Search

Theileria are tick-transmitted parasites that cause often fatal leuko-proliferative diseases in cattle called tropical theileriosis (T. annulata) and East Coast fever (T. parva). However, upon treatment with anti-theilerial drug-transformed leukocytes die of apoptosis indicating that Theileria-induced transformation is reversible making infected leukocytes a powerful example of how intracellular parasites interact with their hosts. Theileria-transformed leukocytes disseminate throughout infected cattle causing a cancer-like disease and here, we discuss how cytokines, noncoding RNAs and oncometabolites can contribute to the transformed phenotype and disease pathology.

Background Tick-borne diseases (TBDs) constitute a major constraint for livestock development in sub-Saharan Africa, with East Coast fever (ECF) being the most devastating TBD of cattle. However, in Burundi, detailed information is lacking on the current prevalence of TBDs and on the associated economic losses from mortality and morbidity in cattle as well as the costs associated with TBD control and treatment. The aim of this study was, therefore, to assess the prevalence and spatial distribution of tick-borne pathogens (TBPs) in cattle across the major agro-ecological zones (AEZs) in Burundi. Methods In a cross-sectional study conducted in ten communes spanning the five main AEZs in Burundi, blood samples were taken from 828 cattle from 305 farms between October and December 2017. Evidence of Theileria parva infection was assessed by antibody level, measured using a polymorphic immunodominant molecule (PIM) antigen-based enzyme-linked immunosorbent assay (ELISA) and by a T. parva-specific p104 gene-based nested PCR. Antibodies against Theileria mutans infection were detected using the 32-kDa antigen-based indirect ELISA, while the 200-kDa antigen and the major surface protein 5 (MSP5)-based indirect ELISA were used to detect antibodies against Babesia bigemina and Anaplasma marginale, respectively. Results The prevalence of T. parva across the ten communes sampled ranged from 77.5 to 93.1% and from 67.8 to 90.0% based on the ELISA and PCR analysis, respectively. A statistically significant difference in infection was observed between calves and adult cattle; however, T. parva infection levels were not significantly associated with sex and breed. The seroprevalence indicating exposure to T. mutans, B. bigemina and A. marginale ranged from 30 to 92.1%, 33.7 to 90% and 50 to 96.2%, respectively. Mixed infections of TBPs were detected in 82.91% of cattle sampled, with 11 different combinations of pathogen species detected . Conclusions The findings indicate that T. parva, A. marginale and B. bigemina infections are endemic in Burundi. Knowledge of the spatial distribution of TBPs will facilitate the design of effective targeted strategies to control these diseases. There is a need for further investigations of the distribution of tick vectors and the population structure of TBPs in order to identify the key epidemiological factors contributing to TBD outbreaks in Burundi.

Key Points Multimerization of p67C Ag as nanoparticle increases its immunogenicity. Vaccine efficacy of p67C Ag increases delivered as nanoparticles., Visual Abstract, 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.

Background East Coast fever (ECF) caused by Theileria parva is endemic in Rwanda. In this study, the antigenic and genetic diversity of T. parva coupled with immunization and field challenge were undertaken to provide evidence for the introduction of ECF immunization in Rwanda. Methods Blood collected from cattle in the field was screened for T. parva using ELISA and PCR targeting the p104 gene. Tp1 and Tp2 gene sequences were generated from field samples and from Gikongoro and Nyakizu isolates. Furthermore, multilocus genotype data was generated using 5 satellite markers and an immunization challenge trial under field conditions using Muguga cocktail vaccine undertaken. Results Out of 120 samples, 44 and 20 were positive on ELISA and PCR, respectively. Antigenic diversity of the Tp1 and Tp2 gene sequences revealed an abundance of Muguga, Kiambu and Serengeti epitopes in the samples. A further three clusters were observed on both Tp1 and Tp2 phylogenetic trees; two clusters comprising of field samples and vaccine isolates and the third cluster comprising exclusively of Rwanda samples. Both antigens exhibited purifying selection with no positive selection sites. In addition, satellite marker analysis revealed that field samples possessed both shared alleles with Muguga cocktail on all loci and also a higher proportion of unique alleles. The Muguga cocktail (Muguga, Kiambu and Serengeti) genotype compared to other vaccine isolates, was the most represented in the field samples. Further low genetic sub-structuring (FST = 0.037) coupled with linkage disequilibrium between Muguga cocktail and the field samples was observed. Using the above data to guide a field immunization challenge trial comprising 41 immunized and 40 control animals resulted in 85% seroconversion in the immunized animals and an efficacy of vaccination of 81.7%, implying high protection against ECF. Conclusions Antigenic and genetic diversity analysis of T. parva facilitated the use of Muguga cocktail vaccine in field conditions. A protection level of 81.7% was achieved, demonstrating the importance of combining molecular tools with field trials to establish the suitability of implementation of immunization campaigns. Based on the information in this study, Muguga cocktail immunization in Rwanda has a potential to produce desirable results.

Equine theileriosis, a tick-transmitted disease caused by the hemoprotozoan parasites Theileria equi and Theileria haneyi, affects equids throughout tropical and subtropical regions of the world. It is a significant regulatory concern in non-endemic countries, where testing for equine theileriosis is required prior to horse import to prevent parasite entry. Within endemic areas, infection causes significant morbidity and mortality, leading to economic losses. No vaccine for equine theileriosis is available, and current drug treatment protocols are inconsistent and associated with significant side effects. Recent work has revealed substantial genetic variability among equine theileriosis organisms, and analysis of ribosomal DNA from affected animals around the world indicates that the organisms can be grouped into five distinct clades. As these diverse parasites are capable of infecting a wide range of both tick and mammalian hosts, movement of different equine Theileria species between endemic countries, and eventually into non-endemic countries, is a significant concern. Furthermore, the substantial genetic variability of these organisms will likely render currently utilized importation diagnostic tests unable to detect all equine Theileria spp. To this end, more complete characterization of these diverse parasites is critical to the continued global control of equine theileriosis. This review discusses current knowledge of equine Theileria spp. in this context, and highlights new opportunities and challenges for workers in this field.

The live infection and treatment (ITM) vaccination procedure using the trivalent Muguga cocktail is increasingly being used to control East Coast fever, with potential implications for Theileria parva population genetic structure in the field. Transmission of the Kiambu V T. parva component to unvaccinated cattle has previously been described in Uganda. We monitored the T. parva carrier state in vaccinated and control animals on a farm in West Kenya where an ITM stabilate derived from the Kenyan T. parva Marikebuni stock was evaluated for field efficacy. A nested PCR-based Marikebuni-specific marker identified a carrier state in nine of ten vaccinated animals, detectable for a period of two years. We used 22 variable number tandem repeat (VNTR) markers to determine multilocus genotypes (MLGs) of 19 T. parva schizont-infected lymphocyte isolates derived from cattle and field ticks. Two isolates from unimmunized cattle were identical to the Marikebuni vaccination stock. Two cattle isolates were identical to a Muguga cocktail component Kiambu V. Seven isolates from ticks exhibited MLGs that were identical to the Serengeti/Muguga vaccine stocks. Six cattle and two tick-derived stocks exhibited unique MLGs. The data strongly suggest transmission of immunizing genotypes, from Marikebuni vaccine-induced carrier cattle to unimmunized cattle. It is possible that genotypes similar to those in the Muguga cocktail are present in the field in Western Kenya. An alternative hypothesis is that these parasites may have originated from vaccine trial sites in Eastern Uganda. If correct, this suggests that T. parva stocks used for immunization can potentially be disseminated 125 km beyond the immediate vaccination site. Regardless of their origin, the data provide evidence that genotypes similar to those in the Muguga cocktail are circulating in the field in East Africa, alleviating concerns about dissemination of 'alien' T. parva germplasm through live vaccination.

