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Theileria parvacauses East Coast fever (ECF), one of the most important and lethal tick-borne diseases of cattle in sub-Saharan Africa. ECF is a considerable burden to the livestock industry, causing annual losses exceeding US $300 million. Currently, diagnosis of T. parva infections relies mainly on clinical signs, serology, and microscopic identification of parasites in either blood or lymph fluid samples. However, some of these tests might not indicate ongoing infection and they all lack the sensitivity to detect low-level infections. Molecular tests such as nested and quantitative PCR assays offer high sensitivity for detection of T. parva. However, these tests remain highly complex technologies that are impractical to use in resource-limited settings where economic losses due to the disease have the most significant impact. A field-deployable, point-of-care test will be of significant value in the treatment and control of ECF in endemic areas. For this purpose, we have developed a CRISPR-Cas12a-based pen-side tool that can sensitively and specifically detect T. parva based on the p104 gene. We describe a streamlined, field-applicable diagnostic tool comprising a 20 min recombinase polymerase amplification (RPA) reaction followed by a 60 min CRISPR-Cas12a reaction using a FAM/Biotin lateral flow strip readout. We tested two different RPA primer pairs and four different CRISPR-RNAs (crRNAs). The p104-based assay displayed high sensitivity, detecting as low as one infected lymphocyte per three microliters of blood and universally detecting eight different T. parva strains without detecting DNA from other Theileria spp. such as Theileria mutans and Theileria lestoquardi. This work opens the way for a field-applicable diagnostic tool for the sensitive point-of-care early diagnosis of T. parva infections in cattle., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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. 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. 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. 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., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

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., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Theileria parva is an economically important, intracellular, tick-transmitted parasite of cattle. A live vaccine against the parasite is effective against challenge from cattle-transmissible T. parva but not against genotypes originating from the African Cape buffalo, a major wildlife reservoir, prompting the need to characterize genome-wide variation within and between cattle- and buffalo-associated T. parva populations. Here, we describe a capture-based target enrichment approach that enables, for the first time, de novo assembly of nearly complete T. parva genomes derived from infected host cell lines. This approach has exceptionally high specificity and sensitivity and is successful for both cattle- and buffalo-derived T. parva parasites. De novo genome assemblies generated for cattle genotypes differ from the reference by ~54K single nucleotide polymorphisms (SNPs) throughout the 8.31 Mb genome, an average of 6.5 SNPs/kb. We report the first buffalo-derived T. parva genome, which is ~20 kb larger than the genome from the reference, cattle-derived, Muguga strain, and contains 25 new potential genes. The average non-synonymous nucleotide diversity (πN) per gene, between buffalo-derived T. parva and the Muguga strain, was 1.3%. This remarkably high level of genetic divergence is supported by an average Wright's fixation index (FST), genome-wide, of 0.44, reflecting a degree of genetic differentiation between cattle- and buffalo-derived T. parva parasites more commonly seen between, rather than within, species. These findings present clear implications for vaccine development, further demonstrated by the ability to assemble nearly all known antigens in the buffalo-derived strain, which will be critical in design of next generation vaccines. The DNA capture approach used provides a clear advantage in specificity over alternative T. parva DNA enrichment methods used previously, such as those that utilize schizont purification, is less labor intensive, and enables in-depth comparative genomics in this apicomplexan parasite., Competing Interests: The authors have declared that no competing interests exist.

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., (© 2020 Blackwell Verlag GmbH.)

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.

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Theileria parva is a parasitic protozoan that causes East Coast fever (ECF), an economically important disease of cattle in eastern, central and southern Africa. In South Sudan, ECF is considered a major constraint for livestock development in regions where the disease is endemic. To obtain insights into the dynamics of T. parva in South Sudan, population genetic analysis was performed. Out of the 751 samples included in this study, 178 blood samples were positive for T. parva by species-specific PCR, were collected from cattle from four regions in South Sudan (Bor = 62; Juba = 45; Kajo keji = 41 and Yei = 30) were genotyped using 14 microsatellite markers spanning the four chromosomes. The T. parva Muguga strain was included in the study as a reference. Linkage disequilibrium was evident when populations from the four regions were treated as a single entity, but, when populations were analyzed separately, linkage disequilibrium was observed in Bor, Juba and Kajo keji. Juba region had a higher multiplicity of infection than the other three regions. Principal components analysis revealed a degree of sub-structure between isolates from each region, suggesting that populations are partially distinct, with genetic exchange and gene flow being limited between parasites in the four geographically separated populations studied. Panmixia was observed within individual populations. Overall T. parva population genetic analyses of four populations in South Sudan exhibited a low level of genetic exchange between the populations, but a high level of genetic diversity within each population., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

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., (© 2016 Blackwell Verlag GmbH.)

