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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., This work was supported by the Ministerio de Ciencia e Innovación, Spain (project BFU2008-01244/BMC) and the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Spain (project FAU2008-00014-00-00). D. Carreón was partially supported by a grant-in-aid from Conacyt, Mexico and the EU grant TB-STEP 212414. M. Villar was funded by the JAE-DOC program (CSIC-FSE), Spain. J.A. Moreno-Cid is a recipient of a JCCM fellowship, Spain.

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., This research was supported by FOMIX–TAMPS-2008-C17-107392, the Spanish Ministerio de Ciencia e Innovación (MICINN) project BFU2008-01244/BMC, the Consejería de Educación y Ciencia, JCCM, Spain (project PEII09-0118-8907), the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Spain (project FAU2008-00014-00-00), and Probiovet S.L., Spain. M. Villar and M. Canales were funded by JAE-DOC program (CSIC-FSE) and Junta de Comunidades de Castilla-La Mancha (Program FSE 2007-2013), Spain, respectively. J.A. Moreno-Cid is a recipient of a JCCM fellowship.

Cattle ticks, Rhipicephalus (Boophilus) spp., infestations are a major veterinary problem that impacts cattle production worldwide. Recently, vaccination has emerged as an alternative to control tick infestations that has shown the advantage of being cost-effective, avoids environmental contamination and prevents selection of drug-resistant ticks resulting from repeated acaricides applications. However, the success of vaccination is highly dependent on the availability of effective vaccines at affordable prices. Recombinant DNA technology has provided the mean for producing antigens in large quantities making possible the development and commercialization of anti-tick vaccines. In the early 1990s, a technology was developed to produce a commercial anti-tick vaccine based on the recombinant BM86 (rBM86) antigen isolated from R. (Boophilus) microplus and intracellularly expressed in Pichia pastoris. However, the technological process for its production was expensive. Herein we conducted an economical analysis of this technology in comparison with other reported processes. The results allowed the identification of critical steps in the technology and addressed research for bioprocess optimization. As a result, two alternative bioprocesses were developed to produce anti-tick vaccines based on the rBM86 protein secreted in P. pastoris and by displaying antigenic peptides on the Escherichia coli membrane (BM95-MSP1a antigen). Vaccine production costs were reduced from US$0.92/dose for the intracellular rBM86 to US$0.72/dose and US$0.56/dose for the secreted rBM86 and BM95-MSP1a, respectively., This research was supported by the Wellcome Trust under the Animal Health in the Developing World initiative through project 0757990 entitled “Adapting recombinant anti-tick vaccines to livestock in Africa”, the Consejería de Educación y Ciencia, JCCM, Spain (project PEII09-0118-8907), and the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Spain (project FAU2008-00014-00-00). J.A. Moreno-Cid is a recipient of a JCCM fellowship.

Laboratory bench-scale and pilot plant scale-up processes were established and characterized for Pichia pastoris disruption in a bead mill after fermentation in methanol. Combinations of retention times per pass and number of passes were evaluated. Heat generation during wet milling was evaluated and the thermal balance of the operation yielded a clear correlation between generated heat and retention time. Under these conditions, approximately 66% and 34% of the heat generated during the process was removed by the refrigerant and the product, respectively. Rheological properties of the process fluids were determined and a change from Newtonian to a non-Newtonian behaviour was observed as disruption progressed. The relation between changes in rheology and product temperature rises suggested that the most effective method for P. pastoris cell disruption is by a single pass of 6 min through the mill., This work was funded in part by The Welcome Trust project 075799.

Graphical abstract, 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.

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., This research was supported by the Wellcome Trust under the ‘Animal Health in the Developing World’ initiative through project 0757990 entitled ‘Adapting recombinant anti-tick vaccines to livestock in Africa’.

