The use of RNA interference (RNAi) to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick, Boophilus microplus, has not been reported. We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens: Bm86, Bm91 and subolesin. Gene-specific double-stranded (dsRNA) was injected into two tick stages, freshly molted unfed and engorged females, and specific gene silencing was confirmed by real time PCR. Gene silencing occurred in injected unfed females after they were allowed to feed. Injection of dsRNA into engorged females caused gene silencing in the subsequently oviposited eggs and larvae that hatched from these eggs, but not in adults that developed from these larvae. dsRNA injected into engorged females could be detected by quantitative real-time RT-PCR in eggs 14 days from the beginning of oviposition, demonstrating that unprocessed dsRNA was incorporated in the eggs. Eggs produced by engorged females injected with subolesin dsRNA were abnormal, suggesting that subolesin may play a role in embryonic development. The injection of dsRNA into engorged females to obtain gene-specific silencing in eggs and larvae is a novel method which can be used to study gene function in tick embryogenesis.
Ehrlichia ruminantium, an obligate intracellular bacterium transmitted by ticks of the genus Amblyomma, causes heartwater disease in ruminants. The gene coding for the major antigenic protein MAP1 is part of a multigene family consisting of a cluster containing 16 paralogs. In the search for differentially regulated genes between E. ruminantium grown in endothelial and tick cell lines that could be used in vaccine development and to determine if differences in the map1 gene cluster exist between different isolates of E. ruminantium, we analyzed the map1 gene cluster of the Senegal and Gardel isolates of E. ruminantium. Both isolates contained the same number of genes, and the same organization as found in the genome sequence of the Welgevonden isolate (H. Van Heerden, N. E. Collins, K. A. Brayton, C. Rademeyer, and B. A. Allsopp, Gene 330:159-168, 2004). However, comparison of two subpopulations of the Gardel isolate maintained in different laboratories demonstrated that recombination between map1-3 and map1-2 had occurred in one subpopulation with deletion of one entire gene. Reverse transcription-PCR on E. ruminantium derived mRNA from infected cells using gene-specific primers revealed that all 16 map1 paralogs were transcribed in endothelial cells. In one vector (Amblyomma variegatum) and several nonvector tick cell lines infected with E. ruminantium, transcripts were found for between 4 and 11 paralogs. In all these cases the transcript for the map1-1 gene was detected and was predominant. Our results indicate that the map1 gene cluster is relatively conserved but can be subject to recombination, and differences in the transcription of map1 multigenes in host and vector cell environments exist.Ehrlichia ruminantium (formerly Cowdria ruminantium [12]) is the causative agent of heartwater, a rickettsial disease transmitted by ticks of the genus Amblyomma which causes major economic losses in wild and domestic ruminants. The disease is endemic in sub-Saharan Africa and also is present on some Caribbean islands (33), where it poses a risk of spreading to the American mainland. Feeding ticks transmit E. ruminantium to vertebrate hosts in their saliva and/or by gut regurgitation (8,19). Phylogenetic studies have revealed a close relationship between E. ruminantium, Ehrlichia canis, and Ehrlichia chaffeensis (30,39).In infections with these ehrlichial agents, the serological response is mainly directed against outer-membrane proteins of approximately 30 kDa. The genes coding for these proteins have been designated the major antigenic protein 1 (map1) in E. ruminantium (32,40), the outer membrane protein p28 (omp-1) in E. chaffeensis, and the p30 outer membrane protein (p30) in E. canis (27,28,30,35,36,42,43). The OMP-1 and P30 protein families are each encoded by a multigene family consisting of 22 genes arranged in a cluster between a hypothetical transcriptional regulator (upstream) and the secA gene (downstream). The 5Ј end of the cluster contains paralogs with short intergenic spaces, whereas the paralogs at t...
