Multilocus genotyping of Theileria parva isolates associated with a live vaccination trial in Kenya provides evidence for transmission of immunizing parasites into local tick and cattle populations.

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.

Mitochondrial and nuclear multilocus phylogeny of Rhipicephalus ticks from Kenya.

The morphological diversity of African ticks of the genus Rhipicephalus and subgenus Boophilus have been studied in detail. However, their taxonomy remains poorly resolved with limited molecular studies performed to improve inter-species discrimination. Herein, ribosomal cytochrome c oxidase I (COI), 12S ribosomal DNA (12S rDNA) and nuclear ribosomal DNA internal transcriber spacer 2 (ITS2) were analyzed in Rhipicephalus tick populations in Kenya. While the morphological and molecular criteria separated R. e. evertsi, R. pulchellus and R. appendiculatus from other members of the genus, except the morphologically similar sibling species R. zambeziensis, this was not the case for other tick populations. COI sequences of Rhipicephalus ticks from Ruma National Park (RNP) in Southwestern Kenya, that were morphologically similar to R. praetextatus/R. simus, a formed distinct clade and barcode gap group. 12S rDNA haplotypes of this population were 99% identical to a GenBank accession of R. muhsamae which is thought to be endemic in West and Central Africa. However, the ITS2 locus indicated that the RNP samples were genetically closest to ticks identified morphologically as R. praetextatus. The COI and 12S rDNA haplotype sequences of R. praetextatus clustered closely with R. simus reference sequences though the two species occurred in distinct barcode gap groups. Our results suggest that the R. simus/R. praetextatus/R. muhsamae comprise a closely related tick species complex found across sub-Saharan Africa and includes the yet to be described RNP population. More studies on the biology, ecology and genomics of all life stages of tick species in the complex may clarify their taxonomic status. A continent-wide study that combines morphology, DNA marker sequencing and emerging methods, such as mass spectrometry and whole-genome resequencing may reveal the diversity and distribution of taxa within the genus Rhipicephalus in sub-Saharan Africa.

THEILERIOSIS IN MOUNTAIN BONGO REPATRIATED TO KENYA: A CLINICAL AND MOLECULAR INVESTIGATION.

Mountain bongo (Tragelaphus euryceros isaaci) from Kenya were exported to zoological institutions in North America and Europe in the 1970s and 1980s. In the following 20-30 years bongo numbers declined in Kenya and the Mountain Bongo Repatriation Project was launched. This resulted in 18 adult bongo, descendants of the original translocated bongo, being repatriated from the United States to Kenya in 2004. These newly arrived bongo were inadvertently exposed to heavy tick infestation on release in a conservancy on the slopes of Mount Kenya. Mortality and morbidity occurred during the third week after arrival. Theileria sp. infection was apparent from the history, clinical signs, and necropsy findings, and Theileria-like parasites were detected microscopically in samples from sick and dead animals. Four bongo died before the outbreak was controlled. In order to identify the Theileria parasite conclusively, molecular amplification techniques were used. A combination of reverse line blotting, with small subunit ribosomal RNA (SSU rRNA) polymerase chain reaction (PCR) amplification and nucleotide sequencing, identified the protozoan parasite Theileria taurotragi, suggesting this as the most probable cause of mortality and morbidity in the repatriated bongo.

Genetic diversity and population structure of Theileria parva in South Sudan.

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.

Genes encoding two Theileria parva antigens recognized by CD8+ T-cells exhibit sequence diversity in South Sudanese cattle populations but the majority of alleles are similar to the Muguga component of the live vaccine cocktail.

East Coast fever (ECF), caused by Theileria parva infection, is a frequently fatal disease of cattle in eastern, central and southern Africa, and an emerging disease in South Sudan. Immunization using the infection and treatment method (ITM) is increasingly being used for control in countries affected by ECF, but not yet in South Sudan. It has been reported that CD8+ T-cell lymphocytes specific for parasitized cells play a central role in the immunity induced by ITM and a number of T. parva antigens recognized by parasite-specific CD8+ T-cells have been identified. In this study we determined the sequence diversity among two of these antigens, Tp1 and Tp2, which are under evaluation as candidates for inclusion in a sub-unit vaccine. T. parva samples (n = 81) obtained from cattle in four geographical regions of South Sudan were studied for sequence polymorphism in partial sequences of the Tp1 and Tp2 genes. Eight positions (1.97%) in Tp1 and 78 positions (15.48%) in Tp2 were shown to be polymorphic, giving rise to four and 14 antigen variants in Tp1 and Tp2, respectively. The overall nucleotide diversity in the Tp1 and Tp2 genes was π = 1.65% and π = 4.76%, respectively. The parasites were sampled from regions approximately 300 km apart, but there was limited evidence for genetic differentiation between populations. Analyses of the sequences revealed limited numbers of amino acid polymorphisms both overall and in residues within the mapped CD8+ T-cell epitopes. Although novel epitopes were identified in the samples from South Sudan, a large number of the samples harboured several epitopes in both antigens that were similar to those in the T. parva Muguga reference stock, which is a key component in the widely used live vaccine cocktail.

