In vitro identification of neutralizing epitopes of Rhipicephalus microplus serpin 17 (RmS-17).

BACKGROUND: Rhipicephalus microplus poses a significant problem for livestock worldwide and is primarily controlled with synthetic acaricides. The continuous use of acaricides results in the selection of resistance and causes environmental harm. Vaccination presents an alternative solution to this problem, although searching for the suitable antigen is still a work in progress. Salivary proteins hold promise for inclusion in vaccine formulation due to their roles in modulating host responses, assisting blood feeding and pathogen transmission. Serpins are a class of proteinase inhibitors and are among the molecules found in tick saliva that modulate host blood coagulation, inflammation, and adaptive immune responses. Previous studies have demonstrated the potential of R. microplus serpin 17 (RmS-17) to interfere with the host's defenses, and antibodies have been shown to neutralize its effects. This makes RmS-17 an putative target for vaccine development. METHODS: Epitope mapping of RmS-17 was achieved using in silico approach combining linear B-cell epitope and antigenicity predictor. In addition, epitope mapping using overlapping peptides in an ELISA screening was used. The serpin tridimensional structure and the epitopes spatial location within the molecule were determined. Peptides were synthetized based on the predictions and used for the production of rabbit anti-sera. Purified IgG's were used to assess the antibodies capacity to neutralize RmS-17. RESULTS: Through in silico mapping, nine potential B cell epitope regions were screened, with p1RmS-17 and p2RmS-17 selected for the experiment based on antigen prediction. In the ELISA screening using overlapping peptides, eight antibody-binding regions were identified, and p3RmS-17 and p4RmS-17 were chosen. Antibodies raised against p3RmS-17 and p4RmS-17 partially neutralized RmS-17 activity. CONCLUSION: It was found that antibodies against a single epitope are sufficient to partially neutralize RmS-17 activity. These findings support the possibility of using an epitope-based vaccine for immunization against R. microplus.

Construction of multi-epitope vaccine against the Rhipicephalus microplus tick: an immunoinformatics approach.

Rhipicephalus microplus, known as the hard tick, is a vector for the parasites Babesia spp. and Anaplasma marginale, both of which can cause significant financial losses to the livestock industry. There is currently no effective vaccine for R. microplus tick infestations, despite the identification of numerous prospective tick vaccine candidates. As a result, the current research set out to develop an immunoinformatics-based strategy using existing methods for designing a multi-epitope based vaccination that is not only effective but also safe and capable of eliciting cellular and humoral immune responses. First, R. microplus proteins Bm86, Subolesin, and Bm95 were used to anticipate and link B and T-cell epitopes (HTL and CTL) to one another. Antigenicity testing, allergenicity assessment, and toxicity screening were just a few of the many immunoinformatics techniques used to identify potent epitopes. Multi-epitope vaccine design was chosen based on the antigenic score 0.935 that is promising vaccine candidate. Molecular docking was used to determine the nature of the interaction between TLR2 and the vaccine construct. Finally, molecular dynamic simulation was used to assess the stability and compactness of the resulting vaccination based on docking scores. The developed vaccine was shown to be stable, have immunogenic qualities, be soluble, and to have high expression by in silico cloning. These findings suggest that experimental investigation of the multi-epitope based vaccine designed in the current study will produce achievable vaccine candidates against R. microplus ticks, enabling more effective control of infestations.

Immunologic characterization of the Bm05br protein using the Rhipicephalus linnaei (Audouin, 1826) species as a tick model.

The identification and characterization of tick proteins allow us to discover new physiological targets useful for the development of tick control methods. Bm05br (Brazil Rhipicephalus microplus protein 05) is a protein with unknown function identified in the saliva of R. microplus. Rs05br (Brazil Rhipicephalus sanguineus protein 05), a protein with 99 % similarity to Bm05br, was identified in Rhipicephalus linnaei egg, larval, and nymphal stages, as well as in adult saliva. To improve the knowledge about both proteins, immunological characterization was performed, including antigenicity analysis, vaccination trials, and artificial feeding. The sequence and antigenicity analysis of Bm05br and Rs05br proteins showed that R. linnaei could serve as a tick model for cross-protection studies. The recombinant Bm05br protein was immunogenic. Anti-Bm05br antibodies recognized the homologous protein Rs05br in different stages, organs, and in the saliva of R. linnaei. Although rBm05br did not induce a protective response against infestation in R. linnaei in this study, further experiments could be developed taking into account new formulations and animal models for vaccination. These results also serve as a reference for future research on the function of these proteins in R. microplus and R. linnaei physiology, as well as other species of the genus Rhipicephalus.

