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Infestations with cattle ticks, Rhipicephalus (Boophilus) microplus and Rhipicephalus annulatus, economically impact cattle production in tropical and subtropical regions of the world. Vaccines containing the recombinant R. microplus BM86 gut antigen were developed and commercialized to induce an immunological protection in cattle against tick infestations. These vaccines demonstrated that tick control by vaccination is cost-effective, reduces environmental contamination and prevents the selection of drug resistant ticks that result from repeated acaricide applications. The protection elicited by BM86-containing vaccines against tick infestations is mediated by a collaborative action between the complement system and IgG antibodies. The efficacy of the vaccination with BM86 and other tick antigens is always higher for R. annulatus than against R. microplus, suggesting that tick genetic and/or physiological factors may affect tick vaccine efficacy. These factors may be related to BM86 protein levels or tick physiological processes such as feeding and protein degradation that could result in more efficient antibody-antigen interactions and vaccine efficacy. To test this hypothesis, we compared the proteome in R. annulatus and R. microplus female ticks after feeding on BM86-vaccinated and control cattle. The results showed that cattle proteins were under represented in R. annulatus when compared to R. microplus, suggesting that R. annulatus ticks ingested less blood, a difference that increased when feeding on vaccinated cattle, probably reflecting the effect of antibody-BM86 interactions on this process. The results also showed that tick protein degradation machinery was under represented in R. annulatus when compared to R. microplus. BM86 mRNA and protein levels were similar in both tick species, suggesting that lesser protease activity in R. annulatus results in more efficient antibody-antigen interactions and higher vaccine efficacy. These results have important implications for tick vaccine research, indicating that not only genetic differences, but also physiological factors may influence tick vaccine efficacy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Bovine babesiosis, also known as cattle fever, is a tick-borne protozoal disease foreign to the United States. It was eradicated by eliminating the vector species, Rhipicephalus (Boophilus) annulatus and Rhipicephalus (Boophilus) microplus, through the efforts of the Cattle Fever Tick Eradication Program (CFTEP), with the exception of a permanent quarantine zone (PQZ) in south Texas along the border with Mexico. Keeping the U.S. free of cattle fever ticks in a sustainable manner is a critical national agricultural biosecurity issue. The efficacy of a Bm86-based anti-tick vaccine commercialized outside of the U.S. was evaluated against a strain of R. annulatus originated from an outbreak in Texas. Vaccination controlled 99.9 and 91.4% of the ticks 8 weeks and 5.5 months after the initial vaccination, respectively. Computer modeling of habitat suitability within the PQZ typically at risk of re-infestation with R. annulatus from Mexico predicted that at a level of control greater than 40%, eradication would be maintained indefinitely. Efficacy and computer modeling data indicate that the integration of vaccination using a Bm86-based anti-tick vaccine with standard eradication practices within the northwestern half of the PQZ could incentivize producers to maintain cattle on pasture thereby avoiding the need to vacate infested premises. Implementing this epidemiologically proactive strategy offers the opportunity to prevent R. annulatus outbreaks in the U.S., which would represent a significant shift in the way the CFTEP operates., (Copyright © 2012. Published by Elsevier Ltd.)

