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Abstract Background There have been ongoing efforts to identify anti-tick vaccine targets to protect cattle from infestation with cattle fever ticks Rhipicephalus (Boophilus) microplus. Two commercial vaccines based on the tick gut protein Bm86 have had variable effectiveness, which has led to poor acceptance, and numerous studies have attempted to identify vaccine antigens that will provide more consistently effective protection. Transcriptomic analysis of R. microplus led to identification of three aquaporin genes annotated to code for transmembrane proteins involved in the transport of water across cell membranes. Previous work showed that vaccination with full-length recombinant aquaporin 1 (RmAQP1) reduced tick burdens on cattle. Targeted silencing of aquaporin 2 (RmAQP2) expression suggested it might also be a good anti-tick vaccination target. Methods Three synthetic peptides from the predicted extracellular domains of RmAQP2 were used to vaccinate cattle. Peptides were conjugated to keyhole limpet hemocyanin (KLH) as an antigenic carrier molecule. We monitored the antibody response with ELISA and challenged vaccinated cattle with R. microplus larvae. Results There was a 25% reduction overall in the numbers of ticks feeding to repletion on the vaccinated cattle. Immune sera from vaccinated cattle recognized native tick proteins on a western blot and reacted to the three individual synthetic peptides in an ELISA. The vaccinated calf with the highest total IgG titer was not the most effective at controlling ticks; ratios of IgG isotypes 1 and 2 differed greatly among the three vaccinated cattle; the calf with the highest IgG1/IgG2 ratio had the fewest ticks. Ticks on vaccinated cattle had significantly greater replete weights compared to ticks on controls, mirroring results seen with RNA silencing of RmAQP2. However, protein data could not confirm that vaccination had any impact on the ability of the tick to concentrate its blood meal by removing water. Conclusions A reduced number of ticks feed successfully on cattle vaccinated to produce antibodies against the extracellular domains of RmAQP2. However, our predicted mechanism, that antibody binding blocks the ability of RmAQP2 to move water out of the blood meal, could not be confirmed. Further study will be required to define the mechanism of action and to determine whether these vaccine targets will be useful components of an anti-tick vaccine cocktail. Graphical Abstract

Abstract Background Ticks are a problem for cattle production mainly in tropical and subtropical regions, because they generate great economic losses. Acaricides and vaccines have been used to try to keep tick populations under control. This has been proven difficult given the resistance to acaricides and vaccines observed in ticks. Resistance to protein rBm86-based vaccines has been associated with the genetic diversity of Bm86 among the ectoparasite’s populations. So far, neither genetic diversity, nor spatial distribution of circulating Bm86 haplotypes, have been studied within the Mexican territory. Here, we explored the genetic diversity of 125 Bm86 cDNA gene sequences from R. microplus from 10 endemic areas of Mexico by analyzing haplotype distribution patterns to help in understanding the population genetic structure of Mexican ticks. Results Our results showed an average nucleotide identity among the Mexican isolates of 98.3%, ranging from 91.1 to 100%. Divergence between the Mexican and Yeerongpilly (the Bm86 reference vaccine antigen) sequences ranged from 3.1 to 7.4%. Based on the geographic distribution of Bm86 haplotypes in Mexico, our results suggest gene flow occurrence within different regions of the Mexican territory, and even the USA. Conclusions The polymorphism of Bm86 found in the populations included in this study, could account for the poor efficacy of the current Bm86 antigen based commercial vaccine in many regions of Mexico. Our data may contribute towards designing new, highly-specific, Bm86 antigen vaccine candidates against R. microplus circulating in Mexico.

Abstract Background Rhipicephalus microplus is a hard tick species that has a high impact on cattle health and production in tropical and subtropical regions. Recently, ribosomal DNA and morphological analysis resulted in the reinstatement of R. australis as a separate species from R. microplus. Both feed on cattle and can transmit bovine pathogens such as Anaplasma and Babesia species. The current treatment with acaricides is becoming increasingly less effective due to the emergence of resistant tick strains. A promising alternative can be found in the form of anti-tick vaccines. The available commercial vaccines can be used to control tick infestation, but the lack of a knockdown effect (> 90% reduction in tick numbers as seen with effective acaricides) hampers its widespread use, hence higher efficacious vaccines are needed. Instead of searching for new protective antigens, we investigated the efficacy of vaccines that contain more than one (partially) protective antigen. For screening vaccine formulations, a previously developed in vitro feeding assay was used in which R. australis larvae are fed sera that were raised against the candidate vaccine antigens. In the present study, the efficacy of the Bm86 midgut antigen and the cytosolic Subolesin (SUB) antigen were evaluated in vitro. Results Antiserum against recombinant Bm86 (rBm86) partially inhibited larval engorgement, whereas antiserum against recombinant SUB (rSUB) did not have any effect on feeding of larvae. Importantly, when larvae were fed a combination of antiserum against rBm86 and rSUB, a synergistic effect on significantly reducing larval infestations was found. Immunohistochemical analysis revealed that the rBm86 antiserum reacted with gut epithelium of R. australis larvae, whereas the antiserum against rSUB stained salivary glands and rectal sac epithelium. Conclusions Combining anti-Bm86 and anti-subolesin antibodies synergistically reduced R. australis larval feeding in vitro. Rhipicephalus australis is a one host tick, meaning that the larvae develop to nymphs and subsequently adults on the same host. Hence, this protective effect could be even more pronounced when larvae are used for infestation of vaccinated cattle, as the antibodies could then affect all three developmental stages. This will be tested in future in vivo experiments.