Structure and function of a family of tick-derived complement inhibitors targeting properdin.

Activation of the serum-resident complement system begins a cascade that leads to activation of membrane-resident complement receptors on immune cells, thus coordinating serum and cellular immune responses. Whilst many molecules act to control inappropriate activation, Properdin is the only known positive regulator of the human complement system. By stabilising the alternative pathway C3 convertase it promotes complement self-amplification and persistent activation boosting the magnitude of the serum complement response by all triggers. In this work, we identify a family of tick-derived alternative pathway complement inhibitors, hereafter termed CirpA. Functional and structural characterisation reveals that members of the CirpA family directly bind to properdin, inhibiting its ability to promote complement activation, and leading to potent inhibition of the complement response in a species specific manner. We provide a full functional and structural characterisation of a properdin inhibitor, opening avenues for future therapeutic approaches.

Genetic diversity of the ATAQ gene in Rhipicephalus microplus collected in Mexico and implications as anti-tick vaccine.

The cattle tick Rhipicephalus microplus has a large impact on cattle production due to its bloodsucking habit and transmission of pathogens that cause babesiosis and anaplasmosis. Application of acaricides constitutes the major control method but is often accompanied by serious drawbacks, including environmental contamination and an increase in acaricide resistance by ticks. The recent development of anti-tick vaccines has provided positive results in the post-genomic era, owing to the rise of reverse vaccinological and bioinformatics approaches to analyze and identify candidate protective antigens for use against ticks. The ATAQ protein is considered a novel antigen for the control of the cattle tick R. microplus; it is expressed in midguts and Malpighian tubules of all ticks from the Rhipicephalus genus. However, genetic diversity studies are required. Here, the ATAQ gene was sequenced of seven R. microplus tick isolates from different regions in Mexico to understand the genetic diversity. The results showed that sequence identity among the Mexican isolates ranged between 98 and 100% and 97.8-100% at the nucleotide and protein levels, respectively. Alignments of deduced amino acid sequences from different R. microplus ATAQ isolates in Mexico revealed a high degree of conservation. However, the Mexican isolates differed from the R. microplus "Mozambique" strain, at 20 amino acid residues. Finally, the analysis of more R. microplus isolates, and possibly of other Rhipicephalus species, to determine the genetic diversity in the ATAQ locus is essential to suggest this antigen as a vaccine candidate that might control tick infestations.

An inhibitor of complement C5 provides structural insights into activation.

The complement system is a crucial part of innate immune defenses against invading pathogens. The blood-meal of the tick Rhipicephalus pulchellus lasts for days, and the tick must therefore rely on inhibitors to counter complement activation. We have identified a class of inhibitors from tick saliva, the CirpT family, and generated detailed structural data revealing their mechanism of action. We show direct binding of a CirpT to complement C5 and have determined the structure of the C5-CirpT complex by cryoelectron microscopy. This reveals an interaction with the peripheral macro globulin domain 4 (C5_MG4) of C5. To achieve higher resolution detail, the structure of the C5_MG4-CirpT complex was solved by X-ray crystallography (at 2.7 Å). We thus present the fold of the CirpT protein family, and provide detailed mechanistic insights into its inhibitory function. Analysis of the binding interface reveals a mechanism of C5 inhibition, and provides information to expand our biological understanding of the activation of C5, and thus the terminal complement pathway.

Multi-scale patterns of tick occupancy and abundance across an agricultural landscape in southern Africa.

