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.

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.

Infectivity of Deinbollia mosaic virus, a novel weed-infecting begomovirus in East Africa.

Weed-infecting begomoviruses play an important role in the epidemiology of crop diseases because they can potentially infect crops and contribute to the genetic diversity of crop-infecting begomoviruses. Despite the important epidemiological role that weed-infecting begomoviruses play, they remain insufficiently studied in Africa. Recently, we identified Deinbollia mosaic virus (DMV), a distinct begomovirus found naturally infecting the weed host Deinbollia borbonica (Sapindaceae) in Kenya and Tanzania. In this study, we investigated the capacity of DMV to infect a restricted host range of Solanaceae and Euphorbiaceae species. Biolistic inoculation of Nicotiana benthamiana with concatemeric DNAs resulted in systemic infection associated with yellow mosaic symptoms, while DNA partial dimers caused asymptomatic systemic infection. DMV was not infectious to cassava (Manihot esculenta Crantz), suggesting host resistance to the virus. Here, we demonstrate the first experimental infectivity analysis of DMV in N. benthamiana and cassava.

Genetic and antigenic diversity of Theileria parva in cattle in Eastern and Southern zones of Tanzania. A study to support control of East Coast fever.

This study investigated the genetic and antigenic diversity of Theileria parva in cattle from the Eastern and Southern zones of Tanzania. Thirty-nine (62%) positive samples were genotyped using 14 mini- and microsatellite markers with coverage of all four T. parva chromosomes. Wright's F index (F(ST) = 0 × 094) indicated a high level of panmixis. Linkage equilibrium was observed in the two zones studied, suggesting existence of a panmyctic population. In addition, sequence analysis of CD8+ T-cell target antigen genes Tp1 revealed a single protein sequence in all samples analysed, which is also present in the T. parva Muguga strain, which is a component of the FAO1 vaccine. All Tp2 epitope sequences were identical to those in the T. parva Muguga strain, except for one variant of a Tp2 epitope, which is found in T. parva Kiambu 5 strain, also a component the FAO1 vaccine. Neighbour joining tree of the nucleotide sequences of Tp2 showed clustering according to geographical origin. Our results show low genetic and antigenic diversity of T. parva within the populations analysed. This has very important implications for the development of sustainable control measures for T. parva in Eastern and Southern zones of Tanzania, where East Coast fever is endemic.

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.

Mycobacterium tuberculosis is the causative agent of tuberculosis in the southern ecological zones of Cameroon, as shown by genetic analysis.

BACKGROUND: Tuberculosis (TB) is a major cause of mortality and suffering worldwide, with over 95% of TB deaths occurring in low- and middle-income countries. In recent years, molecular typing methods have been widely used in epidemiological studies to aid the control of TB, but this usage has not been the case with many African countries, including Cameroon. The aims of the present investigation were to identify and evaluate the diversity of the Mycobacterium tuberculosis complex (MTBC) isolates circulating in two ecological zones of Cameroon, seven years after the last studies in the West Region, and after the re-organization of the National TB Control Program (NTBCP). These were expected to shed light also on the transmission of TB in the country. The study was conducted from February to July 2009. During this period, 169 patients with symptomatic disease and with sputum cultures that were positive for MTBC were randomly selected for the study from amongst 964 suspected patients in the savannah mosaic zone (West and North West regions) and the tropical rainforest zone (Central region). After culture and diagnosis, DNA was extracted from each of the MTBC isolates and transported to the BecA-ILRI Hub in Nairobi, Kenya for molecular analysis. METHODS: Genetic characterization was done by mycobacterial interspersed repetitive unit-variable number tandem repeat typing (MIRU-VNTR) and Spoligotyping. RESULTS: Molecular analysis showed that all TB cases reported in this study were caused by infections with Mycobacterium tuberculosis (98.8%) and Mycobacterium africanum (M. africanum) (1.2%) respectively. We did not detect any M. bovis. Comparative analyses using spoligotyping revealed that the majority of isolates belong to major clades of M. tuberculosis: Haarlem (7.6%), Latin American-Mediterranean (34.4%) and T clade (26.7%); the remaining isolates (31.3%) where distributed among the minor clades. The predominant group of isolates (34.4%) corresponded to spoligotype 61, previously described as the "Cameroon family. Further analysis based on MIRU-VNTR profiles had greater resolving power than spoligotyping and defined additional genotypes in the same spoligotype cluster. CONCLUSION: The molecular characterization of MTBC strains from humans in two ecological regions of Cameroon has shown that M. tuberculosis sensu stricto is the predominant agent of TB cases in the zones. Three decades ago, TB was reported to be caused by M. africanum in 56.0% of cases. The present findings are consistent with a major shift in the prevalence of M. tuberculosis in Cameroon.