The infection and treatment (ITM) live vaccination method for control of Theileria parva infection in cattle is increasingly being adopted, particularly in Maasai pastoralist systems. Several studies indicate positive impacts on human livelihoods. Importantly, the first detailed protocol for live vaccine production at scale has recently been published. However, quality control and delivery issues constrain vaccination sustainability and deployment. There is evidence that the distribution of T. parva is spreading from endemic areas in East Africa, North into Southern Sudan and West into Cameroon, probably as a result of anthropogenic movement of cattle. It has also recently been demonstrated that in Kenya, T. parva derived from cape buffalo can 'breakthrough' the immunity induced by ITM. However, in Tanzania, breakthrough has not been reported in areas where cattle co-graze with buffalo. It has been confirmed that buffalo in northern Uganda national parks are not infected with T. parva and R. appendiculatus appears to be absent, raising issues regarding vector distribution. Recently, there have been multiple field population genetic studies using variable number tandem repeat (VNTR) sequences and sequencing of antigen genes encoding targets of CD8+ T-cell responses. The VNTR markers generally reveal high levels of diversity. The antigen gene sequences present within the trivalent Muguga cocktail are relatively conserved among cattle transmissible T. parva populations. By contrast, greater genetic diversity is present in antigen genes from T. parva of buffalo origin. There is also evidence from several studies for transmission of components of stocks present within the Muguga cocktail, into field ticks and cattle following induction of a carrier state by immunization. In the short term, this may increase live vaccine effectiveness, through a more homogeneous challenge, but the long-term consequences are unknown.

A major risk factor for the spread of livestock diseases and their vectors is the uncontrolled transboundary movement of live animals for trade and grazing. Such movements constrain effective control of tick‐transmitted pathogens, including Theileria parva. Only limited studies have been undertaken to identify ticks and tick‐borne diseases (TTBDs) affecting cattle in central African countries, including Cameroon. We hereby report the collection of baseline data on the prevalence of T. parva in Cameroon through a countrywide cross‐sectional survey, conducted in 2016, involving collection of blood samples from cattle from 63 sites across the five agro‐ecological zones (AEZs) of the country. ELISA‐based surveillance of infected cattle was performed on 479 randomly selected samples and revealed specific antibodies to T. parva in 22.7% and T. mutans in 41.1% of cattle. Screening of 1,340 representative DNA samples for the presence of T. parva identified 25 (1.86%) positives using a p104 antigen gene‐based nested PCR assay. The positives were distributed across agro‐ecological zones I, II, III and V. None of the p104 positive cattle exhibited clinical symptoms of East Coast fever (ECF). Using reverse line blot (RLB), 58 (4.3%) and 1,139 (85%) of the samples reacted with the T. parva and T. mutans oligonucleotide probes, respectively. This represents the first report of T. parva from Cameroon. Surprisingly, no Rhipicephalus appendiculatus ticks, the main vector of T. parva, were identified in a parallel study involving comprehensive morphological and molecular survey of tick species present in the country. Only two of the 25 p104 positive cattle were PCR‐positive for the CD8+ T‐cell target schizont‐expressed antigen gene Tp1. Cloning and sequencing of Tp1 amplicons revealed sequence identity with the reference T. parva Muguga. This new finding raises serious concerns of a potential spread of ECF into the central African region.

East Coast fever is caused by Theileria parva, and poses serious concerns for dairy farmers owing to massive economic losses. In the current study, we compared three methods (DNA extraction kits, FTA-NaOH and FTA-TENT) of DNA extraction to identify the most economical and reliable method. A survey for T. parva prevalence was conducted in dairy cattle in Mbarara, Uganda. Cytochrome C oxidase subunit I (COI) and T. parva-p104 genes were amplified to compare the methods. FTA-NaOH-based polymerase chain reaction (PCR) yielded the best detection rate for both COI gene and p104 gene. Prevalence of T. parva was 45.0% and 83.3% at animal and farm-level, respectively. FTA-NaOH based-PCR is simple, highly sensitive and cost-effective tool for T. parva diagnosis in resource constrained settings.

Theileria parva is the causative agent of East Coast fever and Corridor disease, which are fatal, economically important diseases of cattle in eastern, central and southern Africa. Improved methods of control of the diseases are urgently required. The parasite transforms host lymphocytes, resulting in a rapid, clonal expansion of infected cells. Resistance to the disease has long been reported in cattle from T. parva-endemic areas. We reveal here that first- and second-generation descendants of a single Bos indicus bull survived severe challenge with T. parva, (overall survival rate 57.3% compared to 8.7% for unrelated animals) in a series of five field studies. Tolerant cattle displayed a delayed and less severe parasitosis and febrile response than unrelated animals. The in vitro proliferation of cells from surviving cattle was much reduced compared to those from animals that succumbed to infection. Additionally, some pro-inflammatory cytokines such as IL1β, IL6, TNFα or TGFβ which are usually strongly expressed in susceptible animals and are known to regulate cell growth or motility, remain low in tolerant animals. This correlates with the reduced proliferation and less severe clinical reactions observed in tolerant cattle. The results show for the first time that the inherited tolerance to T. parva is associated with decreased proliferation of infected lymphocytes. The results are discussed in terms of whether the reduced proliferation is the result of a perturbation of the transformation mechanism induced in infected cells or is due to an innate immune response present in the tolerant cattle.

East Coast fever (ECF) in cattle is caused by the protozoan parasite Theileria parva, transmitted by Rhipicephalus appendiculatus ticks. In cattle ECF is often fatal, causing annual losses >$500 million across its range. The African buffalo (Syncerus caffer) is the natural host for T. parva but the transmission dynamics between wild hosts and livestock are poorly understood. This study aimed to determine the prevalence of T. parva in cattle, in a 30 km zone adjacent to the Serengeti National Park, Tanzania where livestock and buffalo co-exist, and to ascertain how livestock keepers controlled ECF and other vector-borne diseases of cattle.\ud \ud A randomised cross-sectional cattle survey and questionnaire of vector control practices were conducted. Blood samples were collected from 770 cattle from 48 herds and analysed by PCR to establish T. parva prevalence. Half body tick counts were recorded on every animal. Farmers were interviewed (n = 120; including the blood sampled herds) using a standardised questionnaire to obtain data on vector control practices. Local workshops were held to discuss findings and validate results.\ud \ud Overall prevalence of T. parva in cattle was 5.07% (CI: 3.70-7.00%), with significantly higher prevalence in older animals. Although all farmers reported seeing ticks on their cattle, tick counts were very low with 78% cattle having none. Questionnaire analysis indicated significant acaricide use with 79% and 41% of farmers reporting spraying or dipping with cypermethrin-based insecticides, respectively. Some farmers reported very frequent spraying, as often as every four days. However, doses per animal were often insufficient.\ud \ud These data indicate high levels of acaricide use, which may be responsible for the low observed tick burdens and low ECF prevalence. This vector control is farmer-led and aimed at both tick- and tsetse-borne diseases of livestock. The levels of acaricide use raise concerns regarding sustainability; resistance development is a risk, particularly in ticks. Integrating vaccination as part of this community-based disease control may alleviate acaricide dependence, but increased understanding of the Theileria strains circulating in wildlife-livestock interface areas is required to establish the potential benefits of vaccination.