Land use influences the prevalence and distribution of ticks due to the intimate relationship of ticks with their environment. This relationship occurs because land use alters two essential tick requirements: vertebrate hosts for blood meals and a suitable microclimate when off-host. Given the risks to human and animal health associated with pathogens transmitted by ticks, there is an ongoing need to understand the impact of environmental drivers on tick distributions. Here, we assessed how landscape features, neighborhood effects, and edges influenced tick occupancy and abundance across an agricultural landscape in southern Africa. We found that Rhipicephalus appendiculatus and Rhipicephalus simus increased in abundance closer to protected savanna, while Haemaphysalis elliptica increased in abundance closer to human habitation. The composition of the landscape surrounding savanna patches also differentially influenced the occupancy of each tick species; H. elliptica was more likely to be found in savanna patches surrounded by subsistence agriculture while R. appendiculatus and R. simus were more likely to be found in savanna surrounded by sugarcane monocultures. At the local scale we found that R. appendiculatus and R. simus avoided savanna edges. The availability of hosts and variation in vegetation structure between commercial agriculture, subsistence agriculture, and savanna likely drove the distribution of ticks at the landscape scale. Understanding how anthropogenic land use influences where ticks occur is useful for land use planning and for assessing public and animal health risks associated with ticks and tick-borne diseases.

Distinct immune response profile during rhipicephalus (boophilus) microplus infestations of guzerat dairy herd according to the maternal lineage ancestry (mitochondrial DNA).

Rhipicephalus (Boophilus) microplus ticks cause major constraints to public and livestock health, and serious economic losses. It is well known that the immune response to infestations with cattle ticks is influenced by the host genetic background leading to distinct immunological profiles between bovine hosts genetically susceptible and resistant. The influence of Bos indicus (Bi) and Bos taurus (Bt) maternal lineage ancestry of mitochondrial DNA in the profile of the immune response of Zebu cattle to ticks remains unknown. The present work evaluated the hematological parameters and the immune response profile in the peripheral blood of a Guzerat dairy herd, further categorized into two maternal lineage ancestry subgroups (Bi-mtDNA and Bt-mtDNA) after experimental infestation with larvae of R. microplus. Our data demonstrated that although hematological and erythrogram analysis showed a similar profile throughout, some cell populations present a distinct profile between the groups. Especially MON, CD335+ and CD8+ T-cells are predominant in Bi-mtDNA. Moreover, an overall picture of R. microplus infestation demonstrated that Bi-mtDNA presented a more efficient and earlier innate immune response. Bi-mtDNA showed a greater number of connections with R. microplus counts and also with the CD25+ activation marker of the immune response. Bi-mtDNA showed greater number of connections, with an important participation of the innate immune while Bt-mtDNA showed a delay in the immune response. Elucidating the mechanisms by which resistant animals prevent heavy tick infestation is a crucial step in the development of predictive biomarkers for tick resistance for use in selective breeding programs, and is also potentially useful for the development of anti-tick vaccines.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies.

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals' leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick's immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female's body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gené's organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes' disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

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.

Gene Expression in the Salivary Gland of Rhipicephalus (Boophilus) microplus Fed on Tick-Susceptible and Tick-Resistant Hosts.

The success of cattle tick fixation largely depends on the secretion of substances that alter the immune response of the host. The majority of these substances are expressed by the parasite salivary gland and secreted in tick saliva. It is known that hosts can mount immune responses against ticks and bovine European breeds, and bovine industrial crossbreeds are more susceptible to infestations than are Bos indicus cattle. To identify candidates for the development of novel control strategies for the cattle tick Rhipicephalus (Boophilus) microplus, a salivary gland transcriptome analysis of engorged females fed on susceptible or resistant hosts was performed. Using RNA-Seq, transcriptomes were de novo assembled and produced a total of 235,451 contigs with 93.3% transcriptome completeness. Differential expression analysis identified 137 sequences as differentially expressed genes (DEGs) between ticks raised on tick-susceptible or tick-resistant cattle. DEGs predicted to be secreted proteins include innexins, which are transmembrane proteins that form gap junction channels; the transporters Na+/dicarboxylate, Na+/tricarboxylate, and phosphate transporter and a putative monocarboxylate transporter; a phosphoinositol 4-phosphate adaptor protein; a cysteine-rich protein containing a trypsin inhibitor-like (TIL) domain; a putative defense protein 3 containing a reeler domain; and an F-actin-uncapping protein LRRC16A with a CARMIL_C domain; these genes were upregulated in ticks fed on tick-susceptible cattle. DEGs predicted to be non-secreted proteins included a small heat shock protein and the negative elongation factor B-like, both acting in a coordinated manner to increase HSP transcript levels in the salivary glands of the ticks fed on tick-susceptible cattle; the 26S protease regulatory subunit 6B and another chaperone with similarity to calnexin, also upregulated in ticks fed on tick-susceptible cattle; an EF-hand calcium binding protein and a serine carboxypeptidase (SCP), both involved in the blood coagulation cascade and upregulated in ticks fed on tick-susceptible cattle; and two ribosomal proteins, the 60S acidic ribosomal protein P2 and the 60S ribosomal protein L19. These results help to characterize cattle tick salivary gland gene expression in tick-susceptible and tick-resistant hosts and suggest new putative targets for the control of tick infestations, as those genes involved in the mechanism of stress response during blood feeding.