Viral metagenomics demonstrates that domestic pigs are a potential reservoir for Ndumu virus.

BACKGROUND: The rising demand for pork has resulted in a massive expansion of pig production in Uganda. This has resulted in increased contact between humans and pigs. Pigs can act as reservoirs for emerging infectious diseases. Therefore identification of potential zoonotic pathogens is important for public health surveillance. In this study, during a routine general surveillance for African swine fever, domestic pigs from Uganda were screened for the presence of RNA and DNA viruses using a high-throughput pyrosequencing method. FINDINGS: Serum samples from 16 domestic pigs were collected from five regions in Uganda and pooled accordingly. Genomic DNA and RNA were extracted and sequenced on the 454 GS-FLX platform. Among the sequences assigned to a taxon, 53% mapped to the domestic pig (Sus scrofa). African swine fever virus, Torque teno viruses (TTVs), and porcine endogenous retroviruses were identified. Interestingly, two pools (B and C) of RNA origin had sequences that showed 98% sequence identity to Ndumu virus (NDUV). None of the reads had identity to the class Insecta indicating that these sequences were unlikely to result from contamination with mosquito nucleic acids. CONCLUSIONS: This is the first report of the domestic pig as a vertebrate host for Ndumu virus. NDUV had been previously isolated only from culicine mosquitoes. NDUV therefore represents a potential zoonotic pathogen, particularly given the increasing risk of human-livestock-mosquito contact.

Cryptosporidium species detected in calves and cattle in Dagoretti, Nairobi, Kenya.

A total of 1,734 cattle faecal samples from 296 dairy-keeping households were collected from urban settings in Nairobi, Kenya. Modified Ziehl-Neelsen staining method and an immunofluorescence assay were used to identify those samples with Cryptosporidium oocyst infection. Oocysts from positive faecal samples were isolated by Sheather's sucrose flotation method and picked from the concentrate using cover slips. Genomic DNA was extracted from 124 of the faecal samples that were positive for Cryptosporidium and was used as template for nested PCR of the 18S rRNA gene. Twenty-five samples (20 %) were PCR-positive for Cryptosporidium, and 24 of the PCR products were successfully cloned and sequenced. Sequence and phylogenetic analysis identified 17 samples (68 %) as Cryptosporidium parvum-like, four samples (16 %) as Cryptosporidium ryanae, three samples (12 %) as Cryptosporidium andersoni and one sample (4 %) as Cryptosporidium hominis. To the best of our knowledge, this is the first genotyping study to report C. parvum-like, C. andersoni and C. hominis in cattle from Kenya. The results of this study show Cryptosporidium infections in calves and cattle may be potential zoonotic reservoirs of the parasite that infects humans.

Population Genetic Analysis and Sub-Structuring of Theileria annulata in Sudan.

Theileria annulata, which causes tropical theileriosis, is a major impediment to improving cattle production in Sudan. Tropical theileriosis disease is prevalent in the north and central regions of Sudan. Outbreaks of the disease have been observed outside the known endemic areas, in east and west regions of the country, due to changes in tick vector distribution and animal movement. A live schizont attenuated vaccination based on tissue culture technology has been developed to control the disease. The parasite in the field as well as the vaccine strain need to be genotyped before the vaccinations are practiced, in order to be able to monitor any breakthrough or breakdown, if any, after the deployment of the vaccine in the field. Nine microsatellite markers were used to genotype 246 field samples positive for T. annulata DNA and the vaccine strain. North and central populations have a higher multiplicity of infection than east and west populations. The examination of principal components showed two sub-structures with a mix of all four populations in both clusters and the vaccine strain used being aligned with left-lower cluster. Only the north population was in linkage equilibrium, while the other populations were in linkage disequilibrium, and linkage equilibrium was found when all samples were regarded as single population. The genetic identity of the vaccine and field samples was 0.62 with the north population and 0.39 with west population. Overall, genetic investigations of four T. annulata populations in Sudan revealed substantial intermixing, with only two groups exhibiting regional origin independence. In the four geographically distant regions analyzed, there was a high level of genetic variation within each population. The findings show that the live schizont attenuated vaccine, Atbara strain may be acceptable for use in all Sudanese regions where tropical theileriosis occurs.

A nested PCR assay exhibits enhanced sensitivity for detection of Theileria parva infections in bovine blood samples from carrier animals.

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.

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.

A novel SINE family occurs frequently in both genomic DNA and transcribed sequences in ixodid ticks of the arthropod sub-phylum Chelicerata.