East Coast Fever (ECF), caused by the tick-borne apicomplexan parasiteTheileria parva, remains one of the most important livestock diseases in sub-Saharan Africa with more than 1 million cattle dying from infection every year. Disease prevention relies on the so-called “Infection and Treatment Method” (ITM), which is costly, complex, laborious, difficult to standardise on a commercial scale and results in a parasite strain-specific, MHC class I-restricted cytotoxic T cell response. We therefore attempted to develop a safe, affordable, stable, orally applicable and potent subunit vaccine for ECF using five differentT. parvaschizont antigens (Tp1, Tp2, Tp9, Tp10 and N36) andSaccharomyces cerevisiaeas an expression platform. Full-length Tp2 and Tp9 as well as fragments of Tp1 were successfully expressed on the surface ofS. cerevisiae.In vitroanalyses highlighted that recombinant yeast expressing Tp2 can elicit IFNγ responses using PBMCs from ITM-immunized calves, while Tp2 and Tp9 induced IFNγ responses from enriched bovine CD8+T cells. A subsequentin vivostudy showed that oral administration of heat-inactivated, freeze-dried yeast stably expressing Tp2 increased total murine serum IgG over time, but more importantly, induced Tp2-specific serum IgG antibodies in individual mice compared to the control group. While these results will require subsequent experiments to verify induction of protection in neonatal calves, our data indicates that oral application of yeast expressing Theileria antigens could provide an affordable and easy vaccination platform for sub-Saharan Africa. Evaluation of antigen-specific cellular immune responses, especially cytotoxic CD8+T cell immunity in cattle will further contribute to the development of a yeast-based vaccine for ECF.

Theileria parva kills over one million cattle annually in sub-Saharan Africa. Parasite genetic complexity, cellular response immunodominance, and bovine MHC diversity have precluded traditional vaccine development. One potential solution is gene gun (GG) immunization, which enables simultaneous administration of one or more DNA-encoded antigens. Although promising in murine, porcine, and human vaccination trials, bovine GG immunization studies are limited. We utilized the model T. parva antigen, polymorphic immunodominant molecule (PIM) to test bovine GG immunization. GG immunization using a mammalian codon optimized PIM sequence elicited significant anti-PIM antibody and cell-mediated responses in 7/8 steers, but there was no difference between immunized and control animals following T. parva challenge. The results suggest immunization with PIM, as delivered here, is insufficient to protect cattle from T. parva. Nonetheless, the robust immune responses elicited against this model antigen suggest GG immunization is a promising vaccine platform for T. parva and other bovine pathogens.

Livestock-wildlife interactions promote the transmission of a wide range of infectious diseases that constraint livestock production. We used a participatory appraisal approach to find out and rank infectious diseases of concern to pastoralists in a zone of intense wildlife-livestock interaction and another zone with limited interactions. Four villages were selected purposively in areas with intensive cattle-wildlife interactions (zone 1), and another two in areas with low to moderate cattle-wildlife interactions (zone 2). Data were collected in focus group discussions (FGDs) using participatory epidemiological methods (PE); each group had 8–13 participants. Results of impact matrix scoring from all sites indicated that malignant catarrhal fever (MCF), anthrax, foot and mouth disease (FMD), contagious bovine pleuropneumonia (CBPP), east coast fever (ECF) and African animal trypanosomiasis (ATT), in decreasing order, had the highest impact on livestock production. A Kruskal–Wallis test revealed a significant difference in FMD annual prevalence between cattle age groups (p 4 years (median score of 32.5, range, 10–50). FMD had the highest impact on milk production, but based on veterinary costs (treatment costs), it was ranked second to CBPP. The study provides information on disease priorities that occur in the target zones in Mara ecosystem and which the local pastoralists must consider when accessing key ecosystem services such as water and pasture. Electronic supplementary material The online version of this article (10.1007/s11250-018-01790-1) contains supplementary material, which is available to authorized users.

Herein we review the epidemiology of ticks and tick-borne diseases (TTBDs), their impact on livestock health and on the economy, control and associated challenges in Uganda. Ticks are leading vectors of economically important pathogens and are widespread in Uganda due to suitable climatic conditions. Besides the physical injury inflicted on the animal host, ticks transmit a number of pathogens that can cause morbidity and mortality of livestock if untreated, resulting in economic losses. Uganda suffers an aggregated annual loss (direct and indirect) of over USD 1.1 billion in the TTBDs complex. East Coast fever (ECF) caused by a protozoan haemoparasite, Theileria parva, is the most prevalent and economically important tick-borne disease (TBD) in Uganda and its vector, the brown ear tick (Rhipicephalus appendiculatus) widely distributed. Other prevalent TBDs in Uganda include anaplasmosis, babesiosis and heartwater. We highlight the role of agro-ecological zones (AEZs) and livestock management system in the distribution of TTBDs, citing warm and humid lowlands as being ideal habitats for ticks and endemic for TBDs. Control of TTBDs is a matter of great importance as far as animal health is concerned in Uganda. Indigenous cattle, which make up over 90% of the national herd are known to be more tolerant to TTBDs and most farms rely on endemic stability to TBDs for control. However, exotic cattle breeds are more capital intensive than indigenous breeds, but the increasing adoption of tick-susceptible exotic cattle breeds (especially dairy) in western and central Uganda demands intensive use of acaricides for tick control and prevention of TBDs. Such acaricide pressure has unfortunately led to selection of acaricide-resistant tick populations and the consequent acaricide resistance observed in the field. Vaccination against ECF, selective breeding for tick resistance and integrated tick control approaches that limit tick exposure, could be adopted to interrupt spread of acaricide resistance. We recommend increasing monitoring and surveillance for TTBDs and for emerging acaricide resistance, improved extension services and sensitization of farmers on tick control measures, appropriate acaricide use and the development and implementation of vaccines for the control of TTBDs as more sustainable and effective interventions. A tick control policy should be developed, taking into account variations of agro-ecological zones, farm circumstances and indigenous technical knowledge, and this should be incorporated into the overall animal health program., This study was financially supported by the the National Agricultural Research Organization (NARO) of Uganda. MC was funded by the Ministerio de Ciencia, Innovación y Universidades, Spain (grant FJC-2018-038277-I).

Theileria parva (T. parva) is a protozoan parasite that causes East Coast fever (ECF). The disease is endemic in Burundi and is a major constraint to livestock development. In this study, the parasite prevalence in cattle in six regions namely; Northern, Southern, Eastern, Western, Central and North Eastern was estimated. Furthermore, the sequence diversity of p67, Tp1 and Tp2 genes was assessed coupled with the population genetic structure of T. parva using five satellite markers. The prevalence of ECF was 30% (332/1109) on microscopy, 60% (860/1431) on ELISA and 79% (158/200) on p104 gene PCR. Phylogenetic analysis of p67 gene revealed that only allele 1 was present in the field samples. Furthermore, phylogenetic analysis of Tp1 and Tp2 showed that the majority of samples clustered with Muguga, Kiambu and Serengeti and shared similar epitopes. On the other hand, genetic analysis revealed that field samples shared only two alleles with Muguga Cocktail. The populations from the different regions indicated low genetic differentiation (FST = 0.047) coupled with linkage disequilibrium and non-panmixia. A low to moderate genetic differentiation (FST = 0.065) was also observed between samples and Muguga cocktail. In conclusion, the data presented revealed the presence of a parasite population that shared similar epitopes with Muguga Cocktail and was moderately genetically differentiated from it. Thus, use of Muguga Cocktail vaccine in Burundi is likely to confer protection against T. parva in field challenge trials.

East Coast fever (ECF) is an often fatal, economically important cattle disease that predominantly affects eastern, central, and southern Africa. ECF is controlled through vaccination by means of simultaneous injection of oxytetracycline and cryogenically preserved stabilate containing live, disease-causing parasites. Storage and transportation of the stabilate requires liquid nitrogen, a commodity that is commonly unreliable in low-resource settings. Here we show that storage of conventionally prepared stabilate at −80 °C for up to 30 days does not significantly affect its ability to infect cultured peripheral blood mononucleated cells or live cattle, suggesting an alternative cold chain that maintains these temperatures could be used to effectively manage ECF.