Factors associated with the efficiency of acaricides on different populations of Rhipicephalus microplus / Fatores associados à eficiência de acaricidas sobre diferentes populações de Rhipicephalus microplus

Tick infestation causes major problems in cattle. Tick parasitism accounts for significant economic losses in many beef and dairy herds in the vast majority of the states in the Brazilian territory including the State of Santa Catarina in the southern region of the country. Tick resistance to several active principles occurs due to a number of factors including the indiscriminate, injudicious and inadequate use of tick insecticides. Considering the great importance of fighting off tick infestations in cattle, we evaluated the efficiency of 8 different topical active principles against the ixodid tick R. microplus and identified the main factors that contribute to the development of ticks that are resistant to acaricides in the farms and bovine herds studied. For such purpose, R. microplus telegionae were collected in 39 farms locates in different municipalities of the West of Santa Catarina, south Brazil. At the time of sample collection, information about the management, the history of the acar used in the herd, the number of annual applications of these pesticides, the frequency of acaricide rotation, and the frequency of technical monitoring for strategic tick control were retrieved. We collected this data in order determine contributing factors to the development resistant R. microplus populations. For the sensitivity profile of these ticks to a number of different acaricides tested, 10 engorged females for each principle and a control were used. The results of our study show that most associations between pyrethroids and organophosphates had an efficiency between 96.6% and 100% in the control of R. microplus except for the combination of Alfacipermetrine 15%, Ethion 16%, and Chlorpyrifos 8.5% that had 93.4% of efficiency. Among the chemicals not associated with any other type of compound, Amitraz 12.5% ​​was effective in 98.3% of the cases. In contrast, Cypermethrin 15% had an efficiency in only 25.8% of the farms/herds analyzed. Based on the results of the present study, we may infer that the methods of control and management used by producers may be related to the low indexes of resistance to topical acaricides in the different populations of ticks in the study area.

A infestação por carrapatos em bovinos gera grandes problemas, sendo esse parasito responsável por elevados prejuízos econômicos em diversos rebanhos de produção de corte e também de leite na maioria das regiões brasileiras, inclusive em Santa Catarina. A resistência dos carrapatos frente aos diversos princípios ativos pode ser decorrente de fatores como o uso indiscriminado de carrapaticidas, bem como a inadequada forma de aplicação dos mesmos. Tendo em vista a grande importância em combater essas infestações, buscou-se avaliar a eficiência de oito diferentes princípios ativos de contato contra o carrapato Rhipicephalus microplus, bem como identificar os principais fatores que possam contribuir com a seleção de carrapatos resistentes nas propriedades avaliadas. Para isso, teleóginas de R. microplus foram coletadas em 39 propriedades de diferentes municípios do Oeste de Santa Catarina. No momento da coleta também foram obtidas informações referentes ao sistema de manejo dos animais, histórico dos carrapaticidas utilizados, número de aplicações anuais, frequência de rodízios de acaricidas, e frequência de acompanhamento técnico para controle estratégico, com a finalidade de evidenciar os diferentes fatores que possam estar contribuindo para o surgimento de eventual resistência das populações do R. microplus. A avaliação de suscetibilidade foi realizada através da técnica de biocarrapaticidograma, utilizando 10 teleóginas ingurgitadas para cada princípio e mais o controle. Dentre os principais resultados obtidos, a maioria das associações entre piretróides e organofosforados demonstraram ter eficiência entre 96,6% e 100%, com exceção da associação de Alfacipermetrina 15%, Ethion 16% e Clorpirifós 8,5% com 93,4% de eficiência. Dentre as bases químicas sem nenhum tipo de associação o Amitraz 12,5% demonstrou ser eficaz em 98,3%, em contrapartida a Cipermetrina 15% teve eficiência em apenas 25,8% das propriedades analisadas. A partir dos resultados obtidos, pode se afirmar que as formas de controle e manejo adotados pelos produtores podem estar relacionadas com os baixos índices de resistência aos carrapaticidas de contato, nas diferentes populações de carrapato na região do estudo.

