Inclusion of Anti-Tick Vaccines into an Integrated Tick Management Program in Mexico: A Public Policy Challenge.

Acaricides are the most widely used method to control the cattle tick Rhipicephalus microplus. However, its use increases production costs, contaminates food and the environment, and directly affects animal and human health. The intensive use of chemical control has resulted in the selection of genes associated with resistance to acaricides, and consumers are increasingly less tolerant of food contamination. This scenario has increased the interest of different research groups around the world for anti-tick vaccine development, in order to reduce the environmental impact, the presence of residues in food, and the harmful effects on animal and human health. There is enough evidence that vaccination with tick antigens induces protection against tick infestations, reducing tick populations and acaricide treatments. Despite the need for an anti-tick vaccine in Mexico, vaccination against ticks has been limited to one vaccine that is used in some regions. The aim of this review is to contribute to the discussion on tick control issues and provide a reference for readers interested in the importance of using anti-tick vaccines encouraging concerted action on the part of Mexican animal health authorities, livestock organizations, cattle producers, and academics. Therefore, it is suggested that an anti-tick vaccine should be included as a part of an integrated tick management program in Mexico.

A One Health Epidemiological Approach for Understanding Ticks and Tick-Borne Pathogens.

Background: The control and prevention of ticks and tick borne diseases (TBDs) is often difficult, since it is necessary to disrupt a complex transmission cycle, involving ticks and vertebrate hosts, which interact in a changing environment, driven by constant environmental and ecological changes. Our view is that factors driving the spread of R. microplus are complex and intrinsically interconnected, something that has often been ignored in control strategies. Results: The aim of this review is to analyze the importance of the epidemiological surveillance of ticks and tick-borne diseases (TTBDs) for Public Health, with the One Health approach; emphasizing the knowledge, importance, and distribution of TTBDs. Conclusions: The key points for surveillance, and raising the scope and limitations of surveillance programs, to delay the emergence of acaricide resistance, to reduce toxic residues in food for human consumption and to protect animal, human, and environmental health, from a One Health perspective will require calling producers, veterinarians, academics, pharmaceutical industry, and decision makers to join efforts in order to mitigate the effects of ticks and TBDs affecting the cattle industry in Mexico.

Morphological and molecular identification of the brown dog tick in Mexico.

Ticks of the Rhipicephalus sanguineus complex are known as the brown dog ticks. This complex groups at least 12 species of ticks that are distributed worldwide. On the American continents, R. sanguineus sensu stricto (s.s.), is distributed in temperate areas, while Rhipicephalus sanguineus sensu lato (s.l.), also called "tropical lineage" is distributed in tropical regions. Previous analyses of brown dog ticks from Mexico have identified the so-called tropical lineage and the country generally has a climate more favorable for these ticks (> 20o C in average). In addition, some pathogens thought to be transmitted by this lineage (such as Ehrlichia canis, and Rickettsia rickettsii) are prevalent in Mexico. Herein we aim to contribute to the study of brown dog ticks by providing morphological identification and molecular analysis of mt 12S rDNA and 16S rDNA sequences from ticks collected from 12 states in Mexico. Our results indicate that the tropical lineage of R. sanguineus s.l., recently redescribed as R. linnaei is widely distributed in Mexico.

Perception of Ticks and Tick-Borne Diseases Worldwide.

In this comprehensive review study, we addressed the challenge posed by ticks and tick-borne diseases (TBDs) with growing incidence affecting human and animal health worldwide. Data and perspectives were collected from different countries and regions worldwide, including America, Europe, Africa, Asia, and Oceania. The results updated the current situation with ticks and TBD and how it is perceived by society with information bias and gaps. The study reinforces the importance of multidisciplinary and international collaborations to advance in the surveillance, communication and proposed future directions to address these challenges.

Identification of microorganisms in partially fed female horn flies, Haematobia irritans.

The horn fly Haematobia irritans (Linnaeus, 1758) (Diptera: Muscidae) is one of the most important ectoparasites of cattle. The parasitism of horn flies interferes with cattle feeding, thus reducing weight gain and milk production. Additionally, horn flies are mechanical vectors of pathogens that cause disease in cattle. The aims of this study were to identify microorganisms in partially fed female horn flies through mining of expressed sequence tags (ESTs) and to characterize microorganism prevalence using real-time RT-PCR. Seven unigenes containing 24 ESTs were homologous to infectious agents. Microorganisms identified in partially fed female horn flies ESTs included Nora virus (3 unigenes; 8 ESTs), Wolbachia endosymbionts (3 unigenes; 3 ESTs), and Mycobacterium bovis (1 unigene; 13 ESTs). These results expanded the repertoire of microorganisms that could cause persistent infections or be mechanically transmitted by horn flies and support further studies on the role of horn flies in the epidemiology of these pathogens in Mexico.

Impact of the Paper by Allen and Humphreys (1979) on Anti-Tick Vaccine Research.

