RESUMO
Anaplasma marginale is an economically important tick-borne pathogen of cattle that causes bovine anaplasmosis. A wide range of geographic strains of A. marginale have been isolated from cattle, several of which have been characterized using genomics and proteomics. While many of these strains have been propagated in tick lines, comparative analyses after propagation in tick cells have not been reported. The overall purpose of this research therefore was to compare the degree of conservation of selected genes after propagation in tick cell culture among A. marginale strains from the U.S. (the Virginia strain) and Brazil (UFMG1 and UFMG2 strains). The genes studied herein included those which encode the proteins HSP70 and SODB involved in heat shock and stress responses, respectively, and two genes that encode major surface proteins MSP4 and MSP5. Strain identities were first confirmed by sequencing the tandem repeats of the msp1a gene which encodes for the adhesin, MSP1a. The results of these studies demonstrated that the genes encoding for both stress response and heat shock proteins were highly conserved among the three A. marginale strains. Antibodies specific for MSP4, MSP5, SODB and HSP70 proteins were used to further characterize the A. marginale strains, and they reacted with all of these strains propagated in tick cell culture, providing further evidence for antigenic conservation. Although antigenic differences were not found among the three A. marginale strains, multi-locus sequence analysis (MLSA) performed with nucleotide sequences of these genes demonstrated that the A. marginale Brazilian and U.S. strains fall in different clades. These results showed that phylogenetically distant strains of A. marginale are antigenically conserved, even after several in vitro passages, supporting the use of some of the above conserved proteins as candidates for universal vaccines.
Assuntos
Anaplasma marginale/isolamento & purificação , Anaplasmose/imunologia , Vetores Aracnídeos/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Doenças dos Bovinos/imunologia , Carrapatos/microbiologia , Anaplasma marginale/classificação , Anaplasma marginale/genética , Anaplasma marginale/crescimento & desenvolvimento , Anaplasmose/microbiologia , Animais , Variação Antigênica , Brasil , Bovinos , Doenças dos Bovinos/microbiologia , Sequência Conservada , Dados de Sequência Molecular , Filogenia , Estados UnidosRESUMO
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.
Assuntos
Vacinas Bacterianas/imunologia , Doenças dos Bovinos/prevenção & controle , Proteínas de Insetos/imunologia , Glicoproteínas de Membrana/imunologia , Rhipicephalus/imunologia , Infestações por Carrapato/veterinária , Animais , Vacinas Bacterianas/administração & dosagem , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/parasitologia , Membrana Celular/genética , Membrana Celular/imunologia , Escherichia coli/genética , Escherichia coli/imunologia , Proteínas de Insetos/genética , Glicoproteínas de Membrana/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Infestações por Carrapato/parasitologia , Infestações por Carrapato/prevenção & controle , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
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.
Assuntos
Antígenos/imunologia , Proteínas de Artrópodes/imunologia , Proteínas de Insetos/imunologia , Ixodes/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas Recombinantes/imunologia , Infestações por Carrapato/veterinária , Vacinação/métodos , Vacinas/imunologia , Animais , Antígenos/administração & dosagem , Proteínas de Artrópodes/administração & dosagem , Cervos , Feminino , Proteínas de Insetos/administração & dosagem , Masculino , Glicoproteínas de Membrana/administração & dosagem , Proteínas Recombinantes/administração & dosagem , Infestações por Carrapato/prevenção & controle , Vacinas/administração & dosagemRESUMO
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.
Assuntos
Antígenos/imunologia , Rhipicephalus/genética , Controle de Ácaros e Carrapatos/métodos , Infestações por Carrapato/veterinária , Vacinação , Animais , Formação de Anticorpos , Antígenos/genética , Proteínas de Artrópodes , Bovinos , Feminino , Técnicas de Silenciamento de Genes , Interferência de RNA , Infestações por Carrapato/prevenção & controle , Vacinação/veterinária , Vacinas/genética , Vacinas/imunologiaRESUMO
The cattle ticks, Rhipicephalus (Boophilus) spp., affect cattle production in tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The recombinant Rhipicephalus microplus Bm86 antigen has been shown to protect cattle against tick infestations. However, variable efficacy of Bm86-based vaccines against geographic tick strains has encouraged the research for additional tick-protective antigens. Herein, we describe the analysis of R. microplus glutathione-S transferase, ubiquitin (UBQ), selenoprotein W, elongation factor-1 alpha, and subolesin (SUB) complementary DNAs (cDNAs) by RNA interference (RNAi) in R. microplus and Rhipicephalus annulatus. Candidate protective antigens were selected for vaccination experiments based on the effect of gene knockdown on tick mortality, feeding, and fertility. Two cDNA clones encoding for UBQ and SUB were used for cattle vaccination and infestation with R. microplus and R. annulatus. Control groups were immunized with recombinant Bm86 or adjuvant/saline. The highest vaccine efficacy for the control of tick infestations was obtained for Bm86. Although with low immunogenic response, the results with the SUB vaccine encourage further investigations on the use of recombinant subolesin alone or in combination with other antigens for the control of cattle tick infestations. The UBQ peptide showed low immunogenicity, and the results of the vaccination trial were inconclusive to assess the protective efficacy of this antigen. These experiments showed that RNAi could be used for the selection of candidate tick-protective antigens. However, vaccination trials are necessary to evaluate the effect of recombinant antigens in the control of tick infestations, a process that requires efficient recombinant protein production and formulation systems.