Analysis of Bm86 conserved epitopes: is a global vaccine against Cattle Tick Rhipicephalus microplus possible? / Análise de epítopos conservados da Bm86: é possível uma vacina global contra Carrapato-do-Boi Rhipicephalus microplus?

Abstract The cattle tick Rhipicephalus microplus causes significant economic losses in agribusiness. Control of this tick is achieved mainly through the application of chemical acaricides, often resulting in contamination of animal food products and of the environment. Another major concern associated with acaricide use is the increasing reports of resistance of this tick vector against the active ingredients of many commercial products. An alternative control method is vaccination. However, the commercially available vaccine based on a protein homologous to Bm86 exhibits variations in efficacy relative to the different geographical locations. This study aimed to identify antigenic determinants of the sequences of proteins homologous to Bm86. Phylogenetic analyses were performed to determine the extent of divergence between different populations of R. microplus to identify the sequence that could be used as a universal vaccine against the multiple geographically distinct populations of R. microplus and related tick species. Considering the extensive sequence and functional polymorphism observed among strains of R. microplus from different geographical regions, we can conclude that it may be possible to achieve effective vaccination against these cattle ticks using a single universal Bm86-based antigen.

Resumo O carrapato Rhipicephalus microplus é responsável por perdas significativas no agronegócio. O controle deste carrapato é feito principalmente por meio da aplicação de acaricidas químicos, geralmente resultando na contaminação de produtos de origem animal e do meio ambiente. Outra preocupação importante associada ao uso de acaricidas é o crescente aumento de relatos sobre a resistência deste carrapato a princípios ativos de vários produtos comerciais. Uma alternativa de controle é por meio de vacinação. Porém, a vacina comercializada contendo proteína homóloga à Bm86, apresenta variações de eficácia em relação às diferentes localizações geográficas. Este estudo buscou identificar determinantes antigênicos das sequencias de proteínas homólogas a Bm86. As análises filogenéticas foram feitas para determinar a extensão da divergência entre diferentes populações de R. microplus com o objetivo de identificar a sequência que poderia ser usada como vacina universal contra as múltiplas populações geograficamente distintas de R. microplus e espécies de carrapatos relacionados. Considerando-se a extensa sequência e o polimorfismo observados entre linhagens de R. microplus de diferentes regiões geográficas, podemos concluir que pode ser possível obter uma vacinação efetiva contra esses carrapatos bovinos utilizando um único antígeno universal baseado em Bm86.

Analysis of Bm86 conserved epitopes: is a global vaccine against Cattle Tick Rhipicephalus microplus possible?

The cattle tick Rhipicephalus microplus causes significant economic losses in agribusiness. Control of this tick is achieved mainly through the application of chemical acaricides, often resulting in contamination of animal food products and of the environment. Another major concern associated with acaricide use is the increasing reports of resistance of this tick vector against the active ingredients of many commercial products. An alternative control method is vaccination. However, the commercially available vaccine based on a protein homologous to Bm86 exhibits variations in efficacy relative to the different geographical locations. This study aimed to identify antigenic determinants of the sequences of proteins homologous to Bm86. Phylogenetic analyses were performed to determine the extent of divergence between different populations of R. microplus to identify the sequence that could be used as a universal vaccine against the multiple geographically distinct populations of R. microplus and related tick species. Considering the extensive sequence and functional polymorphism observed among strains of R. microplus from different geographical regions, we can conclude that it may be possible to achieve effective vaccination against these cattle ticks using a single universal Bm86-based antigen.

Comparison of the differential regulation of T and B-lymphocyte subsets in the skin and lymph nodes amongst three cattle breeds as potential mediators of immune-resistance to Rhipicephalus microplus.