The classic paper by Allen and Humphreys "Immunisation of guinea pigs and cattle against ticks" Nature, 1979, 280: 491-493 led to a surge in the development of tick vaccines as a nonchemical method for prevention of tick infestations in susceptible hosts living in tick-endemic regions. Although observations of host resistance to ticks had been documented since the beginning of the last century, it was not until publication of this paper that the proof of concept of anti-tick vaccines was developed. The described experimental methods directly impacted further investigations on the discovery and evaluation of new anti-tick vaccines.

Babesiosis and Theileriosis in North America.

Babesia and Theileria are apicomplexan parasites that cause established and emerging diseases in humans, domestic and wild animals. These protozoans are transmitted by Ixodid ticks causing babesiosis or theileriosis, both characterized by fever, hemolytic anemia, jaundice, and splenomegaly. In North America (NA), the most common species affecting humans is B. microti, which is distributed in the Northeastern and Upper Midwestern United States (US), where the tick vector Ixodes scapularis is established. In livestock, B. bovis and B. bigemina are the most important pathogens causing bovine babesiosis in tropical regions of Mexico. Despite efforts toward eradication of their tick vector, Rhipicephalus microplus, B. bovis and B. bigemina present a constant threat of being reintroduced into the southern US and represent a continuous concern for the US cattle industry. Occasional outbreaks of T. equi, and T. orientalis have occurred in horses and cattle, respectively, in the US, with significant economic implications for livestock including quarantine, production loss, and euthanasia of infected animals. In addition, a new species, T. haneyi, has been recently discovered in horses from the Mexico-US border. Domestic dogs are hosts to at least four species of Babesia in NA that may result in clinical disease that ranges from subclinical to acute, severe anemia. Herein we review the pathogenesis, diagnosis, and epidemiology of the most important diseases caused by Babesia and Theileria to humans, domestic and wild animals in Canada, the US, and Mexico.

Functional genomics of the horn fly, Haematobia irritans (Linnaeus, 1758).

BACKGROUND: The horn fly, Haematobia irritans (Linnaeus, 1758) (Diptera: Muscidae) is one of the most important ectoparasites of pastured cattle. Horn flies infestations reduce cattle weight gain and milk production. Additionally, horn flies are mechanical vectors of different pathogens that cause disease in cattle. The aim of this study was to conduct a functional genomics study in female horn flies using Expressed Sequence Tags (EST) analysis and RNA interference (RNAi). RESULTS: A cDNA library was made from whole abdominal tissues collected from partially fed adult female horn flies. High quality horn fly ESTs (2,160) were sequenced and assembled into 992 unigenes (178 contigs and 814 singlets) representing molecular functions such as serine proteases, cell metabolism, mitochondrial function, transcription and translation, transport, chromatin structure, vitellogenesis, cytoskeleton, DNA replication, cell response to stress and infection, cell proliferation and cell-cell interactions, intracellular trafficking and secretion, and development. Functional analyses were conducted using RNAi for the first time in horn flies. Gene knockdown by RNAi resulted in higher horn fly mortality (protease inhibitor functional group), reduced oviposition (vitellogenin, ferritin and vATPase groups) or both (immune response and 5'-NUC groups) when compared to controls. Silencing of ubiquitination ESTs did not affect horn fly mortality and oviposition while gene knockdown in the ferritin and vATPse functional groups reduced mortality when compared to controls. CONCLUSIONS: These results advanced the molecular characterization of this important ectoparasite and suggested candidate protective antigens for the development of vaccines for the control of horn fly infestations.

Cytauxzoonosis in North America.

Cytauxzoonosis is an emerging tick-borne disease of domestic and wild felids produced by infection of Cytauxzoon felis, an apicomplexan protozoan similar to Theileria spp. Transmitted by Amblyomma americanum, lone star tick, and Dermacentor variabilis, American dog tick, infection of C. felis in cats is severe, characterized by depression, lethargy, fever, hemolytic crisis, icterus, and possibly death. Cytauxzoonosis occurs mainly in the southern, south-central, and mid-Atlantic United States in North America, in close association with the distribution and activity of tick vectors. Infection of C. felis, although severe, is no longer considered uniformly fatal, and unless moribund, every attempt to treat cytauxzoonosis cats should be made. Herein we review cytauxzoonosis, including its etiology, affected species, its life cycle and pathogenesis, clinical signs, diagnosis, and epidemiology, emphasizing clinical pathology findings in cats infected with this important emerging tick-borne disease in North and South America.

Differential expression of genes in salivary glands of male Rhipicephalus (Boophilus)microplus in response to infection with Anaplasma marginale.