East Coast fever, a tick-borne cattle disease caused by theTheileria parvaparasite, 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 Boran cattle demonstrating heritable tolerance to infection byT. parva(h2= 0.65, s.e. 0.57). Through a linkage analysis we identify a 6 Mb genomic region onBos tauruschromosome 15 that is significantly associated with survival outcome followingT. parvaexposure. Testing this locus in an independent cohort of animals replicates this association with survival followingT. parvainfection. A stop gained polymorphism 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 byT. parva.Author SummaryMore than a million cattle die of East Coast fever in Africa each year, the impact of which disproportionately falls onto low-income, smallholder farmers. The lack of a widely accessible vaccine, heavy reliance on chemicals to control the tick vector and inadequate drug treatments means that new approaches for controlling the disease are urgently required. Through a genetic study of an extended pedigree of Boran cattle that are more than three times less likely to succumb to the disease than matched controls, we identify a region on chromosome 15 of the cattle genome associated with a high level of tolerance to the disease. We show that a variant in this region is also associated with survival in an independent cohort, and is linked to rates of cell expansion during infection. This genetic variant can therefore support marker-assisted selection, allowing farmers to breed tolerant cattle and offers a route to introduce this beneficial DNA to non-native breeds, enabling reduced disease incidence and increased productivity, which would be of benefit to millions of rural smallholder farmers across Africa.

Ticks and tick-borne diseases (TBDs) significantly affect cattle production and the livelihoods of communities in pastoralist areas. Data on protozoan and rickettsial pathogens in ticks infesting cattle in Uganda is scanty; while it is an indicator of the likelihood of disease transmission and occurrence. A cross-sectional study was conducted amongst cattle in the Karamoja Region, northeastern Uganda, from July through September 2017, to determine the tick species diversity, identify protozoan and rickettsial pathogens in the ticks, and characterise pathogenic species by sequence and phylogenetic analyses. About 50 % of the ticks detected from each predilection site on each animal were collected from 100 purposively-selected cattle from 20 randomly-selected herds. Twelve tick species belonging to the genera Amblyomma, Rhipicephalus and Hyalomma were identified, the most abundant being Amblyomma lepidum (93.9 %), followed by Amblyomma variegatum (2.0 %) and Rhipicephalus evertsi evertsi (1.0 %). Tick species that have not been reported in recent studies amongst cattle in Uganda were found, namely Rhipicephalus pravus, Rhipicephalus praetextatus and Rhipicephalus turanicus. The ticks were grouped into 40 pools, by species and location, and the reverse line blot (RLB) hybridisation assay was used to detect pathogens from the ticks. The most frequently detected tick-borne parasites were Theileria mutans, Theileria velifera and Theileria parva, each observed in 25 % (10/40) of the tick pools. Tick-borne pathogens, namely Babesia rossi, Babesia microti and Theileria sp. (sable) that are not common to, or not known to infect, cattle were identified from ticks. The gene encoding Ehrlichia ruminantium pCS20 region, the Ehrlichia and Anaplasma 16S rRNA gene, and T. parva p67 sporozoite antigen gene were amplified, cloned and sequenced. Seven novel E. ruminantium pCS20 variants were identified, and these grouped into two separate clusters with sequences from other parts of Africa and Asia. The T. parva p67 sequences were of the allele type 1, and parasites possessing this allele type are commonly associated with East Coast fever in eastern Africa. Analysis of the Ehrlichia and Anaplasma 16S rRNA gene sequences showed that they were closely related to Rickettsia africae and to a new Ehrlichia species variant recently found in China. Our R. africae 16S rRNA sequences grouped with R. africae isolates from Nigeria, Egypt and Benin. The information on tick species diversity and pathogens in the various tick species provides an indicator of potential transmission amongst cattle populations, and to humans, and can be useful to estimate disease risk and in control strategies.

International audience; Tick-borne diseases (TBD) occur in many temperate countries and are economically important in most tropical and subtropical areas, affecting dairy and beef cattle, as well as small ruminants. Four major tick-borne diseases have been detected in eastern and southern Africa: East Coast fever (ECF) caused by Theileria parva, Theiler 1904, anaplasmosis caused by either Anaplasma marginale, Theiler 1910, Anaplasma centrale, Theiler 1911, or Anaplasma ovis, Bevan 1912, babesiosis caused by Babesia bovis, Babes 1988 and Babesia bigemina, Smith & Kilborne 1893, and heartwater caused by Ehrlichia ruminantium Cowdry 1925. A cross-sectional survey was undertaken to determine the antibody prevalence of these TBDs and to identify the risk factors for TBD infections in the Union of the Comoros. In 2016 and 2017, 903 individual animal serum samples were collected from 429 separate farms, where the farmers answered individual questionnaires. The antibody prevalence of anaplasmosis, babesiosis (B. bigemina) and heartwater was determined by enzyme-linked immunosorbent assays (ELISA) and the antibody prevalence of ECF was assessed using an immunofluorescence antibody test (IFAT). The relationship between TBD seropositivity and livestock-related variables was assessed by multivariate analyses with standard logistic regression models. The results showed that these four TBDs were present in the Union of the Comoros with a global antibody prevalence of 15% (95% CI [12.7%; 17.3%]) for anaplasmosis, 9.2% (95% CI [6.5%, 11.9%]) for B. bigemina babesiosis, 5.3% (95% CI [3.2%, 7.4%]) for ECF and 4.6% (95% CI [3.2%, 6%]) for heartwater. We compared these findings with the abundance and distribution of several tick species known to be TBD vectors and we found a significant correlation between Rhipicephalus appendiculatus and ECF, and between Amblyomma variegatum and heartwater. We also found that two major variables were significantly correlated with B. bigemina antibody prevalence ("island" and "breeding area"), four variables were significantly correlated with anaplasmosis antibody seroprevalence ("island", "number of cattle per farmer", "number of farmers per village" and "breeding area"), two were significantly correlated with ECF antibody prevalence ("number of farmers in village" and "presence of ticks"), and three were significantly correlated with heartwater ("island", "number of cattle per farmer" and "number of farmers in the village"). Our findings confirmed livestock exposure to the four targeted TBDs of major concern for livestock development. Consequently, raising farmers' awareness and setting up a period of quarantine should be considered a priority.

Weather conditions can impact infectious disease transmission, causing mortalities in humans, wild and domestic animals. Although rainfall in dry tropical regions is highly variable over the year, rainfall is thought to play an important role in the transmission of tick-borne diseases. Whether variation in rainfall affects disease-induced mortalities, is, however, poorly understood. Here, we use long-term data on monthly rainfall and Boran cattle mortality (1998–2017) to investigate associations between within-year variation in rainfall and cattle mortalities due to East Coast fever (ECF), anaplasmosis and babesiosis in Laikipia, Kenya, using ARIMAX modelling. Results show a negative correlation between monthly rainfall and cattle mortality for ECF and anaplasmosis, with a lag effect of 2 and 6 months, respectively. There was no association between babesiosis-induced mortalities and monthly rainfall. The results of this study suggest that control of the tick-borne diseases ECF and anaplasmosis to reduce mortalities should be intensified during rainy periods after the respective estimated time lags following dry periods.

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.