Although varying natural resistance to ticks between highly resistant Brahman (Bos taurus indicus), resistant Bonsmara (5/8 B. t. indicus x 3/8 B. t. taurus) and susceptible Holstein-Friesian (B. t. taurus) breeds is documented in skin and blood, little information is available describing draining lymph nodes. To elucidate the cellular dynamics during Rhipicephalus microplus induced immune responses, this study analysed immune factors from these cattle breeds using histology, immunohistochemistry and flow cytometry. Following the collection of skin and lymph node samples before artificial tick infestation, cattle were infested with R. microplus larvae. Subsequent sampling coincided with the tick larvae and adult developmental stages. A significant influx of CD20+ B-lymphocytes in the dermis all cattle breeds was observed while CD3+ T-lymphocytes were significantly increased for more tick resistant breeds. Eosinophil infiltration in germinal centres of lymph nodes was significant for all cattle breeds while tingible body macrophages were significantly increased for adult infested Brahman animals. A negligible fluctuation in CD20+ and CD79α+ B-lymphocyte numbers was present in the lymph node of more resistant cattle breeds, while susceptible animals showed a decrease in B-lymphocytes after infestation, followed by an increase between larvae to adult infested time points. Increased variability of γd T-lymphocyte populations in lymph nodes was correlated with tick susceptibility. In addition, a more stable T helper lymphocyte population was identified in the lymph nodes for the Brahman cattle breed. Results suggest the association of tick susceptibility with differential B-lymphocyte regulation in lymph node tissues, increased variability of WC1+ γδ T-lymphocyte populations in the lymph node as well as a decrease in T helper lymphocytes in the lymph node.

Immunological control of ticks and tick-borne diseases that impact cattle health and production.

The cattle industry is one of the most important agroeconomic activities in Mexico. The national herd is estimated to include approximately 33.5. million head of cattle. Ticks and tick-borne diseases are principal factors with a negative impact on cattle health and production. The most economically important tick species parasitizing cattle in Mexico are Rhipicephalus microplus, R. annulatus, and Amblyomma mixtum. Parasitism by ticks affects cattle health and production directly. Morbidity and mortality caused by tick-borne diseases augment the detrimental effect of tick infestation in cattle. Bovine babesiosis and anaplasmosis are the most important tick-borne diseases of cattle, which are caused by infectious agents transmitted by R. microplus and R. annulatus. However, there are no prophylactic therapies to control bovine babesiosis and anaplasmosis. Chemical control is the most common way to treat animals against ticks, and the use of acaricides can also help manage tick-borne diseases. However, the evolution of resistance to acaricides among cattle tick populations renders chemical control ineffective; which represents a challenge for sustainable ticks and tick-borne diseases control. The only anti-tick vaccine commercially available globally is based on the recombinant antigen Bm86. Because of its mode of immunity against R. microplus and R. annulatus, the Bm86-based vaccine also decreases the exposition of bovines to babesiosis and anaplasmosis. Research with Bm86-based vaccines documented high efficacy against R. annulatus, the efficacy levels against R. microplus varies according to the geographic origin of tick populations, and there is not effect against other ticks species such as Amblyomma spp. The impact of ticks and tick-borne diseases, the problem of chemical control due to acaricide resistance, and progress with anti-tick vaccine research efforts in Mexico are reviewed herein.

Local immune response to larvae of Rhipicephalus microplus in Santa Gertrudis cattle.

This study investigated the local immune response at larval attachment sites in Santa Gertrudis cattle with low and high levels of tick resistance. Skin samples with tick larvae attached were collected from Santa Gertrudis cattle at the end of a period of 25 weekly infestations, when the animals manifested highly divergent tick-resistant phenotypes. There was a tendency for more CD3+ , CD4+ , CD8+ , CD25+ , γδ T cells and neutrophils to concentrate at larval tick attachment site in susceptible cattle than in resistant cattle but the differences were significant only for γδ T cells and CD4+ cells. Most of the cattle developed intra-epidermal vesicles at the larval attachment site but the predominant cell within or around the vesicles was the neutrophil in susceptible animals and eosinophil in the resistant animals. The monoclonal antibodies (mAbs) specific for CD45 and CD45 RO antigens reacted with skin leucocytes from a higher number of susceptible cattle than resistant cattle. Our data suggest that some of the cellular responses mounted at larval attachment site are not involved in tick protection. The mAbs specific for CD45 and CD45 RO directly, or a test for CD45 genotype might be developed as markers of tick susceptibility or resistance.

