Stephen Mwaura

AvGI, an index of genes transcribed in the salivary glands of the ixodid tick Amblyomma variegatum.

Random clones from a cDNA library made from mRNA purified from dissected salivary glands of feeding female Amblyomma variegatum ticks were subjected to single pass sequence analysis. A total of 3992 sequences with an average read length of 580 nucleotides have been used to construct a gene index called AvGI that consists of 2109 non-redundant sequences. A provisional gene identity has been assigned to 39% of the database entries by sequence similarity searches against a non-redundant amino acid database and a protein database that has been assigned gene ontology terms. Homologs of genes encoding basic cellular functions including previously characterised enzyme activities, such as stearoyl CoA saturase and protein phosphatase, of ixodid tick salivary glands were found. Several families of abundant cDNA sequences that may code for protein components of tick cement and A. variegatum proteins which may contribute to anti-haemostatic and anti-inflammatory responses, and, one with potential immunosuppressive activity, were also identified. Interference with the function of such proteins might disrupt the life cycle of A. variegatum and help to control this ectoparasite or to reduce its ability to transmit disease causing organisms. AvGI represents an electronic knowledge base, which can be used to launch investigations of the biology of the salivary glands of this tick species. The database may be accessed via the World Wide Web at http://www.tigr.org/tdb/tgi.shtml.

The persistence of Theileria parva infection in cattle immunized using two stocks which differ in their ability to induce a carrier state: analysis using a novel blood spot PCR assay

An improved Theileria parva DNA detection assay based on the polymerase chain reaction (PCR) using primers derived from the 104 kDa antigen (p104) gene was developed to detect parasite DNA in blood spots on filter paper. The specificity of the assay was validated using DNA from a wide range of cattle-derived and buffalo-derived stocks of T. parva. DNA of T. annulata, T. buffeli, T. lestoquardi, T. mutans and T. taurotragi was not amplified using the p104 primers. The detection threshold of the assay was approximately 1-2 parasites/microl of infected blood. PCR amplification using the p104 primers was applied to sequential samples from groups of cattle experimentally infected with either the T. parva Marikebuni stock that induces a long-term carrier state or the Muguga stock, which does not induce a carrier state. The study extended for up to 487 days post-infection and PCR data from defined time points were compared with parasitological microscopy and serological data, together with xenodiagnosis by experimental application of ticks. Microscopy first detected piroplasms between days 13 and 16 after infection whereas all cattle became PCR +ve between days 9 and 13. Animals infected with the Muguga stock of T. parva had parasite DNA in the peripheral blood, which could be detected by PCR, for between 33 and 129 days post-infection in different animals. By contrast parasite DNA in the blood of cattle infected with the Marikebuni stock could be detected consistently from day 9 up to 487 days, when the study terminated. The data suggest that the nature and persistence of the carrier state may differ markedly between different T. parva parasite stocks.

Quantification of Theileria parva in Rhipicephalus appendiculatus (Acari: Ixodidae) Confirms Differences in Infection Between Selected Tick Strains

ABSTRACT 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.

Differential transcription of two highly divergent gut-expressed Bm86 antigen gene homologues in the tick Rhipicephalus appendiculatus (Acari: Ixodida).

The transcriptional control of gene expression is not well documented in the Arthropoda. We describe transcriptional analysis of two exceptionally divergent homologues (Ra86) of the Bm86 gut antigen from Rhipicephalus appendiculatus. Bm86 forms the basis of a commercial vaccine for the control of Rhipicephalus (Boophilus) microplus. The R. appendiculatus Ra86 proteins contain 654 and 693 amino acids, with only 80% amino acid sequence identity. Reverse‐transcription PCR of gut cDNA showed transcription of only one genotype in individual female ticks. PCR amplification of 3′ untranslated sequences from genomic DNA indicated that both variants could be encoded within a single genome. When both variants were present, one of the two Ra86 genotypes was transcriptionally dominant.

Exposure of vaccinated and naive cattle to natural challenge from buffalo-derived Theileria parva

Highlights • The Muguga cocktail was tested in cattle in a buffalo-only location.• Infection with buffalo-derived T. parva caused Corridor disease in cattle.• At the conservancy, the cocktail did not protect cattle against Corridor disease.• Efficacious vaccines can support integrative livestock/wildlife management.

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.

Production and dose determination of the Infection and Treatment Method (ITM) Muguga cocktail vaccine used to control East Coast fever in cattle.

The Infection and Treatment Method (ITM) of vaccination against the apicomplexan parasite Theileria parva has been used since the early 1970s and is still the only commercially available vaccine to combat the fatal bovine disease, East Coast fever (ECF). The disease is tick-transmitted and results in annual economic losses of at least

Immunization of cattle with Ra86 impedes Rhipicephalus appendiculatus nymphal-to-adult molting

300 million per year. While this vaccine technology has been available for over 40 years, few attempts have been made to standardize the production process and characterize the vaccine. The latest batch was produced in early 2008 at the International Livestock Research Institute (ILRI). The vaccine production involves the use of cattle free from parasites routinely monitored throughout the production process, and a pathogen-free tick colony. This paper describes the protocol used in the recent production, and the process improvements, including improved quality control tools, that had not been employed in previous ITM productions. The paper also describes the processes involved in determining the appropriate field dose, which involved a three-step in vivo study with various dilutions of the vaccine stabilate. The vaccine was shown to be safe and viable after production, and a suitable field dose was identified as 1 ml of a 1:100 dilution.

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

Commercial vaccines based on the tick gut protein Bm86 have been successful in controlling the one-host tick Rhipicephalus (Boophilus) microplus and provide heterologous protection against certain other non-target ixodid tick species. This cross protection, however, does not extend to the three-host tick R. appendiculatus, the vector of the protozoan parasite Theileria parva. When transmitted to cattle, T. parva causes the often fatal disease East Coast fever. Here, we used insect cell-expressed recombinant versions of the R. appendiculatus homologs of Bm86, named Ra86, to vaccinate cattle. We measured multiple fitness characteristics for ticks that were fed on cattle Ra86-vaccinated or unvaccinated. The Ra86 vaccination of cattle significantly decreased the molting success of nymphal ticks to the adult stage. Modeling simulations based on our empirical data suggest that repeated vaccinations using Ra86 could reduce tick populations over successive generations. Vaccination with Ra86 could thus form a component of integrated control strategies for R. appendiculatus leading to a reduction in use of environmentally damaging acaricides.

Recombinant Rhipicephalus appendiculatus gut (Ra86) and salivary gland cement (Trp64) proteins as candidate antigens for inclusion in tick vaccines: protective effects of Ra86 on infestation with adult R. appendiculatus

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