We screened cattle and goats from the districts of Chama, Monze and Mumbwa in Zambia for animal African trypanosomes, Babesia bigemina and Theileria parva using PCRs; 38.1% of the samples tested positive for at least one of the parasite species. The most common parasite was Trypanosoma vivax (19.8%). Its incidence was significantly higher in goats than in cattle, (P<0.05). B. bigemina was found in samples from all the three areas, making it the most widespread of the parasites in Zambia. Among the tested samples, 12.0% of the positive samples were mixed infections. There were significant differences in the infection rates of T. vivax (Mumbwa had a significantly higher infection rate [39.6%, P<0.0001]), Th. parva (Monze had the only cases [P<0.0004]) and B. bigemina (Monze had a significantly higher infection rate [40.5%, P<0.0001]). According to the hematocrit values, the packed cell volume (%) among the cattle with mixed infections was significantly lower than that of the other cattle. The presence of multiple parasite species and mixed infections among the Zambian cattle and goat populations is of both clinical and economic importance to livestock farming. The absence of trypanosomosis among the samples from Monze can be attributed to tsetse eradication efforts that took place around Lake Kariba. This shows that the prevention and control of these parasitic diseases can have a significant impact on the disease status, which can translate directly into the improvement of the livestock sector in Zambia.

Molecular classification and systematics of the Theileria is based on the analysis of the 18S rRNA gene. Reverse line blot or conventional sequencing approaches have disadvantages in the study of 18S rRNA diversity and a next-generation 454 sequencing approach was investigated. The 18S rRNA gene was amplified using RLB primers coupled to 96 unique sequence identifiers (MIDs). Theileria positive samples from African buffalo (672) and cattle (480) from southern Africa were combined in batches of 96 and sequenced using the GS Junior 454 sequencer to produce 825711 informative sequences. Sequences were extracted based on MIDs and analysed to identify Theileria genotypes. Genotypes observed in buffalo and cattle were confirmed in the current study, while no new genotypes were discovered. Genotypes showed specific geographic distributions, most probably linked with vector distributions. Host specificity of buffalo and cattle specific genotypes were confirmed and prevalence data as well as relative parasitemia trends indicate preference for different hosts. Mixed infections are common with African buffalo carrying more genotypes compared to cattle. Associative or exclusion co-infection profiles were observed between genotypes that may have implications for speciation and systematics: specifically that more Theileria species may exist in cattle and buffalo than currently recognized. Analysis of primers used for Theileria parva diagnostics indicate that no new genotypes will be amplified by the current primer sets confirming their specificity. T. parva SNP variants that occur in the 18S rRNA hypervariable region were confirmed. A next generation sequencing approach is useful in obtaining comprehensive knowledge regarding 18S rRNA diversity and prevalence for the Theileria, allowing for the assessment of systematics and diagnostic assays based on the 18S gene., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

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<0.01) and (FST=0.101, p<0.01) respectively. Significant pairwise population genetic differentiations (p<0.05) were observed with FST values ranging from 0.048 to 0.173 between the eastern and northern, eastern and western populations respectively. The principal component analysis (PCA) showed a high level of genetic and geographic sub-structuring among populations. Linkage disequilibrium was observed when populations from all the study AEZs were treated as a single population and when analysed separately. On the overall, the significant genetic diversity and geographic sub-structuring exhibited among the study T. parva isolates has critical implications for ECF control., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

This study was carried out to assess the distribution, abundance of different tick genera and prevalence of Theileria parva infection in Tarime zebu cattle kept in selected wards of Serengeti and Tarime districts in Mara region. Adult ticks were identified and counted from half body parts of 360 animals which were extensively managed in communal land with natural pastures. Concurrently, blood samples were collected and thereafter DNA extracted and a nested polymerase chain reaction (nPCR) was done using primers specific for p104 gene to detect the presence of T. parva DNA. Ticks were identified into four groups: Amblyomma genus, Boophilus sub-genus of Rhipicephalus genus, other species of Rhipicephalus, and Hyalomma genus. Rhipicephalus genus accounted for 71.8 % of the total ticks, whereas Amblyomma, Boophilus sub-genus of Rhipicephalus genus and Hyalomma constituted 14.1, 14.0 and 0.1 %, respectively. There were more animals (p < 0.05) infested with ticks in Tarime district (96.1 %) than in Serengeti (61.7 %). The average counts of ticks were higher in adult animals (p < 0.05) than in young animals. The overall prevalence of T. parva was 27.7 % and was higher (p < 0.05) in Serengeti (38.3 %) than in Tarime district (16.7 %). However, all animals tested positive for T. parva did not show any clinical signs of East Coast fever (ECF), suggesting the existence of subclinical infection in Tarime zebu. These results suggest that Tarime cattle can tolerate ECF infection and are likely to serve as potential carriers of T. parva to other less-tolerant cattle breeds in mixed herds. Since Tarime cattle are preferred by most farmers with mixed herds, routine screening for T. parva is highly recommended to minimize introduction of infected cattle into an immunologically naive population.

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.

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.