Advances in tick vaccinology in Brazil: from gene expression to immunoprotection.

Rhipicephalus (Boophilus) microplus has substantial economic impact on the cattle breeding industry and, chemical control and tick resistance development are the major concern. There is a worldwide search for new options, and control using vaccines has been the main focus nowadays. Studies performed in Brazil found that Bm86-based immunization of bovines reduced the infestation of R. (B.) microplus of vaccinated bovines by 45% to 60%. Native Boophilusmicroplus tripsin inhibitors (BmTIs) with trypsin-, kallikrein-, and elastase-inhibiting activities have been used as immunogens in bovines reaching 72.8.% of efficacy. The reverse vaccinology approach has also been used for antigen search using transcriptome analysis to identify and characterize potential antigens. Study has generated more than 600 million sequences using RNA-seq of larvae, nymphs, salivary glands, intestines, and ovaries of the tick R. (B) microplus. Based on the set of transcripts obtained using this strategy, a total of 20,326 protein sequences have been identified. A pipeline analysis built in house identified the protein sequences that were most likely to be immunogenic based on the overall structural characteristic analysis.

Experimental efficacy of a vaccine against Rhipicephalus australis.

Despite several decades of chemical use the cattle tick, Rhipicephalus australis, still represents an important threat for breeders of tick susceptible cattle under tropical conditions. The intense use of acaricides has lead to the development of multi-resistant strains of ticks and alternative means of control need to be developed to maintain control. A vaccine against ticks, based on the Boophilus microplus Bm86 protein, is one of the promising alternative means of tick control. In this investigation, an experimental vaccine was developed based on the orthologous R. australis Bm86 sequence identified from local R. australis strains and a recombinant protein expressed in Escherichia coli. The efficacy of this vaccine against a local strain of R. australis was assessed under controlled conditions. Vaccinated cattle presented 40.3% fewer ticks than unvaccinated cattle and tick reproductive performance was also affected by the vaccine. Ticks that engorged on vaccinated animals laid 51.2% less egg mass and tick fertility decreased by 18.8% compared to the control group. The use of the vaccine reduced the tick population by 74.2% at each generation. The tick vaccine can be used in an integrated tick control program.

Effect of recombinant glutathione S-transferase as vaccine antigen against Rhipicephalus appendiculatus and Rhipicephalus sanguineus infestation.

The ticks Rhipicephalus appendiculatus and Rhipicephalus sanguineus are the main vectors of Theileria parva and Babesia spp. in cattle and dogs, respectively. Due to their impact in veterinary care and industry, improved methods against R. appendiculatus and R. sanguineus parasitism are under development, including vaccines. We have previously demonstrated the induction of a cross-protective humoral response against Rhipicephalus microplus following vaccination with recombinant glutathione S-transferase from Haemaphysalis longicornis tick (rGST-Hl), suggesting that this protein could control tick infestations. In the present work, we investigated the effect of rGST-Hl vaccine against R. appendiculatus and R. sanguineus infestation in rabbits. In silico analysis revealed that GST from H. longicornis, R. appendiculatus and R. sanguineus have >80% protein sequence similarity, and multiple conserved antigenic sites. After the second vaccine dose, rGST-Hl-immunized rabbits showed elevated antibody levels which persisted until the end of experiment (75 and 60 days for R. appendiculatus and R. sanguineus, respectively). Western blot assays demonstrated cross-reactivity between anti-rGST-Hl antibodies and native R. appendiculatus and R. sanguineus GST extracts from ticks at different life stages. Vaccination with rGST-Hl decreased the number, weight, and fertility of engorged R. appendiculatus adults, leading to an overall vaccine efficacy of 67%. Interestingly, histological analysis of organ morphology showed damage to salivary glands and ovaries of R. appendiculatus adult females fed on vaccinated animals. In contrast, rGST-Hl vaccination did not affect R. appendiculatus nymphs, and it was ineffective against R. sanguineus across the stages of nymph and adult. Taken together, our results show the potential application of rGST-Hl as an antigen in anti-tick vaccine development, however indicating a broad difference in efficacy among tick species.