Deinbollia mosaic virus: a novel begomovirus infecting the sapindaceous weed Deinbollia borbonica in Kenya and Tanzania.

Four isolates of a bipartite begomovirus from naturally infected Deinbollia borbonica plants exhibiting yellow mosaic symptoms in Kenya and Tanzania were molecularly characterised. The DNA-A was most closely related to that of tomato leaf curl Mayotte virus (AM701764; 82%), while the DNA-B shared the highest nucleotide sequence identity with that of East African cassava mosaic virus (AJ704953) at 65%. Based on the current ICTV species demarcation criterion for the genus Begomovirus (≥91% sequence identity for the complete DNA-A), we report the full-length genome sequence of this novel bipartite begomovirus. The results reveal additional diversity and reservoir hosts of begomoviruses in East Africa.

Analyses of mitochondrial genes reveal two sympatric but genetically divergent lineages of Rhipicephalus appendiculatus in Kenya.

BACKGROUND: The ixodid tick Rhipicephalus appendiculatus transmits the apicomplexan protozoan parasite Theileria parva, which causes East coast fever (ECF), the most economically important cattle disease in eastern and southern Africa. Recent analysis of micro- and minisatellite markers showed an absence of geographical and host-associated genetic sub-structuring amongst field populations of R. appendiculatus in Kenya. To assess further the phylogenetic relationships between field and laboratory R. appendiculatus tick isolates, this study examined sequence variations at two mitochondrial genes, cytochrome c oxidase subunit I (COI) and 12S ribosomal RNA (rRNA), and the nuclear encoded ribosomal internal transcribed spacer 2 (ITS2) of the rRNA gene, respectively. RESULTS: The analysis of 332 COI sequences revealed 30 polymorphic sites, which defined 28 haplotypes that were separated into two distinct haplogroups (A and B). Inclusion of previously published haplotypes in our analysis revealed a high degree of phylogenetic complexity never reported before in haplogroup A. Neither haplogroup however, showed any clustering pattern related to either the geographical sampling location, the type of tick sampled (laboratory stocks vs field populations) or the mammalian host species. This finding was supported by the results obtained from the analysis of 12S rDNA sequences. Analysis of molecular variance (AMOVA) indicated that 90.8 % of the total genetic variation was explained by the two haplogroups, providing further support for their genetic divergence. These results were, however, not replicated by the nuclear transcribed ITS2 sequences likely because of recombination between the nuclear genomes maintaining a high level of genetic sequence conservation. CONCLUSIONS: COI and 12S rDNA are better markers than ITS2 for studying intraspecific diversity. Based on these genes, two major genetic groups of R. appendiculatus that have gone through a demographic expansion exist in Kenya. The two groups show no phylogeographic structure or correlation with the type of host species from which the ticks were collected, nor to the evolutionary and breeding history of the species. The two lineages may have a wide geographic distribution range in eastern and southern Africa. The findings of this study may have implications for the spread and control of R. appendiculatus, and indirectly, on the transmission dynamics of ECF.

Multi-locus genotyping reveals absence of genetic structure in field populations of the brown ear tick (Rhipicephalus appendiculatus) in Kenya.