Background: Theileriosis, caused by Theileria parva, is an economically important disease in Africa. It is a major constraint to the development of the livestock industry in some parts of eastern, central and southern Africa. In Zambia, theileriosis causes losses of up to 10,000 cattle annually., Methods: Cattle blood samples were collected for genetic analysis of Theileria parva from Isoka and Petauke districts in Zambia. Microsatellite analysis was then performed on all Theileria parva positive samples for PCR using a panel of 9 microsatellite markers. Microsatellite data was analyzed using microsatellite toolkit, GenAlEx ver. 6, Fstat ver. 2.9.3.2, and LIAN computer softwares., Results: The combined percentage of positive samples in both districts determined by PCR using the p104 gene primers was 54.9% (95% CI: 46.7 - 63.1%, 78/142), while in each district, it was 44.8% (95% CI: 34.8 - 54.8%) and 76.1% (95% CI = 63.9 - 88.4%) for Isoka and Petauke districts, respectively. We analyzed the population genetic structure of Theileria parva from a total of 61 samples (33 from Isoka and 28 from Petauke) using a panel of 9 microsatellite markers encompassing the 4 chromosomes of Theileria parva. Wright's F index (FST = 0.178) showed significant differentiation between the Isoka and Petauke populations. Linkage disequilibrium was observed when populations from both districts were treated as a single population. When analyzed separately, linkage disequilibrium was observed in Kanyelele and Kalembe areas in Isoka district, Isoka district overall and in Petauke district. Petauke district had a higher multiplicity of infection than Isoka district., Conclusion: Population genetic analyses of Theileria parva from Isoka and Petauke districts showed a low level of genotype exchange between the districts, but a high level of genetic diversity within each district population, implying genetic and geographic sub-structuring between the districts. The sub-structuring observed, along with the lack of panmixia in the populations, could have been due to low transmission levels at the time of sampling. However, the Isoka population was less diverse than the Petauke population.

Background: Theileria parva is a tick-borne protozoan parasite, which causes East Coast Fever, a disease of cattle in sub-Saharan Africa. Like Plasmodium falciparum, the parasite undergoes a transient diploid life-cycle stage in the gut of the arthropod vector, which involves an obligate sexual cycle. As assessed using low-resolution VNTR markers, the crossover (CO) rate in T. parva is relatively high and has been reported to vary across different regions of the genome; non-crossovers (NCOs) and CO-associated gene conversions have not yet been characterised due to the lack of informative markers. To examine all recombination events at high marker resolution, we sequenced the haploid genomes of two parental strains, and two recombinant clones derived from ticks fed on cattle that had been simultaneously co-infected with two different parasite isolates., Results: By comparing the genome sequences, we were able to genotype over 64 thousand SNP markers with an average spacing of 127 bp in the two progeny clones. Previously unrecognized COs in sub-telomeric regions were detected. About 50% of CO breakpoints were accompanied by gene conversion events. Such a high fraction of COs accompanied by gene conversions demonstrated the contributions of meiotic recombination to the diversity and evolutionary success of T. parva, as the process not only redistributed existing genetic variations, but also altered allelic frequencies. Compared to COs, NCOs were more frequently observed and more uniformly distributed across the genome. In both progeny clones, genomic regions with more SNP markers had a reduced frequency of COs or NCOs, suggesting that the sequence divergence between the parental strains was high enough to adversely affect recombination frequencies. Intra-species polymorphism analysis identified 81 loci as likely to be under selection in the sequenced genomes., Conclusions: Using whole genome sequencing of two recombinant clones and their parents, we generated maps of COs, NCOs, and CO-associated gene conversion events for T. parva. The data comprises one of the highest-resolution genome-wide analyses of the multiple outcomes of meiotic recombination for this pathogen. The study also demonstrates the usefulness of high throughput sequencing typing for detailed analysis of recombination in organisms in which conventional genetic analysis is technically difficult.

A capture operation to ascertain health status in free-ranging buffaloes from six different areas in the Caprivi Strip in the northeast corner of Namibia was conducted in October 2009. Basic information on the ticks and tick-borne pathogens normally found in wildlife from this area are scarce. The objective of this study was to assess the host status of African buffaloes, Syncerus caffer, for ixodid ticks and two selected tick-borne pathogens in the Caprivi Strip, a key area bordering Angola, Zambia, Botswana, and Zimbabwe. Four different tick species have been identified among the 233 collected specimens, and, of 95 tested buffaloes, 54 (57%) were positive for Theileria parva, whereas only 3 (3%) showed evidence of being infected with Ehrlichia ruminantium.

Corridor disease is an acute, fatal disease of cattle caused by buffalo-adapted Theileria parva. This is a nationally controlled disease in South Africa and strict control measures apply for the movement of buffalo, which includes mandatory testing for the presence of T. parva and other controlled diseases. Accurate diagnosis of the T. parva carrier state in buffalo using the official real-time hybridization PCR assay (Sibeko et al. 2008), has been shown to be affected by concurrent infection with T. sp. (buffalo)-like parasites. We describe the Hybrid II assay, a real-time hybridization PCR method, which compares well with the official hybridization assay in terms of specificity and sensitivity. It is, however, not influenced by mixed infections of T. sp. (buffalo)-like parasites and is as such a significant improvement on the current hybridization assay.

