Efficacy of Rhipicephalus (Boophilus) microplus Bm86 against Hyalomma dromedarii and Amblyomma cajennense tick infestations in camels and cattle.

The recombinant Bm86-based tick vaccines have shown their efficacy for the control of cattle ticks, Rhipicephalus (Boophilus) microplus and R. annulatus infestations. However, cattle ticks often co-exist with multi-host ticks such as Hyalomma and Amblyomma species, thus requiring the control of multiple tick infestations for cattle and other hosts. Vaccination trials using a R. microplus recombinant Bm86-based vaccine were conducted in cattle and camels against Hyalomma dromedarii and in cattle against Amblyomma cajennense immature and adult ticks. The results showed an 89% reduction in the number of H. dromedarii nymphs engorging on vaccinated cattle, and a further 32% reduction in the weight of the surviving adult ticks. In vaccinated camels, a reduction of 27% and 31% of tick engorgement and egg mass weight, respectively was shown, while egg hatching was reduced by 39%. However, cattle vaccination with Bm86 did not have an effect on A. cajennense tick infestations. These results showed that Bm86 vaccines are effective against R. microplus and other tick species but improved vaccines containing new antigens are required to control multiple tick infestations.

Bm86 homologues and novel ATAQ proteins with multiple epidermal growth factor (EGF)-like domains from hard and soft ticks.

Tick control on livestock relies principally on the use of acaricides but the development of acaricide resistance and concerns for environmental pollution underscore the need for alternative control methods, for instance through the use of anti-tick vaccines. Two commercial vaccines based on the recombinant Bm86 protein from Rhipicephalus (Boophilus) microplus ticks were developed. Partial protection of the Bm86 vaccine against other Rhipicephalus (Boophilus) and Hyalomma tick species suggests that the efficacy of a Bm86-based vaccine may be enhanced when based on the orthologous recombinant Bm86 antigen. We therefore identified and analysed the Bm86 homologues from species representing the main argasid and ixodid tick genera, including two from the prostriate Ixodes ricinus tick species. A novel protein from metastriate ticks with multiple epidermal growth factor (EGF)-like domains which is structurally related to Bm86 was identified by using a 3' rapid amplification of cDNA ends (3'-RACE) method with a degenerate primer based on a highly conserved region of Bm86 and its orthologues. This second protein was named ATAQ after a part of its signature peptide. Quantitative reverse transcriptase-PCR showed that ATAQ proteins are expressed in both midguts and Malpighian tubules, in contrast to Bm86 orthologues which are expressed exclusively in tick midguts. Furthermore, expression of this protein over the life stages of R. microplus and Rhipicephalus appendiculatus was more continuous compared with Bm86. Although a highly effective vaccine antigen, gene silencing of Bm86 by RNA interference (RNAi) produced only a weak phenotype. Similarly the RNAi phenotype of Rhipicephalus evertsi evertsi females in which the expression of Ree86, ReeATAQ or a combination of both genes was silenced by RNAi did not differ from a mock-injected control group. The vaccine potential of ATAQ proteins against tick infestations is yet to be evaluated.

Genetic diversity of Ehrlichia ruminantium in Amblyomma variegatum ticks and small ruminants in The Gambia determined by restriction fragment profile analysis.

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.

Immunisation of sheep against heartwater in The Gambia using inactivated and attenuated Ehrlichia ruminantium vaccines.

Heartwater (cowdriosis) is a disease of ruminants caused by a rickettsial pathogen Ehrlichia ruminantium and transmitted by ticks of the genus Amblyomma. The purpose of this work was to evaluate the protective efficacies of inactivated and attenuated vaccines to protect sheep against heartwater in The Gambia. An inactivated vaccine, prepared from E. ruminantium (Gardel stock), and a live attenuated vaccine from E. ruminantium (Senegal stock), were evaluated in two independent on-station trials. A local stock of E. ruminantium (Kerr Seringe) was used as challenge material. Inactivated and live attenuated vaccines provided 43% and 100% protection, respectively, against virulent needle challenge. In a subsequent field trial, the attenuated vaccine protected 75% of sheep against virulent tick challenge, which was fatal for all control sheep. Quantification by real-time PCR showed that an immunising dose of approximately 23,000 attenuated E. ruminantium organisms was sufficient. Moreover, restriction fragment length polymorphism (RFLP) analysis indicated that the local Kerr Seringe genotype caused mortality amongst control sheep, whereas fatalities in the vaccinated group could be attributed to a different genotype.

Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference.

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.

Transcription analysis of the major antigenic protein 1 multigene family of three in vitro-cultured Ehrlichia ruminantium isolates.

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.

Autochthonous canine babesiosis in The Netherlands.

Outbreaks of autochthonous babesiosis, caused by Babesia canis, occurred in The Netherlands in the spring and autumn of 2004 affecting 23 dogs. Nineteen animals recovered after treatment, whereas four dogs died. Adult Dermacentor reticulatus ticks collected from these dogs indicate that canine babesiosis could become endemic in The Netherlands.

Simultaneous detection of Anaplasma and Ehrlichia species in ruminants and detection of Ehrlichia ruminantium in Amblyomma variegatum ticks by reverse line blot hybridization.

The detection of Anaplasma and Ehrlichia species is usually based on species-specific PCR assays, since no assay is yet available which can detect and identify these species simultaneously. To this end, we developed a reverse line blot (RLB) assay for simultaneous detection and identification of Anaplasma and Ehrlichia species in domestic ruminants and ticks. In a PCR the hypervariable V1 region of the 16S ribosomal RNA (rRNA) gene was amplified with a set of primers unique for members of the genera Anaplasma and Ehrlichia [Int. J. Syst. Evol. Microbiol. 51 (2001) 2145]. Amplified PCR products from blood of domestic ruminants or Amblyomma variegatum tick samples were hybridized onto a membrane to which eight species-specific oligonucleotide probes and one Ehrlichia and Anaplasma catch-all oligonucleotide probe were covalently linked. No DNA was amplified from uninfected blood, nor from other hemoparasites such as Theileria annulata, or Babesia bigemina. The species-specific probes did not cross-react with DNA amplified from other species. E. ruminantium, A. ovis and another Ehrlichia were identified by RLB in blood samples collected from small ruminants in Mozambique. Finally, A. variegatum ticks were tested after feeding on E. ruminantium infected sheep. E. ruminantium could be detected in adult ticks even if feeding of nymphs was carried out 3.5 years post-infection. In conclusion, the developed species-specific oligonucleotide probes used in an RLB assay can simultaneously detect and identify several Ehrlichia and Anaplasma species. However, as no quantitative data for the detection limit are available yet, only positive results are interpretable at this stage.