Low Genetic Diversity of the Only Clade of the Tick Rhipicephalus microplus in the Neotropics.

This study addresses the variability of the mitochondrial cytochrome oxidase subunit I (COI) and 16S rDNA (16S), and nuclear internal transcriber spacer ITS2 (ITS2) genes in a set of field-collected samples of the cattle tick, Rhipicephalus microplus (Canestrini, 1888), and in geo-referenced sequences obtained from GenBank. Since the tick is currently considered to be a complex of cryptic taxa in several regions of the world, the main aims of the study are (i) to provide evidence of the clades of the tick present in the Neotropics, (ii) to explore if there is an effect of climate traits on the divergence rates of the target genes, and (iii) to check for a relationship between geographical and genetic distance among populations (the closest, the most similar, meaning for slow spread). We included published sequences of Rhipicephalus annulatus (Nearctic, Afrotropical, and Mediterranean) and R. microplus (Afrotropical, Indomalayan) to fully characterize the Neotropical populations (total: 74 16S, 44 COI, and 49 ITS2 sequences included in the analysis). Only the clade A of R. microplus spread in the Nearctic-Neotropics. Both the K and Lambda's statistics, two measures of phylogenetic signal, support low divergence rates of the tested genes in populations of R. microplus in the Neotropics. These tests demonstrate that genetic diversity of the continental populations does not correlate either with the geographic distance among samples or with environmental variables. The low variability of these genes may be due to a combination of factors like (i) the recent introduction of the tick in the Neotropics, (ii) a large, effective, and fast exchange of populations, and (iii) a low effect of climate on the evolution rates of the target genes. These results have implications for the ecological studies and control of cattle tick infestations.

One Health Approach to Identify Research Needs on Rhipicephalus microplus Ticks in the Americas.

We aim to provide a harmonized view of the factors that affect the survival and promote the spread of R. microplus in the Neotropics, approaching its different facets of biology, ecology, distribution, and control. We review the interactions among environmental niche, landscape fragmentation, vegetal coverage (abiotic traits), and the biotic aspects of its ecology (abundance of domesticated or wild competent hosts), proposing emerging areas of research. We emphasize a holistic view integrating an economically and ecologically sustainable control of infestations and transmitted pathogens by R. microplus in the Neotropics. Examples of research link the trends of climate, the composition of the community of hosts, the landscape features, and a tailored management based on ecological grounds. Our view is that factors driving the spread of R. microplus are complex and deeply interrelated, something that has been seldom considered in control strategies. The effects of climate may affect the dynamics of wildlife or the landscape composition, promoting new patterns of seasonal activity of the tick, or its spread into currently free areas. In this paper we encourage a One Health approach highlighting the main aspects governing the components of the tick's life cycle and its interactions with livestock and wild animals.

Experimental vaccination in rabbits using the peptide RmS-17 antigen reduces the performance of a Mexican Rhipicephalus microplus tick strain.

The tick vector Rhipicephalus microplus is considered one of the main problems in cattle production in tropical and subtropical regions. Anti-tick vaccines may form an alternative tick control method to the use of acaricides, and tick salivary proteins, such as Serpins, may be valuable as target antigens for developing anti-tick vaccines. In this study, we synthesized a recombinant peptide derived from Serpin RmS-17 protein using an Escherichia coli expression system and characterized the efficacy of the peptide RmS-17 for the control of R. microplus females infesting rabbits. Twelve New Zealand white rabbits were assigned to three experimental groups and vaccinated with three subcutaneous doses of the peptide RmS-17, recombinant R. microplus Bm86 antigen, and adjuvant/saline alone. The tick challenge was conducted with 120 R. microplus adults (60 females and 60 males) per animal, with the ticks placed inside a cotton sleeve glued to the back of the rabbit. Serum antibody levels (IgG) were assessed by ELISA and confirmed by Western blot; also, the reproductive performance of R. microplus was determined. The results showed that experimental vaccination in rabbits using the peptide RmS-17 antigen had a vaccine efficacy of 79% based on reductions in adult tick number, oviposition, and egg fertility compared to control animals. The peptide RmS-17 vaccinated rabbits developed a strong humoral immune response expressed by high anti-pRmS-17 IgG levels, and the Western blot analysis confirmed that it is immunogenic. The efficacy for the Bm86 vaccine was 62%, which is within the range of efficacy reported previously for Bm86 vaccine. The negative correlation between antibody levels and reduction in tick number strongly suggests that the effect of the vaccine was the result of the antibody response in vaccinated rabbits. In conclusion, this is the first study to evaluate the efficacy of the peptide RmS-17 against R. microplus tick infestation and show it to be immunogenic and protective in a rabbit model.