Livestock diseases have long been a challenge to livestock production and public health in sub-Saharan Africa and Zambia in particular. The Eastern Province of Zambia is one area in Zambia that is not spared by this challenge. Among various livestock diseases affecting cattle in this region, the most prominent are East Coast Fever (ECF) and African Animal Trypanasomiasis (AAT). Since little has been published on the epidemiological trends of these diseases in eastern Zambia, a retrospective epidemiological study was carried out using reports that were submitted to the provincial veterinary office over the past 20 years. This paper assists in evaluating the impact of some of these aid programmes. Data was analysed using Excel(©), SPSS(®), Epi Info(©), and Epi Map(©) software. Apparent prevalence of AAT in cattle had decreased in the study period from estimates as high as 50% in Katete and Petauke district in 1990 and 1992 respectively to just below 3% (Petauke and Katete) in 2008, thereby, reducing the provincial apparent prevalence from 20% in 1992 to just below 3% in 2008. AAT apparent prevalence dropped from estimates as high as 17% in Chadiza district and 6% in Chipata district in 1990 to just below 1% in 2008 thereby reducing the provincial mean prevalence of East Coast Fever from 6% (1990) to 1% (2008). The inclusion of donor assistance in disease control programmes for both AAT and ECF appeared to have a significant impact on the prevalence of both diseases., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A real-time PCR assay based on TaqMan probe chemistry was developed for the detection of Theileria parva DNA in blood samples. It uses a Theileria genus-specific PCR primer set and a T. parva-specific probe to amplify and hybridize with a species-specific part of the 18S rRNA gene of the parasite. The test was evaluated using positive and negative reference blood samples and shown to be specific for T. parva. Analytical sensitivity was determined by testing a dilution series of T. parva positive blood. It was shown to be able to detect parasitaemia as low as 2 × 10(-6)%. The Taqman assay results were also compared with that obtained with the real-time hybridization probe PCR assay, which is currently employed as the official test for the diagnosis of T. parva infections in buffalo and cattle and was shown to be equally sensitive. A panel of 1164 field samples was screened using both assays and 164 samples tested positive in both tests, indicating a good correlation., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Theileria parva, the most important bovine theilerial species in sub-Saharan Africa, causes widespread mortality and morbidity in endemic areas. A survey was conducted using buffy-coat specimens from 60 apparently healthy adult communally herded Nguni-type cattle at the northeastern edge of the Hluhluwe-iMfolozi Park to determine, by means of PCR and Reverse Line Blot (RLB) hybridisation, the occurrence of Theileria and Babesia species. The presence of Trypanosoma species was determined using PCR-RFLP. Results showed that 6.7 % of the specimens were positive for Theileria parva. This significant finding suggests that cattle in South Africa, and not only African buffaloes (Syncerus caffer), may be subclinical carriers of T. parva. Other species identified were T. mutans (83.3%), T. velifera (70.0%), Theileria sp. (sable) (46.8%) and T taurotragi (1.7%). Two specimens (3.3%) were positive for Babesia bovis and single specimens (1.7%) positive for B. bigemina and B. rossi, respectively. Mixed infections, of up to 4 species, were common (65.0%). Only 1 specimen was found to be positive for Trypanosoma vivax, and 2 for T theileri, of which only the first species is pathogenic.

We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria parva, the causative agent of East Coast fever (ECF), an economically important cattle disease in eastern, central and southern Africa. These assays target the polymorphic immunodominant molecule (PIM) and p150 LAMP genes. The primer set for each gene target consists of six primers, and each set recognises eight distinct regions on the target gene to give highly specific detection of T. parva. The detection limit of each primer set is 1fg, which is equivalent to one copy of the PIM and p150 T. parva genes. These PIM and p150 LAMP primer sets amplify DNA of T. parva isolates from cattle and buffalo from different countries including Kenya, South Africa, Tanzania, Rwanda, Uganda and Burundi, indicating their ability to detect T. parva from different countries. With the advantages of simplicity, rapidity and cost effectiveness, these LAMP assays are good candidates for molecular epidemiology studies and for monitoring control programs in ECF-endemic, resource poor countries., (Copyright 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Theileria parva causes East Coast fever, an economically important disease of cattle in sub-Saharan Africa. We describe a nested polymerase chain reaction (nPCR) assay for the detection of T. parva DNA in cattle blood spotted onto filter paper using primers derived from the T. parva-specific 104-kDa antigen (p104) gene. The sensitivity of this assay was compared to a previously described p104-based PCR and also the reverse line blot (RLB) technique, using serial dilutions of blood from a calf with known T. parva piroplasm parasitaemia. The relative sensitivities of the three assays were 0.4, 1.4 and 4 parasites/microl corresponding to blood parasitaemias of 9.2 x 10(-6)%, 2.8 x 10(-5)% and 8.3 x 10(-5)%, respectively. The three assays were applied to samples from two calves infected with the T. parva Muguga stock. Parasite DNA was consistently detectable by the two p104 PCR assays until 48 and 82 days post-infection, respectively, and thereafter sporadically. RLB detected parasite DNA in the two infected calves until days 43 and 45. Field samples from 151 Kenyan cattle exhibited 37.7% positivity for T. parva by regular p104 PCR and 42.3% positivity using p104 nPCR. Among 169 cattle blood samples from Southern Sudan, 36% were positive for T. parva using nPCR. The nPCR assay represents a highly sensitive tool for detection and monitoring of asymptomatic carrier state infections of T. parva in the blood of cattle.