Rhipicephalus appendiculatus is an important tick vector of several pathogens and parasitizes domestic and wild animals across eastern and southern Africa. However, its inherent genetic variation and population structure is poorly understood. To investigate whether mammalian host species, geographic separation and resulting reproductive isolation, or a combination of these, define the genetic structure of R. appendiculatus, we analyzed multi-locus genotype data from 392 individuals from 10 geographic locations in Kenya generated in an earlier study. These ticks were associated with three types of mammalian host situations; (1) cattle grazing systems, (2) cattle and wildlife co-grazing systems (3) wildlife grazing systems without livestock. We also analyzed data from 460 individuals from 10 populations maintained as closed laboratory stocks and 117 individuals from five other species in the genus Rhipicephalus. The pattern of genotypes observed indicated low levels of genetic differentiation between the ten field populations (FST=0.014±0.002) and a lack of genetic divergence corresponding to the degree of separation of the geographic sampling locations. There was also no clear association of particular tick genotypes with specific host species. This is consistent with tick dispersal over large geographic ranges and lack of host specificity. In contrast, the 10 laboratory populations (FST=0.248±0.015) and the five other species of Rhipicephalus (FST=0.368±0.032) were strongly differentiated into distinct genetic groups. Some laboratory bred populations diverged markedly from their field counterparts in spite of originally being sampled from the same geographic locations. Our results demonstrate a lack of defined population genetic differentiation in field populations of the generalist R. appendiculatus in Kenya, which may be a result of the frequent anthropogenic movement of livestock and mobility of its several wildlife hosts between different locations.

Heterogeneity in the prevalence and intensity of bovine trypanosomiasis in the districts of Amuru and Nwoya, Northern Uganda.

BACKGROUND: Livestock trypanosomiasis, transmitted mainly by tsetse flies of the genus Glossina is a major constraint to livestock health and productivity in the sub-Saharan Africa. Knowledge of the prevalence and intensity of trypanosomiasis is important in understanding the epidemiology of the disease. The objectives of this study were to (a) assess the prevalence and intensity of trypanosome infections in cattle, and (b) to investigate the reasons for the heterogeneity of the disease in the tsetse infested districts of Amuru and Nwoya, northern Uganda. METHODS: A cross-sectional study was conducted from September, 2011 to January, 2012. Blood samples were collected from 816 cattle following jugular vein puncture, and screened for trypanosomes by HCT and ITS-PCR. A Pearson chi-squared test and logistic regression analyses were performed to determine the association between location, age, sex, and prevalence of trypanosome infections. RESULTS: Out of the 816 blood samples examined, 178 (22 %) and 338 (41 %) tested positive for trypanosomiasis by HCT and ITS-PCR, respectively. Trypanosoma vivax infection accounted for 77 % of infections detected by ITS-PCR, T. congolense (16 %), T. brucei s.l (4 %) and mixed (T. vivax/ T. congolense/T.brucei) infections (3 %). The risk of trypanosome infection was significantly associated with cattle age (χ (2) = 220.4, df = 3, P < 0.001). The highest proportions of infected animals were adult males (26.7 %) and the least infected were the less than one year old calves (2.0 %). In addition, the risk of trypanosome infection was significantly associated with sex (χ (2) = 16.64, df = 1, P < 0.001), and males had a significantly higher prevalence of infections (26.8 %) than females (14.6 %). CONCLUSION: Our results indicate that the prevalence and intensity of trypanosome infections are highly heterogeneous being associated with cattle age, location and sex.

Prevalence of Theileria equi and Babesia caballi as well as the identification of associated ticks in sympatric Grevy's zebras (Equus grevyi) and donkeys (Equus africanus asinus) in northern Kenya.

The role of equine piroplasmosis as a factor in the population decline of the Grevy's zebra is not known. We determined the prevalence of Babesia caballi and Theileria equi in cograzing Grevy's zebras (Equus grevyi) and donkeys (Equus africanus asinus) in northern Kenya and identified the associated tick vectors. Blood samples were taken from 71 donkeys and 16 Grevy's zebras from March to May 2011. A nested PCR reaction using 18s ribosomal (r)RNA primers on 87 blood spots showed 72% (51/71; 95% confidence interval [CI] 60.4-81.0%) of donkeys and 100% (16/16; 95% CI, 77.3-100%) of Grevy's zebras were T. equi positive. No samples were positive for B. caballi. Sequence comparison using the National Center for Biotechnology Information's basic local alignment search tool identified homologous 18s rRNA sequences with a global geographic spread. The T. equi-derived sequences were evaluated using Bayesian approaches with independent Metropolis-coupled Markov chain Monte Carlo runs. The sequences clustered with those found in Sudan, Croatia, Mongolia, and the US, with statistical support greater than 80% for the two main clades. Hyalomma tick species were found on both donkeys and Grevy's zebras, whereas Rhipicephalus pulchellus was found exclusively on Grevy's zebras and Hyalomma marginatum rupfipes on donkeys. The prevalence of T. equi was 100% in Grevy's zebras and 72% in donkeys with common tick vectors identified. Our results suggest that donkeys and Grevy's zebras can be asymptomatic carriers and that piroplasmosis is endemic in the study area.