The published literature on schizont-“transforming,” or pathogenic theileriosis, in African wild artiodactyls is dated and based on limited information. Here the authors review the taxonomy, diagnosis, epidemiology, hematology, pathology, and aspects of control in various species. Molecular studies based on 18S and 16S rRNA gene sequences have shown that African wild artiodactyls are commonly infected with diverse Theileria spp., as well as nontheilerial hemoprotozoa and rickettsia-like bacteria, and coinfections with pathogenic and nonpathogenic Theileria species are often recorded. Although theileriosis is still confusingly referred to as cytauxzoonosis in many species, the validity of a separate Cytauxzoon genus in artiodactyls is debated. The epidemiology of theileriosis is complex; the likelihood of fatal disease depends on the interplay of parasite, vertebrate host, tick vector, and environmental factors. Roan calves ( Hippotragus equinus) and stressed animals of all host species are more susceptible to fatal theileriosis. Even though regenerative anemia is common, peripheral blood piroplasm parasitemia does not correlate with disease severity. Other than anemia, common macroscopic lesions include icterus, hemorrhages (mucosal, serosal, and tissue), fluid effusions into body cavities, lung edema, and variably sized raised cream-colored foci of leukocyte infiltration in multiple organs. Histopathologic findings include vasocentric hyperproliferation and lysis of atypical leukocytes with associated intracellular schizonts, parenchymal necrosis, hemorrhage, thromboembolism, and edema. Immunophenotyping is required to establish the identity of the schizont-transformed leukocytes in wild ungulates. Throughout the review, we propose avenues for future research by comparing existing knowledge on selected aspects of theileriosis in domestic livestock with that in African wild artiodactyls.

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.

Theileria parva is the causative agent of East Coast fever (ECF), a tick-borne disease that kills over a million cattle each year in sub-Saharan Africa. Immune protection against T. parva involves a CD8+ cytotoxic T cell response to parasite-infected cells. However, there is currently a paucity of knowledge regarding the role played by innate immune cells in ECF pathogenesis and T. parva control., Theileria parva is the causative agent of East Coast fever (ECF), a tick-borne disease that kills over a million cattle each year in sub-Saharan Africa. Immune protection against T. parva involves a CD8+ cytotoxic T cell response to parasite-infected cells. However, there is currently a paucity of knowledge regarding the role played by innate immune cells in ECF pathogenesis and T. parva control. Here, we demonstrate an increase in intermediate monocytes (CD14++ CD16+) with a concomitant decrease in the classical (CD14++ CD16−) and nonclassical (CD14+ CD16+) subsets at 12 days postinfection (dpi) during lethal infection but not during nonlethal T. parva infection. Ex vivo analyses of monocytes demonstrated upregulation of interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) mRNA and increased nitric oxide production during T. parva lethal infection compared to nonlethal infection at 10 dpi. Interestingly, no significant differences in peripheral blood parasite loads were observed between lethally and nonlethally infected animals at 12 dpi. In vitro stimulation with T. parva schizont-infected cells or Escherichia coli lipopolysaccharide (LPS) resulted in significant upregulation of IL-1β production by monocytes from lethally infected cattle compared to those from nonlethally infected animals. Strikingly, monocytes from lethally infected animals produced significant amounts of IL-10 mRNA after stimulation with T. parva schizont-infected cells. In conclusion, we demonstrate that T. parva infection leads to alterations in the molecular and functional phenotypes of bovine monocytes. Importantly, since these changes primarily occur in lethal infection, they can serve as biomarkers for ECF progression and severity, thereby aiding in the standardization of protection assessment for T. parva candidate vaccines.

Highlights • Three doses of p67C antigen generated stronger immune responses than two doses. • Antibody titers and CD4+ T-cell proliferation correlated with protection against ECF. • The number of doses could not be reduced from three to two without compromising the protection., East Coast fever (ECF) is a lymphoproliferative disease caused by the tick-transmitted protozoan parasite Theileria parva. ECF is one of the most serious cattle tick-borne diseases in Sub-Saharan Africa. We have previously demonstrated that three doses of the C-terminal part of the sporozoite protein p67 (p67C) adjuvanted with ISA206VG confers partial protection against ECF at a herd level. We have tested the efficacy of two doses of this experimental vaccine, as reducing the vaccination regimen would facilitate its deployment in the field. We reconfirm that three antigen doses gave a significant level of protection to severe disease (46%, ECF score

Climate change is predicted to increase incidence of vector-borne diseases in humans, however, little is known about the impact of such diseases in livestock. In the absence of historical data with which to examine the inter-relation between climate and disease, participatory epidemiological (PE) methods were used with Maasai pastoralists of Monduli District, northern Tanzania to establish local observations on two major vector-borne diseases of cattle, namely East Coast fever (ECF) and African animal trypanosomiasis (AAT). Data collection involving gender segregated groups (10 men groups and 9 women groups) occurred in 10 randomly selected villages between November 2014 and March 2015. ECF and AAT were ranked amongst the top 5 most important cattle diseases with strong agreement across informant groups (Kendall's W=0.40 for men and 0.45 for women; p0.01). Matrix scoring for both men and women groups confirmed that Masaai easily recognize these diseases. All groups associated ECF with the wet and cool dry seasons. AAT was more variable throughout the year, with more cases reported in the long dry season. Likewise, pastoralists reported differences in seasonal occurrence of disease vectors (Rhipicephalus appendiculatus and Glossina spp.) by village. Comparing 2014-1984, participant groups consistently reported declines in rainfall, vegetation cover and quality pasture, as well as increases in severe droughts. Experiences with ECF/AAT and vector abundance between these time periods was more variable across villages, and likely relates to changes in climate and animal management practices over the last 30 years. This baseline study is the first to document the inter-relation between climate and cattle vector-borne disease from the pastoralist perspective. Findings from this study reveal a complex interplay between human, animal and environmental factors, understanding of which is urgently required to devise approaches to mitigate effects of climate change in these vulnerable areas.

Background The apicomplexan parasite Theileria parva causes a livestock disease called East coast fever (ECF), with millions of animals at risk in sub-Saharan East and Southern Africa, the geographic distribution of T. parva. Over a million bovines die each year of ECF, with a tremendous economic burden to pastoralists in endemic countries. Comprehensive, accurate parasite genome annotation can facilitate the discovery of novel chemotherapeutic targets for disease treatment, as well as elucidate the biology of the parasite. However, genome annotation remains a significant challenge because of limitations in the quality and quantity of the data being used to inform the location and function of protein-coding genes and, when RNA data are used, the underlying biological complexity of the processes involved in gene expression. Here, we apply our recently published RNAseq dataset derived from the schizont life-cycle stage of T. parva to update structural and functional gene annotations across the entire nuclear genome. Results The re-annotation effort lead to evidence-supported updates in over half of all protein-coding sequence (CDS) predictions, including exon changes, gene merges and gene splitting, an increase in average CDS length of approximately 50 base pairs, and the identification of 128 new genes. Among the new genes identified were those involved in N-glycosylation, a process previously thought not to exist in this organism and a potentially new chemotherapeutic target pathway for treating ECF. Alternatively-spliced genes were identified, and antisense and multi-gene family transcription were extensively characterized. Conclusions The process of re-annotation led to novel insights into the organization and expression profiles of protein-coding sequences in this parasite, and uncovered a minimal N-glycosylation pathway that changes our current understanding of the evolution of this post-translational modification in apicomplexan parasites.