Theileria parva is the etiologic agent of East Coast fever, an economically important disease of cattle in sub-Saharan Africa. This protozoan parasite is biologically transmitted by Rhipicephalus appendiculatus (Neumann) (Acari: Ixodidae). An understanding of the vector-parasite interaction may aid the development of improved methods for controlling transmission. We developed quantitative polymerase chain reaction (qPCR) and nested PCR (nPCR) assays targeting the T. parva-specific p104 gene to study T. parva pathogenesis in two strains of R. appendiculatus that had previously been selected to be relatively more (Kiambu) or less (Muguga) susceptible to infection. Nymphs from both strains were fed simultaneously to repletion on acutely infected calves. Nymphs from the Kiambu strain showed significantly higher engorgement weights compared with Muguga strain nymphs. Immediately after engorgement qPCR confirmed that nymphal Kiambu ticks had significantly higher parasite loads at repletion than Muguga nymphs. By 12 d postengorgement, parasites were below quantifiable levels but could be detected by nPCR in 83-87% (Muguga and Kiambu, respectively) of nymphs. After the molt, adult feeding on naïve cattle stimulated parasite replication in the salivary glands. PCR detected significantly more infected ticks than microscopy, and there was a significant difference between the two tick strains both in the proportion of ticks that develop salivary gland infections, and in the number of parasites within infected salivary glands. These data confirm that although both tick strains were competent vectors, Kiambu is both a significantly more susceptible and a more efficient host for T. parva than Muguga. The mechanisms that contribute to the levels of susceptibility and efficiency are unknown; however, this study lays the groundwork for a comparison of the transcriptome of these tick strains, the next step toward discovering the genes involved in the tick-parasite interaction.

Corridor disease, caused by the tick-borne protozoan parasite Theileria parva, is a controlled disease in South Africa. The Cape buffalo is the reservoir host and uninfected buffalo have become sought-after by the game industry in South Africa, particularly for introduction into Corridor disease-free areas. A real-time polymerase chain reaction (PCR) test for detection of T. parva DNA in buffalo and cattle was developed to improve the sensitivity and specificity of the official diagnostic test package in South Africa. Oligonucleotide primers and hybridization probes were designed based on the 18S ribosomal RNA (rRNA) gene. Amplification of control DNA using Theileria genus-specific primers resulted in detection of T. taurotragi and T. annulata, in addition to T. parva. A T. parva-specific forward primer was designed which eliminated amplification of all other Theileria species, except for Theileria sp. (buffalo); however only the T. parva product was detected by the T. parva-specific hybridization probe set. The real-time PCR assay requires less time to perform, is more sensitive than the other molecular assays previously used in T. parva diagnostics and can reliably detect the parasite in carrier animals with a piroplasm parasitaemia as low as 8.79 x 10(-4)%.

Theileria parva causes widespread morbidity and mortality in cattle in endemic regions. An outbreak of theileriosis occurred on a farm near Ladysmith in KwaZulu-Natal, South Africa, which is not a declared Corridor disease-infected area. A survey of Red Brangus cattle from all age groups and areas of the farm was performed. Transmission of the parasite from infected animals on the farm to susceptible animals by tick transmission and tick-stabilate injection, was attempted. The survey indicated high numbers of animals with antibody titres to T. parva but only 6 infected animals, based on real-time PCR and RLB analysis. The transmission experiments failed to transmit the parasite. The study shows the difficulty in elucidating a source of infection and determining the dynamics of new infections in a herd where multiple possible sources are present and treatment with tetracyclines has taken place.

Theileria parva is an intracellular protozoan parasite transmitted by ticks that causes a fatal lymphoproliferative disease of cattle known as East Coast Fever. Vaccination against the disease currently relies on inoculation of the infective sporozoite stage of the parasite and simultaneous treatment with long-acting formulations of oxytetracycline. Sporozoites are maintained as frozen stabilates of triturated infected ticks and the method requires accurate titration of stabilates to determine appropriate dose rates. Titration has traditionally been undertaken in cattle and requires large numbers of animals because of individual variation in susceptibility to infection. An alternative tissue culture-based method is laborious and time consuming. We have developed a flow cytometric method for quantifying the infectivity of sporozoite stabilates in vitro based on the detection of intracellular parasite antigen. The method allows clear identification of parasitized cells with a high degree of sensitivity and specificity. Analysis of infected cells between 48 and 72 h post-infection clearly defines the potential transforming capability of different stabilates.