Protective efficacy of the peptide Subolesin antigen against the cattle tick Rhipicephalus microplus under natural infestation.

The cattle tick Rhipicephalus microplus affect animal health, welfare, and cattle production in tropical and subtropical zones of the world. Anti-tick vaccines have been an effective alternative for cattle tick control instead of traditional chemical products. To date, Subolesin antigen has shown efficacy for the control of tick infestation in cattle, and previous studies showed that one peptide derived from this protein has demonstrated to elicit a strong and specific humoral immune response. Based on these findings, herein we characterized the efficacy of the peptide Subolesin for the control of cattle tick, R. microplus infestation under field conditions. Twenty-four female calves were assigned to four experimental groups and immunized with three subcutaneous doses of the peptide Subolesin, Bm86, both antigens (dual vaccine) and adjuvant/saline alone, respectively. Serum antibody levels (IgG) were assessed by ELISA and confirmed by Western blot; also, reproductive performance of naturally infested R. microplus was determined. The results showed that immunizations with the experimental antigens reduced tick infestations with vaccine's efficacy of 67 % (peptide Subolesin), 56 % (Bm86), and 49 % (dual vaccine) based on adult tick numbers, oviposition, and egg fertility between vaccinated and control animals. Peptide Subolesin-immunized calves developed a strong humoral immune response expressed by high anti-pSubolesin IgG levels, and the Western blot analysis confirmed that it is immunogenic. Cattle receiving Bm86 and dual vaccine showed less protection, although Bm86 was within the range reported previously. The negative correlation between antibody levels and reduction of naturally infested R. microplus strongly suggested that the effect of the vaccine was the result of the antibody response in immunized cattle. In conclusion, it was demonstrated that the peptide Subolesin induced a specific immune response in cattle under field conditions, resulting in reduced R. microplus populations in subsequent generations. Finally, integrated tick control must consider anti-tick vaccines as a cost-effective, sustainable, and successful tool for controlling cattle tick infestations.

Changes in Serum Biomarkers of Oxidative Stress in Cattle Vaccinated with Tick Recombinant Antigens: A Pilot Study.

Tick vaccination is an environmentally friendly alternative for tick control, pathogen infection, and transmission. Tick vaccine protection is sometimes incomplete, which may be due to problems in the stability, conformation, and activity of antibodies. This might be related to oxidative stress, but more studies are needed about the possible relationships between oxidative stress and immune function. The objective of this study was to evaluate and compare various serum biomarkers of antioxidant response and oxidative damage in cattle vaccinated with two recombinant antigens, the chimera of Subolesin- BM95 (homologue antigen of BM86)-MSP1a and BM86, and a control consisting in the adjuvant of the vaccines. Cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of the plasma (FRAP), trolox equivalent antioxidant capacity (TEAC), total thiol concentrations, and uric acid were evaluated in serum to determine the antioxidant response. To evaluate oxidative status, ferrous oxidation-xylenol orange (FOX), total oxidant status (TOS), advanced oxidation protein products (AOPP) and hydrogen peroxide (H2O2) concentrations in serum were determined. In addition, correlations between biomarkers of oxidative stress and antibody titers were evaluated. A significant decrease in all antioxidant biomarkers, with exception of thiol, and also a decrease in the oxidant markers TOS, AOPP and H2O2 was observed in cattle vaccinated with BM86, that also showed the highest antibody titers response whereas no significant differences in any of the biomarkers were detected in the Subolesin-Bm95-MSP1a and control groups. In addition, the dynamics of Cuprac and H2O2 with time showed significant differences between the groups. Although this is a pilot study and the results should be interpreted with caution and corroborated by studies involving a large number of animals, our results indicate that, in our experimental conditions, those vaccines able to induce a lower oxidative stress produce a higher concentration of antigen-specific antibodies. Overall, the results of the study provided information on the behavior of different biomarkers related to antioxidant defense, and the oxidative damage in cattle in response to vaccination.