The Bm86 antigen, as originally identified in Boophilus microplus, is the basis of commercial tick vaccines against this tick species. The potential for using this antigen or homologues of the antigen in vaccination against other tick species has been assessed. We have conducted vaccine trials in cattle using the B. microplus-derived recombinant Bm86 vaccine (TickGARD) using pairs of vaccinated calves and control calves. These were infested with B. microplus and Boophilus decoloratus larvae simultaneously. For both species, the numbers of engorged female adult ticks, their weight and egg-laying capacity were all reduced, leading to a reduction in reproductive capacity of 74% for B. microplus and 70% for B. decoloratus. Hyalomma anatolicum anatolicum ticks were fed both as immatures as well as adults on vaccinated calves and non-vaccinated controls. There was an overall 50% reduction in the total weight of nymphs engorging on vaccinated calves, and a suggestion of a subsequent effect on feeding adults. For Hyalomma dromedarii there was a 95% reduction in the number of nymphs engorging and a further 55% reduction in weight of those ticks surviving. Rhipicephalus appendiculatus and Amblyomma variegatum ticks were fed simultaneously both as immatures and subsequently as adults. There was no evidence for a significant vaccination effect. Finally, the amino acid sequence of a Bm86 homologue found in H. a. anatolicum unequivocally demonstrated the conservation of this molecule in this species. Our strategy for the development of multivalent anti-tick vaccines is discussed in relation to these findings.
In West Africa, losses due to heartwater disease are not known because the incidence/prevalence has not been well studied or documented. To develop a diagnostic tool for molecular epidemiology, three PCR-based diagnostic assays, a nested pCS20 PCR, a nested map1 PCR and a nested reverse line blot (RLB) hybridization assay, were evaluated to determine their ability to detect infection in vector ticks, by applying them simultaneously to A. variegatum field ticks to detect Ehrlichia ruminantium, the causative agent of heartwater. The nested pCS20 PCR assay which amplified the pCS20 gene fragment showed the highest detection performance with a detection rate of 16.6%; the nested map1 PCR, which amplified the gene encoding the major antigenic protein1 (map1 gene) showed a detection rate of 11% and the RLB, based on the 16S rDNA sequence of anaplasma and ehrlichial species, detected 6.2%. The RLB, in addition, demonstrated molecular evidence of Ehrlichia ovina, Anaplasma marginale and Anaplasma ovis infections in The Gambia. Subsequently, the pCS20 assay was applied to study the prevalence and distribution of E. ruminantium tick infection rates at different sites in five divisions of The Gambia. The rates of infection in the country ranged from 1.6% to 15.1% with higher prevalences detected at sites in the westerly divisions (Western, Lower River and North Bank; range 8.3-15.1%) than in the easterly divisions (Central River and Upper River; range 1.6-7.5%). This study demonstrated a gradient in the distribution of heartwater disease risk for susceptible livestock in The Gambia which factor must be considered in the overall design of future upgrading programmes.
Understanding genetic diversity of Ehrlichia ruminantium in host and vector populations is an important prerequisite to controlling heartwater by vaccination in traditional livestock systems in sub-Saharan Africa. We carried out a study in two phases: (i) evaluating the usefulness of the PCR-RFLP assay based on the map1 coding sequence of E. ruminantium as a discriminatory tool to characterise genetic diversity, (ii) applying the technique to field samples from Amblyomma variegatum ticks and small ruminants to characterise genotypic diversity of the organism in three main agroecological zones of The Gambia, Sudano-Guinean (SG), Western Sudano-Sahelian (WSS) and Eastern Sudano-Sahelian (ESS). Restriction fragment length polymorphisms were observed among different strains of E. ruminantium supporting the usefulness of the PCR-RFLP technique for studying genetic diversity of the organism. Restriction enzyme map1 profile analysis indicated the presence in The Gambia of multiple genotypes (at least 11) of E. ruminantium with sites in the WSS and SG zones showing comparatively high number of diverse genotypes. Profiles similar to the Kerr Seringe genotype (DQ333230) showed the highest distribution frequency, being present at sites in all three agroecological zones, thereby making the strain a suitable candidate for further characterisation in cross-protection studies. An additional three genotypes showed relatively high distribution frequency and were present in all three zones making them equally important for isolation and subsequent characterisation. The study demonstrated the occurrence of mixed infections with E. ruminantium genotypes in ruminants and ticks.
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