A multi-variate logistic regression analysis was performed on two sets of data on the prevalence of Theileria annulata in Northern Sudan and Theileria parva in Southern Sudan, to determine the potential risk factors that might affect the distribution of the infections in those regions. The logistic regression model was fit with the tested risk factors for each disease, separately. The results indicated that locations, management systems and age could be held as risk factors for T. annulata infection in Northern Sudan, while for T. parva locations and seasons could be held as risk factors in Southern Sudan. The results of this study will assist in the development of more effective control strategies for smallholder dairy farms in the country.

A herd-based study was carried out in Central Equatoria State, Southern Sudan, to study epidemiological aspects of tick-borne diseases. Six herds of cattle situated in three different locations were selected and investigated every 3 months during the year 2005. Blood smears for Giemsa staining and blood spots on filter paper for deoxyribonucleic acid extraction were collected from 600 apparently healthy indigenous cattle. A total of 69 (11.5%) samples showed the presence of piroplasms in Giemsa-stained blood smears, and polymerase chain reaction increased the detection limit to 297 (49.5%). Using reverse line blot, it was possible to detect and differentiate eight different piroplasms namely, Theileria parva (71.2%), Theileria mutans (73%), Theileria velifera (45.3%), Theileria taurotragi (2.7%), Theileria buffeli (0.5%), Theileria annulata (0.2%), Babesia bovis (1.7%), and Babesia bigemina (0.3%). Mixed infections were detected in 406 samples (67.7%) accounting for 17 different combinations. High infection of Theileria parva was reported among young calves compared to older cattle. The highest prevalence of Theileria parva was reported in the rainy season (October). The implications of these results on the epidemiology of tick-borne diseases are discussed with emphasis on East Coast fever.

An epidemiological analysis based on three country wide surveys was carried out to determine the prevalence of infections with Theileria spp. in Rwanda. In the 1998 dry season, a total of 264 blood samples were submitted to Theileria spp. characterisation using the 18S species-specific PCR-RFLP assay. The same samples together with 634 samples (317 samples/season) collected during the 2002 dry season and the 2003 wet season were further analysed using the p104 Theileria parva specific PCR. The results from the 18S characterisation showed the presence of four Theileria spp., namely T. parva, T. mutans, T. taurotragi and T. velifera in the field. Half of the animals had multiple Theileria spp. infections. T. parva was the most prevalent and a high correlation (94%) was found between the prevalence results using the 18S and the p104 PCR assays. The prevalence of T. parva infections was stable over time and over season but decreased significantly from the high land to the low land areas. This unexpected trend cannot be explained alone by ecology or the dynamics of the tick population in the different zones, many other components such as breed type, tick control practices and grazing system are likely to play a role. Another important finding was the fact that young animals are infected early in life in all regions except in the high land zone indicating the existence of a particular epidemiological situation in this part of the country.

We evaluated sexual recombination in the apicomplexan parasite Theileria parva using genome-wide marker analysis of haploid sporozoite populations obtained from infected Rhipicephalus appendiculatus ticks. Analysis of 231 parasite clones derived by in vitro infection of bovine lymphocytes revealed 48 distinct combinations of 64 polymorphic marker loci. One genotype accounted for more than 75% of the clones, and the population was highly inbred with respect to this. The occurrence of frequent recombination was evident from reassortment of contiguous markers in blocks, with some recombination occurring within blocks. Analysis of four polymorphic loci encoding antigens targeted by protective cytotoxic-T-lymphocyte responses confirmed that these loci reassort, both within and between chromosomes, suggesting that recombination may influence immune recognition. Marker analysis of a panel of 142 clones derived from the population after an additional passage through a calf and the same tick colony revealed 18 genotypes, with the original dominant genotype accounting for 75% of the population and a higher level of inbreeding with respect to it in the remaining clones. Selected marker analysis of genomic DNA from these stabilates and the two preceding generations of the isolate, each derived from distinct tick colonies, revealed shifts in population structure with each generation, suggesting that the tick vector may impose nonrandom selective pressure on the parasite.

Theileria sp. (China) causes severe limitations on the development of the livestock industry in the north-west of China. An enzyme-linked immunosorbent assay (ELISA) based on merozoite homogenate of the parasite for diagnosis of infection has been established; however, cross-reactivity with other small ruminant-infecting piroplasms could not be excluded. Thus, a prerequisite for epidemiological surveys and diagnosis was the establishment of a recombinant protein-based ELISA. To this end, serum from Theileria sp. (China)-infected sheep was used to screen a Theileria lestoquardi expression library, resulting in the identification of a specifically reacting clone with a high identity to the heat shock protein 70 (HSP70) of Theileria parva and Theileria annulata and thus named TlHSP70. An HSP70 homologue was also confirmed to be expressed by Theileria sp. (China) merozoites (TcHSP70). A part of the TlHSP70 protein, found to be conserved in TcHSP70, was recombinantly expressed and used to establish an ELISA. A total of 260 field serum samples tested resulted in a sensitivity and specificity of 94.3 and 89.5%, respectively, in comparison with the merozoite homogenate ELISA. The potentials of the application of the test in epidemiological surveys to map out the prevalence of the disease and for routine diagnostics are described.