East Coast fever (Theileria parva infection in cattle) was eradicated from South Africa in the mid-1900. However, another form named Corridor disease (CD), associated with T. parva carrier buffaloes exists and outbreaks have increased in endemic areas. The occurrence of a CD carrier state in cattle under field situations has not been demonstrated but remains a subject of controversy. The current study investigated the T. parva carrier state following a severe outbreak in cattle introduced onto a game ranch. Monitoring of the outbreak included clinical signs, mortality, microscopy, serology, real-time PCR and xenodiagnoses. The herd of cattle received block treatment using oxytetracyclines (OTC) by the farmer during the outbreak. Cattle were sampled early during the outbreak and twice within the following 75 days. All buffaloes were tested for a T. parva carrier state. Two batches of questing adult R. appendiculatus were collected at the time of disease occurrence and a year later. These ticks were fed on susceptible cattle under controlled conditions and monitored for disease transmission. Ticks infected with a buffalo-derived stock of T. parva were fed on one bovine under controlled conditions and simultaneously injected with OTC, simulating the infection and treatment method of vaccination and was used as a positive control. Clean R. appendiculatus nymphs were fed on four recovered PCR positive cattle from the outbreak and on the positive control animal. The adult ticks were tested for infectivity by xenodiagnoses on susceptible bovines. For the initial outbreak the CD prevalence was 62.3% with a mortality rate of 29.5%. However, the outbreak was contained by block OTC treatment of the herd since only 3.4% cattle subsequently died until the end of the investigations. Adult ticks fed on one field bovine and the laboratory established T. parva carrier both transmitted fatal infections to susceptible cattle. Ticks fed on two field cattle transmitted T. taurotragi and one failed to transmit any infection. Questing adult R. appendiculatus collected during the outbreak transmitted fatal CD to two bovines while ticks collected a year later transmitted T. taurotragi. These findings demonstrated the effectiveness of disease control either by cattle treatment using OTC simulating the ITM or by intensive cattle dipping following the outbreak or by both interventions. The potential risk of creating carrier cattle by OTC treatment during CD outbreaks should be considered, supporting the continued control measures of segregation of cattle and buffalo herds.

East Coast Fever (ECF), caused by the tick-borne apicomplexan parasite Theileria parva, is a leading cause of morbidity and mortality in cattle of sub-Saharan Africa. The infection and treatment method (ITM) is currently the only vaccine available to control T. parva. Although ITM elicits levels of protection, its widespread adoption is limited by costs, laborious production process, and antibiotic co-treatment requirement, necessitating the development of a more sustainable vaccine. To this end, efforts have been concentrated in the identification of new T. parva vaccine antigens and in the development of suitable platforms for antigen expression. In this study, we investigated the molecular and antigenic properties of T. parva antigen Tp9 expressed by mammalian cells. Data indicate that Tp9 contains a signal peptide that is weakly functional in mammalian cells. Thus, Tp9 secretion from mammalian cells increased 10-fold after the native signal peptide was replaced with the human tissue plasminogen activator signal peptide (tPA). Sera from all T. parva-immune cattle recognized this recombinant, secreted Tp9. Additionally, PBMC from ITM-immunized cattle produced significant (p < 0.05) amounts of IFNγ following ex vivo exposure to Tp9, but this response varied between cattle of different MHC class I and class II genotypes. In addition, depletion experiments demonstrated that IFNγ to Tp9 was primarily produced by CD4+ T cells. Molecular analysis demonstrated that Tp9 presents a signal peptide that is weakly functional in mammalian cells, suggesting that it remains within lymphocytes during infection. Tp9 secretion from mammalian cells was substantially increased when the tPA secretion signal sequence was substituted for the native secretion signal sequence. Using full-length, recombinant Tp9 secreted from mammalian cells, we demonstrated that T. parva-immune cattle develop both humoral and cellular immune responses to this antigen. Collectively, these results provide rationale for further evaluation of Tp9 as a component of a T. parva subunit vaccine.

The infection and treatment (ITM) procedure remains the only available method of immunization against Theileria parva infection. One constraint to deployment is the perception that the carrier state induced by ITM could result in enhanced disease problems. More than one million cattle have been ITM vaccinated in pastoralist systems in Tanzania over the last 2 decades. We present the results of a longitudinal study of six groups of cattle in Maasai villages in northern Tanzania exposed to natural tick challenge for between 2 weeks and 14 years post-vaccination. The p104 nested PCR revealed a higher frequency of T. parva carriers among vaccinates (30%) compared with controls (8%) (OR = 4.89, p = .000), with the highest frequency of carriers found in calves vaccinated 6 months previously, although carrier state was also detected in cattle vaccinated >10 years prior to the study. Variable number tandem repeat genotype analysis revealed 6 MS7 alleles with sizes ranging from 150 bp to 500 bp, but only two alleles were detected in cattle vaccinated >4 years earlier, relative to five alleles detected in recently vaccinated cattle and controls. In terms of heterozygosity, diversity was maximal in calves vaccinated within the last 2 weeks (h = 0.776) but lowest in cattle vaccinated 4 years earlier (h = 0.375). The analysis suggested close genetic relatedness of parasites in vaccinated and unvaccinated groups and up to 96% of variation was within rather than between the groups. These results confirm that ITM leads to a long-term T. parva carrier state in cattle and the detection of vaccine component VNTR in co-grazing unvaccinated cattle suggests potential vaccine transmission by ticks. However, vaccination stocks did not totally replace local genotypes, at least in cattle populations. These findings should mitigate concerns that ITM modifies T. parva field populations in a way that enhances disease in the medium term.

Background The Infection and Treatment Method (ITM) of vaccination is the only immunization procedure currently available to protect cattle against East Coast fever (ECF), a tick-transmitted disease responsible for losses of several hundreds of millions of dollars per year in sub-Saharan Africa. The vaccine comprises a homogenized preparation of infected ticks packaged in straws and stored in liquid nitrogen. The current manufacturing protocol results in straws containing 30–40 doses (ILRI 0804), which is impractical for immunizing small herds as found in dairy and smallholder farming systems. The ILRI 0804 SD stabilate was prepared as a 1:5 dilution of the parent stabilate, with the aim of producing vaccine stabilate straws containing between four to eight doses and thus suitable for smallholder farming systems. Infectivity of the diluted stabilate was assessed and the protective efficacy of the diluted stabilate was determined by performing experimental and field immunizations. Results Two groups of six cattle were inoculated with 1 ml of the diluted stabilate at 1:20 (equivalent to the recommended field dose for ILRI 0804, assuming no loss of sporozoite viability during thawing and refreezing) and 1:14 (assuming 30–35% loss of sporozoite viability). Schizonts were detected in all 12 animals, showing viability of sporozoites. Ten animals from the infectivity study and two control animals not previously exposed to T. parva were challenged with the parental ILRI 0804 stabilate. The results show that the two control animals displayed severe ECF reactions and were treated 14 days after challenge. Of the previously infected animals, only one underwent a severe reaction following challenge, a result in accord with the challenge experiments performed previously with the parent stabilate [Ticks Tick-Borne Dis 7:306-314, 2016]. The animal that displayed a severe reaction had no detectable schizonts and did not seroconvert following the initial inoculation with ILRI 0804 SD. In addition, 62 animals immunized under field conditions showed a mean seroconversion rate of 82%. Conclusion The results presented in this article demonstrate that it is possible to prepare straws suitable for use in smallholder herds by thawing, diluting and refreezing already packaged vaccine.

Chemotherapy of East Coast fever, a lymphoproliferative cancer-like disease of cattle causing significant economic losses in Africa, is largely dependent on the use of buparvaquone, a drug that was developed in the late 1980's. The disease is caused by the tick-borne protozoan pathogen Theileria parva. Buparvaquone can be used prophylactically and it is also active against tropical theileriosis, caused by the related parasite Theileria annulata. Recently, drug resistance was reported in T. annulata, and could occur in T. parva. Using a 3H-thymidine incorporation assay we screened 796 open source compounds from the Medicines for Malaria Venture (MMV) to discover novel chemicals with potential inhibitory activity to T. parva. We identified nine malaria box compounds and eight pathogen box compounds that inhibited the proliferation of F100TpM, a T. parva infected lymphocyte cell line. However, only two compounds, MMV008212 and MMV688372 represent promising leads with IC50 values of 0.78 and 0.61 μM, respectively, and CC50 values > 5 μM. The remaining compounds exhibited a high degree of toxicity (CC50 values 10 μM. Our preliminary data suggest that it may be possible to repurpose compounds from the cancer field as well as MMV as novel anti-T. parva molecules., Graphical abstract Image 1, Highlights • 17 of 796 MMV compounds inhibited proliferation of T parva infected lymphocytes. • 15 of the 17 hit compounds also inhibited proliferation of control cells. • MMV008212 and MMV688372 represent promising leads, therapeutic index >8. • Dasatinib was as active and safe as buparvaquone in vitro, therapeutic index >2000.

