in cases of mixed infections.

Lymphocytes and macrophages are infected by Theileria equi, but T cells and B cells are not required to establish infection in vivo.

Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence.

Population attributable fractions of farm vector tick (Rhipicephalus appendiculatus) presence on Theileria parva infection seroprevalence under endemic instability.

The primary objective of this study was to assess the impact of Rhipicephalus appendiculatus tick presence (exposure variable) on Theileria parva infection seroprevalence (outcome variable) in a group of cattle belonging to a farm using population attributable fractions (PAF). The analyses were based on a representative sample of 80 traditional smallholder mixed farms. The farms were selected by first stratifying the population administratively and implementing a multistage random sampling in Mbeere district in Kenya. The PAFs were estimated using the stratified, Bruzzi, and sequential partitioned PAF approaches. A secondary objective was, thus, to evaluate the impact of the approaches on the PAF estimates. The stratified and Bruzzi approaches estimated proportion of T. parva infection cases directly attributable to the exposure after controlling for confounding by agro-ecological zone (AEZ). The sequential partitioned PAF approach estimated a PAF associated with exposure after adjusting for any effect that the AEZ may have had by influencing the prevalence of the exposure. All analyses were carried out at the farm level where a farm was classified as infested if the tick was found on cattle on a farm, and infected if at least one animal on a farm was positive for T. parva antibodies. Variance estimation for PAFs was implemented using 'delete-a-group' jackknife re-sampling method. The stratified PAF (26.7% [95% CI: 9.0%, 44.4%]) and Bruzzi PAF (26.4% [95% CI: 9.6%, 43.2%]) were consistent in estimating a relatively low impact of farm vector tick presence with a relatively high level of uncertainty. The partitioned PAF (15.5% [95% CI: 1.5%, 29.6%]) suggested that part of the impacts estimated using the stratified PAF and Bruzzi approaches was driven by AEZ effects. Overall, the results suggested that under endemic instability in Mbeere district, (1) presence of R. appendiculatus was not a good indicator of T. parva infection occurrence on a farm; (2) ecological variation could play a role in determining infection impacts. This study provides a preliminary basis for evaluating the potential value and utility of estimating PAFs for variables amenable to control in tick-borne diseases (TBDs) epidemiological studies.

Micro- and minisatellite-expressed sequence tag (EST) markers discriminate between populations of Rhipicephalus appendiculatus.

Biological differences, including vector competence for the protozoan parasite Theileria parva have been reported among populations of Rhipicephalus appendiculatus (Acari: Ixodidae) from different geographic regions. However, the genetic diversity and population structure of this important tick vector remain unknown due to the absence of appropriate genetic markers. Here, we describe the development and evaluation of a panel of EST micro- and minisatellite markers to characterize the genetic diversity within and between populations of R. appendiculatus and other rhipicephaline species. Sixty-six micro- and minisatellite markers were identified through analysis of the R. appendiculatus Gene Index (RaGI) EST database and selected bacterial artificial chromosome (BAC) sequences. These were used to genotype 979 individual ticks from 10 field populations, 10 laboratory-bred stocks, and 5 additional Rhipicephalus species. Twenty-nine markers were polymorphic and therefore informative for genetic studies while 6 were monomorphic. Primers designed from the remaining 31 loci did not reliably generate amplicons. The 29 polymorphic markers discriminated populations of R. appendiculatus and also 4 other Rhipicephalus species, but not R. zambeziensis. The percentage Principal Component Analysis (PCA) implemented using Multiple Co-inertia Analysis (MCoA) clustered populations of R. appendiculatus into 2 groups. Individual markers however differed in their ability to generate the reference typology using the MCoA approach. This indicates that different panels of markers may be required for different applications. The 29 informative polymorphic micro- and minisatellite markers are the first available tools for the analysis of the phylogeography and population genetics of R. appendiculatus.

Mixed Theileria infections in free-ranging buffalo herds: implications for diagnosing Theileria parva infections in Cape buffalo (Syncerus caffer).

Buffalo-adapted Theileria parva causes Corridor disease in cattle. Strict control measures therefore apply to the movement of buffalo in South Africa and include mandatory testing of buffalo for the presence of T. parva. The official test is a real-time hybridization PCR assay that amplifies the V4 hypervariable region of the 18S rRNA gene of T. parva, T. sp. (buffalo) and T. sp. (bougasvlei). The effect that mixed T. parva and T. sp. (buffalo)-like infections have on accurate T. parva diagnosis was investigated in this study. In vitro mixed infection simulations indicated PCR signal suppression at 100 to 1000-fold T. sp. (buffalo) excess at low T. parva parasitaemia. Suppression of PCR signal was found in field buffalo with mixed infections. The T. parva-positive status of these cases was confirmed by selective suppression of T. sp. (buffalo) amplification using a locked nucleic acid clamp and independent assays based on the p67, p104 and Tpr genes. The incidence of mixed infections in the Corridor disease endemic region of South Africa is significant, while the prevalence in buffalo outside the endemic area is currently low. A predicted increase of T. sp. (buffalo)-like infections can affect future diagnoses where mixed infections occur, prompting the need for improvements in current diagnostics.

Genotypic diversity, a survival strategy for the apicomplexan parasite Theileria parva.

The tick-borne protozoan parasite Theileria parva causes East Coast fever (ECF), a severe lymphoproliferative disease of cattle that is a major constraint to the improvement of livestock in eastern, central and southern Africa. Studies in cattle experimentally infected with T. parva have shown that the protective cytotoxic T lymphocyte (CTL) response is tightly focused, with individual animals recognizing only one or two dominant antigens, the identity of which varies with MHC class I phenotype. It is well known that cross-protection between T. parva stocks is limited, but precise evaluation of genetic diversity in field populations of the parasite has been hampered by a lack of molecular markers spanning the genome. A recently described panel of satellite markers has provided evidence for substantial genotypic diversity and recombination but does not provide cover for large segments of the genome. To address this deficiency, we undertook to identify additional polymorphic markers covering these regions and we report herein 42 newly identified PCR-RFLP markers distributed across the 4 T. parva chromosomes, as well as 19 new satellite markers for chromosomes 1 and 2. This brings the total number of available polymorphic markers to 141 for the 8.5 Mb genome. We have used these markers to characterise existing parasite stabilates and have also shown that passage of the parasite through naïve cattle and ticks can lead to substantial changes of parasite populations in resulting stabilates. These markers have also been used to show that passage of mixed parasites through an immunised calf results in the removal of the immunising genotype from the parasite population produced by ticks fed on this animal.