East Coast fever (ECF), a tick-borne disease of cattle, is a major constraint to livestock development in Africa in general and southern Zambia in particular. Understanding the transmission patterns of this disease complex is very difficult as shown by previous studies in southern and eastern Zambia due to the interplay of risk factors. In this long-term study, we investigated whether global weather changes had any influence on disease transmission in traditionally kept cattle in southern Zambia. The results from this study show a strong association between increased Theileria parva contacts in cattle and the presence of El Niño, clearly linking a simple climatic index to disease outbreaks. We therefore propose that in southern Zambia, the simple and readily available multiple El Niño Southern oscillation index (MEI) ranks be used in planning ECF control programmes and early warning.

Theileria parva sporozoite stabilates are used in the infection and treatment method of immunization, a widely accepted control option for East Coast fever in cattle. T. parva sporozoites are extracted from infected adult Rhipicephalus appendiculatus ticks either manually, using a pestle and a mortar, or by use of an electric homogenizer. A comparison of the two methods as a function of stabilate infectivity has never been documented. This study was designed to provide a quantitative comparison of stabilates produced by the two methods. The approach was to prepare batches of stabilate by both methods and then subject them to in vitro titration. Equivalence testing was then performed on the average effective doses (ED). The ratio of infective sporozoites yielded by the two methods was found to be 1.14 in favour of the manually ground stabilate with an upper limit of the 95% confidence interval equal to 1.3. We conclude that the choice of method rests more on costs, available infrastructure and standardization than on which method produces a richer sporozoite stabilate.

Four Theileria parva isolates from Muguga area of Kiambu district, Kenya, were used to establish schizont-infected cell lines. Their protein antigens were then separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS page). The isolates were subsequently subjected to protein analysis and characterisation by the western immunoblotting technique. Probing for the polymorphic immunodominant molecule (PIM) was done using monoclonal antibody no. 4. SDS page detected up to 20 protein antigens of molecular mass 35,000-180,000 Da. The western blot analysis revealed a greater heterogeneity in the molecular mass (M(r)) of PIM than previously thought. The M(r) of PIM varied between 80 and 90 kDa. The isolates further revealed different densities of surface epitopes with variable reaction to the monoclonal antibody. The implications of these findings to the epidemiology of east coast fever and immunisation programmes are discussed.

Genetic diversity and structural organisation of the polymorphic immunodominant molecule (PIM) gene of the protozoan parasite Theileria parva was studied in isolates from sympatric and allopatric areas. The analyses revealed a mosaic structure consisting of highly conserved regions shared among some of the isolates from geographically different areas and homologous sequence runs shared among isolates from one area. The specific pattern of diversity in which large insertions and deletions were observed, giving a mosaic structure to the PIM locus, is quite exceptional for single-locus genes. The polymorphic middle region of the gene was characterised by large deletions or insertions in many isolates. There was no correlation between the copy number of the tetrapeptide repeats in this region and the total length of the sequence. The gene was highly polymorphic when compared with sequences from other known T. parva antigenic regions. The findings support the concept that as yet unidentified mechanisms are generating extensive diversity and shaping the PIM locus. The relevance of this finding for diagnosis and the relationship between these mechanisms and the possible role of this protein in host immune responses is discussed.

The ability of PCR to detect infections of Theileria parva, the cause of East Coast Fever, in field-collected tick and bovine samples from Tanzania was evaluated. PCR-detected infection prevalence was high (15/20, 75%) in unfed adult Rhipicephalus appendiculatus ticks that fed as nymphs on an acutely-infected calf, but low (22/836, 2.6%) in unfed adult R. appendiculatus collected from field sites in Tanzania. Tick infection prevalence was comparable to that in previous studies that used salivary gland staining to detect T. parva infection in field-collected host-seeking ticks. Of 282 naturally-exposed zebu calves, seven had PCR-positive buffy coat samples prior to detection of Theileria spp. parasites in stained buffy coat cells or lymph node biopsies. Evidence of Theileria spp. infections was detected in stained smears of lymph node biopsies from 109 calves (38.6%) and buffy coat samples from 81 (28.7%), while buffy coat samples from 66 (23.4%) were PCR-positive for T. parva. Implications of these findings for the sensitivity and specificity of the PCR are discussed.