Theileria parva is a tick-transmitted protozoan parasite that causes a disease called East Coast fever (ECF) in cattle. This important tick borne-disease (TBD) causes significant economic losses in cattle in many sub-Saharan countries, including Tanzania. Cattle immunization using Muguga cocktail has been recommended as an effective method for controlling ECF in pastoral farming systems in Tanzania. However, immunity provided through immunization is partially strain-specific. Therefore, the control of ECF in Tanzania is still a challenge due to inadequate epidemiological information. This study was conducted to assess genetic diversity of Tp1 and Tp2 genes from T. parva isolates that are recognized by CD8 + T-cells in cattle and buffalo. The Tp1 and Tp2 genes are currently under evaluation as candidates for inclusion in a subunit vaccine. A total of 130 blood samples collected from cattle which do not interact with buffalo (98), cattle co-grazing with buffalo (19) and buffalo (13) in Mara, Mbeya, Morogoro, Tanga, and Coast regions in Tanzania were used in this study. Genomic DNA was extracted from the blood samples, Tp1 and Tp2 genes were amplified using nested PCR and the PCR products were purified and sequenced. The partial sequencing of the Tp1 and Tp2 genes from T. parva isolates exhibited polymorphisms in both loci, including the epitope-containing regions. Results for sequence analysis showed that the overall nucleotide polymorphism (π) was 0.7% and 13.5% for Tp1 and Tp2, respectively. The Tajima’s D and Fu’s Fs test showed a negative value for both Tp1 and Tp2 genes, indicating deviations from neutrality due to a recent population expansion. The study further revealed a low to high level of genetic differentiations between populations and high genetic variability within populations. The study also revealed that most samples from the seven populations possessed several epitopes in antigens that were identical to those in the T. parva Muguga reference stock, which is the main component of the widely used live vaccine cocktail. Therefore, different strategic planning and cost-effective control measures should be implemented in order to reduce losses caused by ECF in the study areas.

Theileria parva is a tick-transmitted, apicomplexan protozoan found in buffalo (Syncerus caffer) and cattle in eastern, central and southern Africa. The parasite causes a fatal, lymphoproliferative disease in susceptible cattle. Previous studies have shown that the parasites in buffalo comprise a more heterogeneous population than those in cattle, which has led to the concept that the population of parasites circulating in cattle represents a restricted subpopulation of those in buffalo. The present study was undertaken to identify if and where this restriction may occur in cattle naturally infected with parasites from buffalo, by sequencing the T. parva p67 antigen gene from eight buffalo and 12 acutely infected cattle from the same endemic site in Kenya. From 103 sequences, we detected 44 different alleles. Nine alleles were found in both cattle and buffalo, and 17 and 18 found only in the cattle and buffalo populations respectively. Nucleotide and amino acid sequence analyses revealed a similar level of diversity of parasites in both hosts. Principal coordinates and phylogenetic tree analyses did not reveal any clustering associated with the host animals, and the number and degree of mixed T. parva infections was similar in the respective populations. The results suggest that any restriction in the ability of T. parva from buffalo to survive and be transmitted from cattle occurs after entry into and initial transformation of bovine lymphocytes.

Theileria parva infections in cattle causes huge economic losses in the affected African countries, directly impacting the livelihood of the poor small-holder farmers. The current immunization protocol using live sporozoites in eastern Africa, is among the control measures designed to limit T. parva infections in cattle. However, the ability of the immune protection induced by this immunization to protect against field parasites has been compromised by the diversity of the parasite involving the schizont antigen genes. Previous studies have reported on the antigenic diversity of T. parva parasites from southern and eastern Africa, however, similar reports on T. parva parasites particularly from cattle from southern Africa remains scanty, due to the self-limiting nature of Corridor disease. Thus, we evaluated the diversity of CD8+ T-cell regions of ten schizont antigen genes in T. parva parasites associated with Corridor disease and East Coast fever (ECF) from southern and eastern Africa respectively. Regions of schizont antigen (TpAg) genes containing the CD8+ T-cell epitopes (CTL determinants) were amplified from genomic DNA extracted from blood of T. parva positive samples, cloned and sequenced. The results revealed limited diversity between the two parasite groups from cattle from southern and eastern Africa, defying the widely accepted notion that antigen-encoding loci in cattle-derived parasites are conserved, while in buffalo-derived parasites, they are extensively variable. This suggests that only a sub-population of parasites is successfully transmitted from buffalo to cattle, resulting in the limited antigenic diversity in Corridor disease parasites. Tp4, Tp5, Tp7 and Tp8 showed limited to absence of diversity in both parasite groups, suggesting the need to further investigate their immunogenic properties for consideration as candidates for a subunit vaccine. Distinct and common variants of Tp2 were detected among the ECF parasites from eastern Africa indicating evidence of parasite mixing following immunization. This study provides additional information on the comparative diversity of TpAg genes in buffalo- and cattle-derived T. parva parasites from cattle from southern and eastern Africa.

Tremendous progress has been made over the last ten years on East Coast fever (ECF) research. Publication of a reference genome sequence of Theileria parva, the causative agent of ECF, has led to a more thorough characterization of the genotypic and antigenic diversity of the pathogen. It also facilitated identification of antigens that are targets of bovine major histocompatibility complex class I restricted cytotoxic T-lymphocytes (CTLs), induced by a live parasite-based infection and treatment method (ITM) vaccine. This has led to improved knowledge of epitope-specific T-cell responses to ITM that most likely contribute to the phenomenon of strain-specific immunity. The Muguga cocktail ITM vaccine, which provides broad-spectrum immunity to ECF is now a registered product in three countries in eastern Africa. Effort is directed at improving and scaling up the production process to make this vaccine more widely available on a commercial basis in the region. Meanwhile, research to develop a subunit vaccine based on parasite neutralizing antibodies and CTLs has been revived through convening of a research consortium to develop proof-of-concept for a next generation vaccine. Many new scientific and technical advances are facilitating this objective. Hence, the next decade promises even more progress toward an improved control of ECF.

A cross-sectional survey was carried out in four counties of Jonglei State, South Sudan, between May and June 2012 to determine the distribution and northern limit of Theileria parva, the causative agent of East Coast fever in cattle, and its tick vector Rhipicephalus appendiculatus, as a prerequisite to the deployment of relevant control strategies. A total of 1636 ticks, 386 serum samples and 399 blood samples were collected from indigenous, apparently healthy, cattle of different age groups. Tick species were identified morphologically, and the identity of R. appendiculatus was confirmed by DNA barcoding. Overall, the T. parva infection rate in R. appendiculatus was 25% as shown by nested PCR. ELISA was used to assess antibodies to T. parva, and the overall seroprevalence was 22.8%. PCR of the blood samples showed 55 (13.8%) were positive for T. parva. This is the first molecular confirmation of T. parva DNA in areas north of Juba, where it was previously known and established. The northern limit of T. parva was determined as N⁰06.17.792, about 242 Km north from Juba. Implication of this limit on the epidemiology and control of ECF is discussed.