Tick and Host Derived Compounds Detected in the Cement Complex Substance.

Ticks are obligate hematophagous arthropods and vectors of pathogens affecting human and animal health worldwide. Cement is a complex protein polymerization substance secreted by ticks with antimicrobial properties and a possible role in host attachment, sealing the feeding lesion, facilitating feeding and pathogen transmission, and protection from host immune and inflammatory responses. The biochemical properties of tick cement during feeding have not been fully characterized. In this study, we characterized the proteome of Rhipicephalus microplus salivary glands (sialome) and cement (cementome) together with their physicochemical properties at different adult female parasitic stages. The results showed the combination of tick and host derived proteins and other biomolecules such as α-Gal in cement composition, which varied during the feeding process. We propose that these compounds may synergize in cement formation, solidification and maintenance to facilitate attachment, feeding, interference with host immune response and detachment. These results advanced our knowledge of the complex tick cement composition and suggested that tick and host derived compounds modulate cement properties throughout tick feeding.

Species occurrence of ticks in South America, and interactions with biotic and abiotic traits.

The datasets of records of the distribution of ticks and their hosts are invaluable tools to understand the phylogenetic patterns of evolution of ticks and the abiotic traits to which they are associated. Such datasets require an exhaustive collection of bibliographical references. In most cases, it is necessary the confirmation of reliable identification of ticks, together with an update of the scientific names of the vertebrate hosts. These data are not easily available, because many records were published in the so-called "grey literature". Herein, we introduced the Dataset of Ticks in South America, a repository that collates data on 4,764 records of ticks (4,124 geo-referenced) with a special reference to an extra 2,370 records of ticks on cattle, together with a set of abiotic traits, curated from satellite-derived information over the complete target region. The dataset includes details of the phylogenetic relationships of the species of hosts, providing researchers with both biotic and abiotic traits that drive the distribution and evolution of ticks in South America.

Oral Vaccination With a Formulation Combining Rhipicephalus microplus Subolesin With Heat Inactivated Mycobacterium bovis Reduces Tick Infestations in Cattle.

Vaccines are an environmentally friendly alternative to acaracides for the control of tick infestations, to reduce the risk for tick-borne diseases affecting human and animal health worldwide, and to improve animal welfare and production. Subolesin (SUB, also known as 4D8) is the functional homolog of Akirin2 involved in the regulation of development and innate immune response, and a proven protective antigen for the control of ectoparasite infestations and pathogen infection. Oral vaccination combining protein antigens with immunostimulants has proven efficacy with increased host welfare and safety, but has not been used for the control of tick infestations. Here we describe the efficacy of oral vaccination with a formulation combining Rhipicephalus microplus SUB and heat inactivated Mycobacterium bovis (IV) on cattle tick infestations and fertility. The levels of IgG antibody titers against SUB and M. bovis P22, and the expression of selected immune response genes were determined and analyzed as possible correlates of protection. We demonstrated that oral immunization with the SUB+IV formulation resulted in 51% reduction in the number of female ticks and 30% reduction in fertility with an overall efficacy of 65% in the control of R. microplus infestations by considering the cumulative effect on reducing tick survival and fertility in cattle. The akr2, IL-1ß, and C3 mRNA levels together with antibody levels against SUB correlated with vaccine efficacy. The effect of the oral immunization with SUB+IV in cattle on tick survival and fertility is essential to reduce tick infestations, and extended previous results on the effect of R. microplus SUB for the control of cattle tick infestations. These results support the development of oral vaccines formulations for the control of tick infestations and the incidence of tick-borne diseases.

Towards an Effective, Rational and Sustainable Approach for the Control of Cattle Ticks in the Neotropics.