This paper reports the sequence analysis of Bm86 Campo Grande strain comparing it with Bm86 and Bm95 antigens from the preparations TickGardPLUS and GavacTM, respectively. The PCR product was cloned into pMOSBlue and sequenced. The secondary structure prediction tool PSIPRED was used to calculate alpha helices and beta strand contents of the predicted polypeptide. The hydrophobicity profile was calculated using the algorithms from the Hopp and Woods method, in addition to identification of potential MHC class-I binding regions in the antigens. Pair-wise alignment revealed that the similarity between Bm86 Campo Grande strain and Bm86 is 0.2% higher than that between Bm86 Campo Grande strain and Bm95 antigens. The identities were 96.5% and 96.3% respectively. Major suggestive differences in hydrophobicity were predicted among the sequences in two specific regions. O objetivo deste estudo foi analisar a seqüência de Bm86 cepa Campo Grande comparando-a com os antígenos Bm86 e Bm95 das preparações TickGardPLUS e GavacTM, respectivamente. O produto de PCR foi clonado em PMOSBlue e seqüenciado. Para calcular os conteúdos de alfa-hélice e fita beta do polipeptídio previsto, foi utilizada a ferramenta de prognóstico de estrutura secundária PSIPRED. O perfil de hidrofobicidade foi calculado usando os algoritmos de Hopp e Woods, além da identificação das possíveis regiões de ligação com MHC classe I nos antígenos. O alinhamento "pair-wise" revelou que a similaridade entre Bm86 cepa Campo Grande e Bm86 é 0,2% maior que aquela entre Bm86 cepa Campo Grande e Bm95. As identidades foram de 96,5% e 96,3%, respectivamente. Com relação à hidrofobicidade, os resultados sugerem que a maior diferença Made available in DSpace on 2018-05-24T00:41:56Z (GMT). No. of bitstreams: 1 SP19597ID30708.pdf: 482558 bytes, checksum: 38f22872ff8b2297e76a6026f9f190ad (MD5) Previous issue date: 2009-01-16

doi não encontrodo. Rhiphicephalus (Boophilus) microplus é um dos mais importantes artrópodes em medicina veterinária, em consequências das perdas econômicas e problemas de saúde causados na produção de gado. A vacinação representa o melhor método avaliado com custo efetivo para prevenir perdas econômicas e aumentar a duração e qualidade de vida dos animais de produção. Um peptídeo sintético, SBm 7462, derivado da proteína intestinal Bm86 doR. (B.) microplus, tem obtido excelentes resultados no controle de carrapatos. A construção e síntese de uma sequência nucleotidíca baseada neste peptídeo podem ser úteis para o design de uma vacina DNA que tem muitas vantagens sob uma vacina sintética. Um gene, denominado seq1 foi construído repetindo-se três vezes a sequência nucleotidídica do SBm 7462. Ele foi clonado dentro do vetor pCIneo de expressão em mamíferos e injetado em camundongos BALB/c. Quando camundongos foram inoculados com o cassete de expressão, eles não responderam ao ELISA. Eles elevaram os títulos de anticorpos apenas quando inoculados com o peptídeo sintético SBm7462®. E os melhores títulos de imunoglobulinas foram vistos quando o SBm7462® foi administrado subcutaneamente. Rhiphicephalus (Boophilus) microplus is one of the most important arthropods in veterinary medicine due economic losses and health problems caused in cattle production. The vaccination represents optimum method evaluated with effective cost to prevent economic losses and to increase the duration and quality of life of the production animals. A synthetic peptide, SBm 7462, derived from Bm86, has been shown great results in control of ticks. The construction and synthesis of one nucleotide sequence based on this peptide might be useful for design a DNA vaccine that has many advances than peptide vaccine. A gene, called seq1, was constructed with a three repetition of nucleotide sequence of SBm 7462. It was cloned into a pCIneo vector expression in mammals and injected in BALB/c mouse. When mice were inoculated with the expression cassette they did not response in ELISA. They elevated antibody titles only when vaccinated with the synthetic peptide SBm7462®. And, the best titles of immunoglobulins were seen when the SBm7462® was administered subcutaneously.