Red deer (Cervus elaphus) and white-tailed deer (Odocoileus virginianus) are hosts for different tick species and tick-borne pathogens and play a role in tick dispersal and maintenance in some regions. These factors stress the importance of controlling tick infestations in deer and several methods such as culling and acaricide treatment have been used. Tick vaccines are a cost-effective alternative for tick control that reduced cattle tick infestations and tick-borne pathogens prevalence while reducing the use of acaricides. Our hypothesis is that vaccination with vector protective antigens can be used for the control of tick infestations in deer. Herein, three experiments were conducted to characterize (1) the antibody response in red deer immunized with recombinant BM86, the antigen included in commercial tick vaccines, (2) the antibody response and control of cattle tick infestations in white-tailed deer immunized with recombinant BM86 or tick subolesin (SUB) and experimentally infested with Rhipicephalus (Boophilus) microplus, and (3) the antibody response and control of Hyalomma spp. and Rhipicephalus spp. field tick infestations in red deer immunized with mosquito akirin (AKR), the SUB ortholog and candidate protective antigen against different tick species and other ectoparasites. The results showed that deer produced an antibody response that correlated with the reduction in tick infestations and was similar to other hosts vaccinated previously with these antigens. The overall vaccine efficacy was similar between BM86 (E=76%) and SUB (E=83%) for the control of R. microplus infestations in white-tailed deer. The field trial in red deer showed a 25-33% (18-40% when only infested deer were considered) reduction in tick infestations, 14-20 weeks after the first immunization. These results demonstrated that vaccination with vector protective antigens could be used as an alternative method for the control of tick infestations in deer to reduce tick populations and dispersal in regions where deer are relevant hosts for these ectoparasites., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Vaccines containing the Rhipicephalus (Boophilus) microplus BM86 and BM95 antigens protect cattle against tick infestations. Tick subolesin (SUB), elongation factor 1a (EF1a) and ubiquitin (UBQ) are new candidate protective antigens for the control of cattle tick infestations. Previous studies showed that R. microplus BM95 immunogenic peptides fused to the Anaplasma marginale major surface protein (MSP) 1a N-terminal region (BM95-MSP1a) for presentation on the Escherichia coli membrane were protective against R. microplus infestations in rabbits. In this study, we extended these results by expressing SUB-MSP1a, EF1a-MSP1a and UBQ-MSP1a fusion proteins on the E. coli membrane using this system and demonstrating that bacterial membranes containing the chimeric proteins BM95-MSP1a and SUB-MSP1a were protective (>60% vaccine efficacy) against experimental R. microplus and Rhipicephalus annulatus infestations in cattle. This system provides a novel, simple and cost-effective approach for the production of tick protective antigens by surface display of antigenic protein chimera on the E. coli membrane and demonstrates the possibility of using recombinant bacterial membrane fractions in vaccine preparations to protect cattle against tick infestations., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Ixodes ricinus is one the most abundant tick species in Europe and these ticks transmit pathogens causing human and animal diseases. The cattle ticks, Rhipicephalus (Boophilus) spp., affect cattle production in tropical and subtropical regions of the world. Development of vaccines directed against tick proteins may reduce tick infestations and the transmission of tick-borne pathogens. However, a limiting step in tick vaccine development has been the identification of tick protective antigens. Herein, the tick iron metabolism pathway was targeted in an effort to identify new tick protective antigens. Recombinant I. ricinus (IrFER2) and Rhipicephalus microplus (RmFER2) ferritin 2 proteins were expressed in Escherichia coli and used to immunize rabbits and cattle, respectively. Vaccination with IrFER2 reduced I. ricinus tick numbers, weight and fertility in rabbits with an overall vaccine efficacy (E) of 98%. Control of cattle tick, R. microplus and Rhipicephalus annulatus infestations was obtained in vaccinated cattle with overall E of 64% and 72%, respectively. Notably, the efficacy of the RmFER2 vaccine was similar to that obtained with Bm86 against R. microplus. These collective results demonstrated the feasibility of using ferritin 2 to develop vaccines for the control of tick infestations., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The lone star tick, Amblyomma americanum, vectors pathogens of emerging diseases of humans and animals in the United States. Currently, measures are not available for effective control of A. americanum infestations. Development of vaccines directed against tick proteins may reduce tick infestations and the transmission of tick-borne pathogens. However, the limiting step in tick vaccine development has been the identification of tick protective antigens. Herein, we report the application of RNA interference (RNAi) for screening an A. americanum cDNA library for discovery of tick protective antigens that reduce tick survival and weights after feeding. Four cDNA clones, encoding for putative threonyl-tRNA synthetase (2C9), 60S ribosomal proteins L13a (2D10) and L13e (2B7), and interphase cytoplasm foci protein 45 (2G7), were selected for vaccine studies in cattle, along with subolesin, a tick protective protein identified previously. In vaccinated cattle, an overall efficacy (E)>30% was obtained when considering the vaccine effect on both nymphs and adults, but only 2D10, 2G7 and subolesin affected both tick stages. The highest efficacy of control for adult ticks (E>55%) was obtained in cattle vaccinated with recombinant 2G7 or subolesin. These collective results demonstrated the feasibility of developing vaccines for the control of lone star tick infestations. The use of RNAi for identification of tick protective antigens proved to be a rapid and cost-effective tool for discovery of candidate vaccine antigens, and this approach could likely be applied to other parasites of veterinary and medical importance., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