Ticks and transmitted pathogens constitute a major burden for cattle industry in the Neotropics. To address this limitation, the Spanish Ibero-American Program of Science and Technology in Development office (CYTED) supported from 2018 a network of scientists named "LaGar" (CYTED code 118RT0542) aimed at optimizing the control strategies of cattle ticks in the neotropical region. As part of network activities, a meeting and course were organized on 4-8 November 2019 in Querétaro, Mexico to address the objective of developing the infrastructure necessary for an effective, sustainable (i.e., combination of efficacious acaricides with anti-tick vaccines) and rational (i.e., considering tick ecology, seasonal dynamics and cattle-wildlife interactions) control of cattle tick infestations and transmitted pathogens. The course was focused on scientists, students, cattle holders and producers and pharmaceutical/industry representatives. In this way the course addressed the different views presented by participants with the conclusion of producing a research-driven combination of different interventions for the control of tick tick-borne diseases.

Bacterial membranes enhance the immunogenicity and protective capacity of the surface exposed tick Subolesin-Anaplasma marginale MSP1a chimeric antigen.

Ticks are vectors of diseases that affect humans and animals worldwide. Tick vaccines have been proposed as a cost-effective and environmentally sound alternative for tick control. Recently, the Rhipicephalus microplus Subolesin (SUB)-Anaplasma marginale MSP1a chimeric antigen was produced in Escherichia coli as membrane-bound and exposed protein and used to protect vaccinated cattle against tick infestations. In this research, lipidomics and proteomics characterization of the E. coli membrane-bound SUB-MSP1a antigen showed the presence of components with potential adjuvant effect. Furthermore, vaccination with membrane-free SUB-MSP1a and bacterial membranes containing SUB-MSP1a showed that bacterial membranes enhance the immunogenicity of the SUB-MSP1a antigen in animal models. R. microplus female ticks were capillary-fed with sera from pigs orally immunized with membrane-free SUB, membrane bound SUB-MSP1a and saline control. Ticks ingested antibodies added to the blood meal and the effect of these antibodies on reduction of tick weight was shown for membrane bound SUB-MSP1a but not SUB when compared to control. Using the simple and cost-effective process developed for the purification of membrane-bound SUB-MSP1a, endotoxin levels were within limits accepted for recombinant vaccines. These results provide further support for the development of tick vaccines using E. coli membranes exposing chimeric antigens such as SUB-MSP1a.

Prospects for vaccination against the ticks of pets and the potential impact on pathogen transmission.

Diseases transmitted by arthropod vectors such as ticks greatly impact human and animal health. In particular, many diseases of dogs and cats are potentially transmissible to people by arthropod vectors and therefore their control is important for the eradication of vector-borne diseases (VBD). Vaccination is an environmentally friendly alternative for vector control that allows control of several VBD by targeting their common vector. Recent results have shown that it is possible to use vector protective antigens for the control of arthropod vector infestations and pathogen infection. However, as reviewed in this paper, very little progress has been made for the control of ectoparasite infestations and VBD in pets using vaccination with vector protective antigens. The growing interaction between pets and people underlines the importance of developing new interventions for the monitoring and control of VBD.

Artificial feeding of Rhipicephalus microplus female ticks with anti calreticulin serum do not influence tick and Babesia bigemina acquisition.

Ticks are obligate haematophagous ectoparasites considered the principal vectors of disease among animals. Rhipicephalus microplus and R. annulatus ticks are the most important vectors for Babesia bigemina and B. bovis, two of the most important intraerythrocytic protozoan parasites species in cattle, responsible for babesiosis which together with anaplasmosis account for substantial economic losses in the livestock industry worldwide. Anti-tick vaccines are a proved alternative to traditional tick and tick borne diseases control methods but are still limited primarily due to the lack of effective antigens. Subsequently to the identification of antigens the validation is a laborious work often expensive. Tick artificial feeding, is a low cost alternative to test antigens allowing achieving critical data. Herein, R. microplus females were successfully artificially fed using capillary tubes. Calreticulin (CRT) protein, which in a previous study has been identified as being involved in B. bigemina infection in R. annulatus ticks, was expressed as recombinant protein (rCRT) in an E. coli expression system and antibodies raised against rCRT. Anti-rCRT serum was supplemented to a blood meal, offered to partially engorged R. microplus females and their effect in feeding process as well as infection by B. bigemina was analyzed. No significant reductions in tick and egg weight were observed when ticks fed with anti-rCRT serum. Furthermore, B. bigemina infection levels did not show a statistically significant decrease when ticks fed with anti-rCRT antibodies. Results suggest that CRT is not a suitable candidate for cattle vaccination trials.