Reassociation kinetics and flow cytometry data indicate that ixodid tick genomes are large, relative to most arthropods, containing>or=10(9) base pairs. The molecular basis for this is unknown. We have identified a novel small interspersed element with features of a tRNA-derived SINE, designated Ruka, in genomic sequences of Rhipicephalus appendiculatus and Boophilus (Rhipicephalus) microplus ticks. The SINE was also identified in expressed sequence tag (EST) databases derived from several tissues in four species of ixodid ticks, namely R. appendiculatus, B. (R.) microplus, Amblyomma variegatum and also the more distantly related Ixodes scapularis. Secondary structure predictions indicated that Ruka could adopt a tRNA structure that was, atypically, most similar to a serine tRNA. By extrapolation the frequency of occurrence in the randomly selected BAC clone sequences is consistent with approximately 65,000 copies of Ruka in the R. appendiculatus genome. Real time PCR analyses on genomic DNA indicate copy numbers for specific Ruka subsets between 5800 and 38,000. Several putative conserved Ruka insertion sites were identified in EST sequences of three ixodid tick species based on the flanking sequences associated with the SINEs, indicating that some Ruka transpositions probably occurred prior to speciation within the metastriate division of the Ixodidae. The data strongly suggest that Class I transposable elements form a significant component of tick genomes and may partially account for the large genome sizes observed.

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.

Genetic Diversity and Structure of Domestic Cavy (Cavia porcellus) Populations from Smallholder Farms in Southern Cameroon.

Although domestic cavies are widely used in sub-Saharan Africa as a source of meat and income, there are only a few studies of their population structure and genetic relatedness. This seminal study was designed with the main objective to assess the genetic diversity and determine the population structure of cavy populations from Cameroon to guide the development of a cavy improvement program. Sixteen microsatellite markers were used to genotype 109 individuals from five cavy populations (Wouri, Moungo and Nkongsamba in the Littoral region, and Mémé and Fako in the Southwest region of Cameroon). Twelve markers worked in the five populations with a total of 17 alleles identified, with a range of 2.9 to 4.0 alleles per locus. Observed heterozygosity (from 0.022 to 0.277) among populations was lower than expected heterozygosity (from 0.42 to 0.54). Inbreeding rates between individuals of the populations and between individuals in each population were 59.3% and 57.2%, respectively, against a moderate differentiation rate of 4.9%. All the tested loci deviated from Hardy-Weinberg equilibrium, except for locus 3. Genetic distances between populations were small (from 0.008 to 0.277), with a high rate of variability among individuals within each population (54.4%). Three distinct genetic groups were structured. This study has shown that microsatellites are useful for the genetic characterization of cavy populations in Cameroon and that the populations investigated have sufficient genetic diversity that can be used to be deployed as a basis for weight, prolificacy and disease resistance improvement. The genetic of diversity in Southern Cameroon is wide and constitute an opportunity for cavy breeding program.

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.

Population genetic analysis of Theileria parva isolated in cattle and buffaloes in Tanzania using minisatellite and microsatellite markers.

A population genetic study of Theileria parva was conducted on 103 cattle and 30 buffalo isolates from Kibaha, Lushoto, Njombe Districts and selected National parks in Tanzania. Bovine blood samples were collected from these study areas and categorized into 5 populations; Buffalo, Cattle which graze close to buffalo, Kibaha, Lushoto and Njombe. Samples were tested by nested PCR for T. parva DNA and positives were compared for genetic diversity to the T. parva Muguga vaccine reference strain, using 3micro and 11 minisatellite markers selected from all 4 chromosomes of the parasite genome. The diversity across populations was determined by the mean number of different alleles, mean number of effective alleles, mean number of private allele and expected heterozygosity. The mean number of allele unique to populations for Cattle close to buffalo, Muguga, Njombe, Kibaha, Lushoto and Buffalo populations were 0.18, 0.24, 0.63, 0.71, 1.63 and 3.37, respectively. The mean number of different alleles ranged from 6.97 (Buffalo) to 0.07 (Muguga). Mean number of effective alleles ranged from 4.49 (Buffalo) to 0.29 (Muguga). The mean expected heterozygosity were 0.07 0.29, 0.45, 0.48, 0.59 and 0.64 for Muguga, cattle close to buffalo, Kibaha, Njombe, Lushoto and Buffalo populations, respectively. The Buffalo and Lushoto isolates possessed a close degree of diversity in terms of mean number of different alleles, effective alleles, private alleles and expected heterozygosity. The study revealed more diversity in buffalo isolates and further studies are recommended to establish if there is sharing of parasites between cattle and buffaloes which may affect the effectiveness of the control methods currently in use.

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.

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.

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.