The association between mean daily weight gain, Theileria parva infections, clinical East Coast fever and other possible determinants of weight gain were examined in a longitudinal observational study that was conducted in cohorts of female calves from five agro-ecological zone (AEZ)-grazing strata. The strata were upper-midlands (UM) 1 zero-grazing, UM 1 open-grazing, UM 2 zero-grazing, UM 4 zero-grazing and UM 4 open-grazing. In total, 225 calves on 188 smallholder dairy farms were visited within the first 2 weeks of life and thereafter at biweekly intervals up to the age of 6 months between March 1995 and August 1996. During each visit, the calves were weighed and other calf-management practices in the farm during the visit such as housing, feeding and tick control also were recorded. Other events such as morbidity and mortality between or during the visits were also recorded. The overall mean daily weight gains were 0.24-0.29 kg (S.D.=0.17-0.22 kg) and were lower than the recommended targets for smallholder farms of 0.40-0.50 kg. The major tendency in variability of daily weight gains was due to visit-to-visit variation (especially in calves >3 months old). Differences in mean daily gains were associated with AEZ-grazing strata and calf-level factors that included breed of calf, calf sickness, incidence of ECF, feeding of milk, concentrate feeds and minerals and interaction between calf age and AEZ-grazing strata (P<0.05). ECF and other calf sicknesses exerted a temporal effect on calf-growth at the height of illness and immediately after; calves later recovered the lost growth except where other factors such as poor calf nutrition prevailed. Improvement in calf-growth in Murang'a District is achievable and extension services should continue to target individual-calf-level management practices.

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

During the period August-September 1995, an epidemic of East Coast fever occurred at a dairy farm in Morogoro region of eastern Tanzania. Due to an intensive dipping scheme since 1970, a very unstable endemic status had been established in the animals. A breakdown in the dipping scheme caused a major disease outbreak; the dip wash was not changed for 18 months prior to the outbreak and dipping continued in a dip wash of unknown strength. There was also a delay in detecting the disease at an early stage. In total, 180 out of 432 (42%) of the cattle at the farm died--resulting in a loss of Tshs. 26,330,000 (US$ 42,879). The attack risk was nearly 77%. The outbreak points to the importance of adopting integrated strategies for the control of ticks and tick-borne diseases.

The incidence of first contact with the protozoan Theileria parva was determined in two traditional cattle herds in the Southern Province of Zambia during a period of average rainfall in 1997 and 1998, following a drought in the previous two years. Compared to that period, there was a marked increase in the number of rainy season first contacts attributable to transmission by Rhipicephalus appendiceulatus adults. However, there were still more dry season contacts that resulted from nymphal transmission. These results highlight the important role that climate plays in the transmission of theileriosis in the Southern Province of Zambia.

A reverse line blot hybridization (RLB) test was developed to specifically identify six Theileria spp. (T. annulata, T. parva, T. mutans, T. velifera, T. taurotragi, and T. buffeli/orientalis) and three Babesia spp. (B. bovis, B. bigemina, and B. divergens). No cross reaction was observed with other livestock pathogens (such as Anaplasma marginale, A. centrale, A. ovis, Cowdria ruminantium, Trypanosoma brucei, T. congolense, and T. vivax). This method was used to test bovine blood samples collected in Sicily in April and November, 1998. Preliminary results indicated that T. annulata and T. buffeli/orientalis were the main species observed in cattle blood. Babesia species represented 1.8% and 23.5% in April and November, respectively.

Theileria parasites transform bovine leukocytes and induce uncontrolled lymphoproliferation only in the macroschizont stage of their life cycle. The isolation of highly purified stage-specific parasite RNA and proteins is an essential prerequisite when studying the Theileria-host relationship. We therefore improved a protocol based on the cytolytic bacterial toxin aerolysin by taking advantage of the microtubule inhibitor nocodazole. In this report we describe that nocodazole-mediated separation of the parasite from the host cell microtubule network was used with success to improve quantity and quality of purified parasites. We furthermore show that nocodazole is a useful tool to study cell cycle checkpoints due to its capacity to induce reversible cell cycle arrest in Theileria-infected B cells.

Theileria parva is the causal agent of East Coast fever (ECF), a fatal disease of cattle characterized by pyrexia, transient lymphadenopathy and panleukopenia. We have evaluated monoclonal antibodies (mAb) against three distinct antigens (p67, PIM and p32) of the parasite as immunohistological reagents for monitoring the kinetics of infection in cattle. Bovine lymphocytes were stained with the mAb at various intervals after infection in vitro and in vivo. The p67 sporozoite surface antigen was detected in only a small percentage of both, in vitro and in vivo infected cells. In contrast, expression of the polymorphic immunodominant molecule (PIM) of the parasite proved a useful indicator of infection and staining was correlated with the results of Giemsa analysis. PIM was detected from day 3 in in vitro-infected cells, but was not detected until day 5 in vivo after challenge with a 70% lethal dose of stabilized sporozoite. The p32 antigen was expressed only late in infection in vivo and its expression was associated with the development of merozoites. Less than 20% of in vitro-infected cells expressed p32. The immunohistochemical staining with anti-PIM mAb was found to be a useful tool for analysis of T. parva infection kinetics in cattle.