Tick capillary feeding for the study of proteins involved in tick-pathogen interactions as potential antigens for the control of tick infestation and pathogen infection.

BACKGROUND: Ticks represent a significant health risk to animals and humans due to the variety of pathogens they can transmit during feeding. The traditional use of chemicals to control ticks has serious drawbacks, including the selection of acaricide-resistant ticks and environmental contamination with chemical residues. Vaccination with the tick midgut antigen BM86 was shown to be a good alternative for cattle tick control. However, results vary considerably between tick species and geographic location. Therefore, new antigens are required for the development of vaccines controlling both tick infestations and pathogen infection/transmission. Tick proteins involved in tick-pathogen interactions may provide good candidate protective antigens for these vaccines, but appropriate screening procedures are needed to select the best candidates. METHODS: In this study, we selected proteins involved in tick-Anaplasma (Subolesin and SILK) and tick-Babesia (TROSPA) interactions and used in vitro capillary feeding to characterize their potential as antigens for the control of cattle tick infestations and infection with Anaplasma marginale and Babesia bigemina. Purified rabbit polyclonal antibodies were generated against recombinant SUB, SILK and TROSPA and added to uninfected or infected bovine blood to capillary-feed female Rhipicephalus (Boophilus) microplus ticks. Tick weight, oviposition and pathogen DNA levels were determined in treated and control ticks. RESULTS: The specificity of purified rabbit polyclonal antibodies against tick recombinant proteins was confirmed by Western blot and against native proteins in tick cell lines and tick tissues using immunofluorescence. Capillary-fed ticks ingested antibodies added to the blood meal and the effect of these antibodies on tick weight and oviposition was shown. However, no effect was observed on pathogen DNA levels. CONCLUSIONS: These results highlighted the advantages and some of the disadvantages of in vitro tick capillary feeding for the characterization of candidate tick protective antigens. While an effect on tick weight and oviposition was observed, the effect on pathogen levels was not evident probably due to high tick-to-tick variations among other factors. Nevertheless, these results together with previous results of RNA interference functional studies suggest that these proteins are good candidate vaccine antigens for the control of R. microplus infestations and infection with A. marginale and B. bigemina.

Vaccination with proteins involved in tick-pathogen interactions reduces vector infestations and pathogen infection.

Tick-borne pathogens cause diseases that greatly impact animal health and production worldwide. The ultimate goal of tick vaccines is to protect against tick-borne diseases through the control of vector infestations and reducing pathogen infection and transmission. Tick genetic traits are involved in vector-pathogen interactions and some of these molecules such as Subolesin (SUB) have been shown to protect against vector infestations and pathogen infection. Based on these premises, herein we characterized the efficacy of cattle vaccination with tick proteins involved in vector-pathogen interactions, TROSPA, SILK, and Q38 for the control of cattle tick, Rhipicephalus (Boophilus) microplus infestations and infection with Anaplasma marginale and Babesia bigemina. SUB and adjuvant/saline placebo were used as positive and negative controls, respectively. The results showed that vaccination with Q38, SILK and SUB reduced tick infestations and oviposition with vaccine efficacies of 75% (Q38), 62% (SILK) and 60% (SUB) with respect to ticks fed on placebo control cattle. Vaccination with TROSPA did not have a significant effect on any of the tick parameters analyzed. The results also showed that vaccination with Q38, TROSPA and SUB reduced B. bigemina DNA levels in ticks while vaccination with SILK and SUB resulted in lower A. marginale DNA levels when compared to ticks fed on placebo control cattle. The positive correlation between antigen-specific antibody titers and reduction of tick infestations and pathogen infection strongly suggested that the effect of the vaccine was the result of the antibody response in vaccinated cattle. Vaccination and co-infection with A. marginale and B. bigemina also affected the expression of genes encoding for vaccine antigens in ticks fed on cattle. These results showed that vaccines using tick proteins involved in vector-pathogen interactions could be used for the dual control of tick infestations and pathogen infection.

Tick vaccines and the control of tick-borne pathogens.