The gene that encodes the tick protective antigen, 4D8, was cloned from 10 species belonging to 6 genera, and the nucleotide and amino acid sequences were analyzed. 4D8 nucleotide and protein sequences were conserved among these tick species with identity/similarity between 65-98 and 60-98%, respectively. The function of 4D8 was characterized by RNA interference (RNAi) in five tick species. After the ticks were allowed to feed, degeneration of gut, salivary glands and reproductive tissues was observed, and tick survival, weight and oviposition were significantly reduced. 4D8 RNAi effected >90% reduction in oviposition in all tick species tested. Because of the critical role that 4D8 plays during tick feeding and oviposition, which ultimately results in the reduction of tick progeny, we proposed the generic name "subolesin" (Latin, suboles: offspring, progeny) for tick 4D8 proteins and subA for the subolesin-encoding gene.

Antigens protective against Ixodes scapularis infestations were identified by cDNA expression library immunization (ELI) and analysis of expressed sequenced tags (EST). Three cDNAs protective against larval tick infestations, 4F8, with homology to a nucleotidase, and 4D8 and 4E6 of unknown function, were characterized and obtained as recombinant proteins for immunization studies. Vaccination trials with recombinant proteins demonstrated an effect of these antigens against I. scapularis larvae in a mouse model. Herein, we evaluated the effect of recombinant antigens on I. scapularis adult infestations on immunized sheep. Vaccination with recombinant 4D8, 4F8, 4E6 and the combination of all three antigens reduced adult tick infestations by 58, 12, 20, and 16%, respectively, when compared to the control group but was statistically significant for 4D8 and 4F8 only. Oviposition was reduced by 22-49% in all groups immunized with recombinant tick antigens (P<0.05). The overall efficacy of vaccine formulations considering the effect on tick infestations and oviposition averaged 33-71%. These antigens, and especially 4D8, appear to be good candidates for continued development of a vaccine for control of tick infestations.

cDNA expression library immunization (ELI) and analysis of expressed sequenced tags (EST) in a mouse model of tick infestations was used to identified cDNA clones that affected I. scapularis. Three protective antigens against larval tick infestations, 4F8, with homology to a nucleotidase, and 4D8 and 4E6 of unknown function, were selected for further characterization. All three antigens were expressed in all I. scapularis stages and localized in adult tick tissues. 4D8 was shown to be conserved in six other tick species. Based on immunization trials with synthetic polypeptides against larvae and nymphs and on artificial feeding experiments of adults, these antigens, especially 4D8, appear to be good candidates for continued development of a vaccine for control of tick infestations and may be useful in a formulation to target multiple species of ticks.

Identification of antigens that induce an immune response against tick infestations is required for the development of vaccines against these economically important ectoparasites. In order to identify protective antigens, we constructed a cDNA expression library from a continuous Ixodes scapularis cell line (IDE8) that was initially derived from tick embryos. cDNA clones were subjected to several rounds of screening in which mice were immunized with individual pools and then challenge-exposed by allowing I. scapularis larvae to feed on the immunized and control mice. Immunity against tick infestation was determined by the reduction in the ability of the larvae to feed to repletion and molt to the nymphal stage. Individual clones in pools that induced immunity to larval infestations were partially sequenced and grouped according to their putative protein function by comparison with sequence databases. The screening identified several individual antigens that induced a protective immune response against I. scapularis infestations. Our studies demonstrated for the first time that cDNA expression library immunization (ELI) combined with sequence analysis is a powerful and efficient tool for identification of candidate antigens for use in vaccines against ticks.