Theileria parva causes East Coast Fever (ECF) a protozoan infection which manifests as a non-symptomatic syndrome among endemically stable indigenous cattle populations. Knowledge of the current genetic diversity and population structure of T. parva is critical for predicting pathogen evolutionary trends to inform development of effective control strategies. In this study the population genetic structure of 78 field isolates of T. parva from indigenous cattle (Ankole, n=41 and East African shorthorn Zebu (EASZ), n=37) sampled from the different agro ecological zones (AEZs) of Uganda was investigated. A total of eight mini- and micro-satellite markers encompassing the four chromosomes of T. parva were used to genotype the study field isolates. The genetic diversity of the surveyed T. parva populations was observed to range from 0.643±0.55 to 0.663±0.41 among the Central and Western AEZs respectively. The overall Wright's F index showed significant genetic variation between the surveyed T. parva populations based on the different AEZs and indigenous cattle breeds (FST=0.133, p

A participatory epidemiological (PE) study was conducted with livestock keepers in Moroto and Kotido districts, Karamoja Region, Uganda, between October and December 2013 to determine the management options and relative importance of tick-borne diseases (TBDs) amongst transhumant zebu cattle. Data collection involved 24 focus group discussions (each comprising 8–12 people) in 24 settlement areas (manyattas), key informant interviews (30), direct observation, a review of surveillance data, clinical examination, and laboratory confirmation of cases of TBDs. Methods used in group discussions included semi-structured interviews, simple ranking, pairwise ranking, matrix scoring, proportional piling and participatory mapping. The results of pairwise comparison showed the Ngakarimojong-named diseases, lokit (East Coast fever, ECF), lopid (anaplasmosis), loukoi (contagious bovine pleuropneumonia, CBPP), lokou (heartwater) and lokulam (babesiosis), were considered the most important cattle diseases in Moroto in that order, while ECF, anaplasmosis, trypanosomosis (ediit), CBPP and nonspecific diarrhoea (loleo) were most important in Kotido. Strong agreement between informant groups (Kendall’s coefficient of concordance W=0.568 and 0.682; p

The control of Theileria parva, a protozoan parasite that threatens almost 50% of the cattle population in Africa, is still a challenge in many affected countries. Theileria parva field parasites from eastern Africa, and parasites comprising the current live T. parva vaccine widely deployed in the same region have been reported to be genotypically diverse. However, similar reports on T. parva parasites from southern Africa are limited, especially in Corridor disease designated areas. Establishing the extent of genetic exchange in T. parva populations is necessary for effective control of the parasite infection. Twelve polymorphic microsatellite and minisatellite loci were targeted for genotypic and population genetics analysis of T. parva parasites from South Africa, Mozambique, Kenya and Uganda using genomic DNA prepared from cattle and buffalo blood samples. The results revealed genotypic similarities among parasites from the two regions of Africa, with possible distinguishing allelic profiles on three loci (MS8, MS19 and MS33) for parasites associated with Corridor disease in South Africa, and East Coast fever in eastern Africa. Individual populations were in linkage equilibrium (VD L) was observed. Genetic divergence was observed to be more within (AMOVA = 74%) than between (AMOVA = 26%) populations. Principal coordinate analysis showed clustering that separated buffalo-derived from cattle-derived T. parva parasites, although parasites from cattle showed a close genetic relationship. The results also demonstrated geographic sub-structuring of T. parva parasites based on the disease syndromes caused in cattle in the two regions of Africa. These findings provide additional information on the genotypic diversity of T. parva parasites from South Africa, and reveal possible differences based on three loci (MS8, MS19 and MS33) and similarities between buffalo-derived T. parva parasites from southern and eastern Africa.

East Coast fever (ECF) and Corridor disease (CD) caused by cattle- and buffalo-derived T. parva respectively are the most economically important tick-borne diseases of cattle in the affected African countries. The p67 gene has been evaluated as a recombinant subunit vaccine against East Coast fever (ECF), and for discrimination of T. parva parasites causing ECF and Corridor disease (CD). The p67 allele type 1 was first identified in cattle-derived T. parva parasites from east Africa, where parasites possessing this allele type have been associated with ECF. Subsequent characterization of buffalo-derived T. parva parasites from South Africa where ECF was eradicated, revealed the presence of a similar allele type, raising concerns as to whether or not allele type 1 from parasites from the two regions is identical. A 900 bp central fragment of the gene encoding p67 was PCR amplified from T. parva DNA extracted from blood collected from cattle and buffalo in South Africa, Mozambique, Kenya, Tanzania and Uganda, followed by DNA sequence analysis. Four p67 allele types previously described were identified. A subtype of p67 allele type 1 was identified in parasites from clinical cases of CD and buffalo from southern Africa. Notably, p67 allele type 1 sequences from parasites associated with ECF in East Africa and CD in Kenya were identical. Analysis of two p67 B-cell epitopes (TpM12 and AR22.7) revealed amino acid substitutions in allele type 1 from buffalo-derived T. parva parasites from southern Africa. However, both epitopes were conserved in allele type 1 from cattle- and buffalo-derived T. parva parasites from East Africa. These findings reveal detection of a subtype of p67 allele type 1 associated with T. parva parasites transmissible from buffalo to cattle in southern Africa.

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. Electronic supplementary material The online version of this article (10.1186/s13071-018-2904-7) contains supplementary material, which is available to authorized users.

International audience; East Coast fever (ECF), one of the most serious tick-borne diseases in sub-Saharan and eastern Africa, was introduced to the island of Grande Comore in 2002 through zebu import from Tanzania, resulting in at least a 10% loss of livestock. A participatory epidemiology initiative was launched in 2015 to gain a better understanding of ECF epidemiology. Thirty-six villages were investigated involving 36 focus group sessions and 120 individual questionnaires. Farmers' knowledge of ECF and of priority diseases affecting the country was assessed, and the impacts of ECF and other major diseases were compared by a scoring method. The results showed that 69.4% (95% CI [51.3, 87.5%]) of the farmers had good to very good knowledge of ECF. The most important cattle diseases on Grande Comore were considered to be East Coast fever, heartwater, babesiosis, and cutaneous diseases. About 58% of the farmers (95% CI [49.2, 66.8%]) use curative treatments when cattle were sick. Between January and September 2015, the ECF incidence was estimated at 18.5% (95% CI [15.5, 21.4%]), and 87.5% (95% CI [72.7, 100%]) of the cattle infected by ECF died. The ECF incidence estimated in our study was found to be less when compared to that observed in Tanzania even though the climatic conditions in the Union of the Comoros are suitable for the biological vector of ECF, the tick species Rhipicephalus appendiculatus. Access to chemical treatment and its effectiveness against ECF, as well as controlling borders and organizing quarantine, are discussed.

Objectives The objective of this study was to assess whether cytotoxic T cells (CTL) generated by the live vaccine, known as “ITM Muguga cocktail”, which is used for the cattle disease East Cost fever (ECF) in Sub-Saharan Africa, showed a broad reactivity against many different strains of the causative parasite Theileria parva. We also assessed whether immune responses were similar in cattle expressing the same MHC class I haplotypes. Results The antigenic specificity of CTL from MHC class I-matched cattle vaccinated with the Muguga cocktail were different. Three cattle of MHC class I haplotype A18, one A18/A19 and two haploidentical (A18v/A12) animals, showed differential recognition of autologous cells infected with a panel of T. parva isolates. This could have implications in the field where certain strains could break through the vaccine. Furthermore, neither of the haploidentical cattle recognized the CTL epitope (Tp1214–224), presented by the A18 haplotype, in contrast to the third animal, showing differences in immunodominance in animals of the same haplotype A18. This suggests that the CTL specificities following immunization with the Muguga cocktail can vary even between haploidentical individuals and that some parasite strains may break through immunity generated by the Muguga cocktail. Electronic supplementary material The online version of this article (10.1186/s13104-018-3145-8) contains supplementary material, which is available to authorized users.

Graphical abstract, Highlights • 2007 Theileria parva proteins expressed in the sporozoite were identified. • Proteins include known T. parva antigens targeted by antibodies and cytotoxic T cells. • Proteins predicted to be orthologs of Plasmodium falciparum sporozoite surface molecules were identified. • Proteins predicted to be orthologs of P. falciparum invasion organelle proteins were identified. • Proteins that may contribute to the phenomenon of bovine lymphocyte transformation were identified., 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.