Ticks are obligate hematophagous ectoparasites that transmit a wide variety of pathogens to humans and animals. The incidence of tick-borne diseases has increased worldwide in both humans and domestic animals over the past years resulting in greater interest in the study of tick-host-pathogen interactions. Advances in vector and pathogen genomics and proteomics have moved forward our knowledge of the vector-pathogen interactions that take place during the colonization and transmission of arthropod-borne microbes. Tick-borne pathogens adapt from the vector to the mammalian host by differential gene expression thus modulating host processes. In recent years, studies have shown that targeting tick proteins by vaccination can not only reduce tick feeding and reproduction, but also the infection and transmission of pathogens from the tick to the vertebrate host. In this article, we review the tick-protective antigens that have been identified for the formulation of tick vaccines and the effect of these vaccines on the control of tick-borne pathogens.

Vaccinomics, the new road to tick vaccines.

Ticks are a threat to human and animal health worldwide. Ticks are considered to be second worldwide to mosquitoes as vectors of human diseases, the most important vectors of diseases that affect cattle industry worldwide and important vectors of diseases affecting pets. Tick vaccines are a cost-effective and environmentally friendly alternative to protect against tick-borne diseases through the control of vector infestations and reducing pathogen infection and transmission. These premises stress the need for developing improved tick vaccines in a more efficient way. In this context, development of improved vaccines for tick-borne diseases will be greatly enhanced by vaccinomics approaches starting from the study of tick­host­pathogen molecular interactions and ending in the characterization and validation of vaccine formulations. The discovery of new candidate vaccine antigens for the control of tick infestations and pathogen infection and transmission requires the development of effective screening platforms and algorithms that allow the analysis and validation of data produced by systems biology approaches to tick research. Tick vaccines that affect both tick infestations and pathogen transmission could be used to vaccinate human and animal populations at risk and reservoir species to reduce host exposure to ticks while reducing the number of infected ticks and their vectorial capacity for pathogens that affect human and animal health worldwide.

Targeting the tick protective antigen subolesin reduces vector infestations and pathogen infection by Anaplasma marginale and Babesia bigemina.

The ultimate goal of vector vaccines is the control of vector infestations while reducing pathogen infection and transmission to protect against the many diseases caused by vector-borne pathogens. Previously (Vaccine 2011;29:2248-2254), we demonstrated that subolesin vaccination and release of tick larvae after subolesin knockdown by RNA interference (RNAi) were effective for the control of cattle tick, Rhipicephalus (Boophilus) microplus infestations in cattle. In this study, we used the fact that these animals were naturally infected with Anaplasma marginale and Babesia bigemina to evaluate the effect of subolesin vaccination and gene knockdown on tick infection by these cattle tick-transmitted pathogens. Ticks fed on vaccinated cattle had lower subolesin mRNA levels when compared to controls, resembling RNAi results. A. marginale and B. bigemina infection was determined by PCR and decreased by 98% and 99%, respectively in ticks fed on vaccinated cattle and by 97% and 99%, respectively after subolesin knockdown. These results demonstrated that targeting subolesin expression by vaccination or RNAi results in lower subolesin mRNA and pathogen infection levels, probably due to the effect of subolesin downregulation on tick feeding, gene expression and gut and salivary glands tissue development and function. These results suggested that subolesin vaccines could be used for the dual control of tick infestations and pathogen infection, a result that could be relevant for other vectors and vector-borne pathogens.

Control of Rhipicephalus (Boophilus) microplus infestations by the combination of subolesin vaccination and tick autocidal control after subolesin gene knockdown in ticks fed on cattle.

Tick subolesin was shown in immunization trials using the recombinant protein to protect hosts against tick infestations. In this study, we demonstrated that subolesin vaccination and release of ticks after subolesin knockdown by RNA interference (RNAi) could be used for the control of Rhipicephalus (Boophilus) microplus tick infestations in cattle and suggested that the combination of these methods could increase the efficacy of cattle tick control under some circumstances. The greatest tick control was obtained when both release of ticks after subolesin knockdown and vaccination were used concurrently. However, modeling results suggested that vaccine efficacy could be increased if at least 80% of the ticks infesting cattle correspond to subolesin-knockdown ticks. The results of this proof-of-concept trial demonstrated the efficacy of the sterile acarine technique (SAT) through production of subolesin-knockdown larvae by dsRNA injection into replete females for the control of R. microplus tick infestations, alone or in combination with subolesin vaccination.