BACKGROUND: Bovine anaplasmosis, caused by the rickettsial tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), is vectored by Rhipicephalus (Boophilus)microplus in many tropical and subtropical regions of the world. A. marginale undergoes a complex developmental cycle in ticks which results in infection of salivary glands from where the pathogen is transmitted to cattle. In previous studies, we reported modification of gene expression in Dermacentor variabilis and cultured Ixodes scapularis tick cells in response to infection with A. marginale. In these studies, we extended these findings by use of a functional genomics approach to identify genes differentially expressed in R. microplus male salivary glands in response to A. marginale infection. Additionally, a R. microplus-derived cell line, BME26, was used for the first time to also study tick cell gene expression in response to A. marginale infection. RESULTS: Suppression subtractive hybridization libraries were constructed from infected and uninfected ticks and used to identify genes differentially expressed in male R. microplus salivary glands infected with A. marginale. A total of 279 ESTs were identified as candidate differentially expressed genes. Of these, five genes encoding for putative histamine-binding protein (22Hbp), von Willebrand factor (94Will), flagelliform silk protein (100Silk), Kunitz-like protease inhibitor precursor (108Kunz) and proline-rich protein BstNI subfamily 3 precursor (7BstNI3) were confirmed by real-time RT-PCR to be down-regulated in tick salivary glands infected with A. marginale. The impact of selected tick genes on A. marginale infections in tick salivary glands and BME26 cells was characterized by RNA interference. Silencing of the gene encoding for putative flagelliform silk protein (100Silk) resulted in reduced A. marginale infection in both tick salivary glands and cultured BME26 cells, while silencing of the gene encoding for subolesin (4D8) significantly reduced infection only in cultured BME26 cells. The knockdown of the gene encoding for putative metallothionein (93 Meth), significantly up-regulated in infected cultured BME26 cells, resulted in higher A. marginale infection levels in tick cells. CONCLUSIONS: Characterization of differential gene expression in salivary glands of R. microplus in response to A. marginale infection expands our understanding of the molecular mechanisms at the tick-pathogen interface. Functional studies suggested that differentially expressed genes encoding for subolesin, putative von Willebrand factor and flagelliform silk protein could play a role in A. marginale infection and multiplication in ticks. These tick genes found to be functionally relevant for tick-pathogen interactions will likely be candidates for development of vaccines designed for control of both ticks and tick-borne pathogens.

Subolesin expression in response to pathogen infection in ticks.

BACKGROUND: Ticks (Acari: Ixodidae) are vectors of pathogens worldwide that cause diseases in humans and animals. Ticks and pathogens have co-evolved molecular mechanisms that contribute to their mutual development and survival. Subolesin was discovered as a tick protective antigen and was subsequently shown to be similar in structure and function to akirins, an evolutionarily conserved group of proteins in insects and vertebrates that controls NF-kB-dependent and independent expression of innate immune response genes. The objective of this study was to investigate subolesin expression in several tick species infected with a variety of pathogens and to determine the effect of subolesin gene knockdown on pathogen infection. In the first experiment, subolesin expression was characterized in ticks experimentally infected with the cattle pathogen, Anaplasma marginale. Subolesin expression was then characterized in questing or feeding adult ticks confirmed to be infected with Anaplasma, Ehrlichia, Rickettsia, Babesia or Theileria spp. Finally, the effect of subolesin knockdown by RNA interference (RNAi) on tick infection was analyzed in Dermacentor variabilis males exposed to various pathogens by capillary feeding (CF). RESULTS: Subolesin expression increased with pathogen infection in the salivary glands but not in the guts of tick vector species infected with A. marginale. When analyzed in whole ticks, subolesin expression varied between tick species and in response to different pathogens. As reported previously, subolesin knockdown in D. variabilis infected with A. marginale and other tick-borne pathogens resulted in lower infection levels, while infection with Francisella tularensis increased in ticks after RNAi. When non-tick-borne pathogens were fed to ticks by CF, subolesin RNAi did not affect or resulted in lower infection levels in ticks. However, subolesin expression was upregulated in D. variabilis exposed to Escherichia coli, suggesting that although this pathogen may induce subolesin expression in ticks, silencing of this molecule reduced bacterial multiplication by a presently unknown mechanism. CONCLUSIONS: Subolesin expression in infected ticks suggested that subolesin may be functionally important for tick innate immunity to pathogens, as has been reported for the akirins. However, subolesin expression and consequently subolesin-mediated innate immunity varied with the pathogen and tick tissue. Subolesin may plays a role in tick innate immunity in the salivary glands by limiting pathogen infection levels, but activates innate immunity only for some pathogen in the guts and other tissues. In addition, these results provided additional support for the role of subolesin in other molecular pathways including those required for tissue development and function and for pathogen infection and multiplication in ticks. Consequently, RNAi experiments demonstrated that subolesin knockdown in ticks may affect pathogen infection directly by reducing tick innate immunity that results in higher infection levels and indirectly by affecting tissue structure and function and the expression of genes that interfere with pathogen infection and multiplication. The impact of the direct or indirect effects of subolesin knockdown on pathogen infection may depend on several factors including specific tick-pathogen molecular interactions, pathogen life cycle in the tick and unknown mechanisms affected by subolesin function in the control of global gene expression in ticks.

Identification and characterization of Rhipicephalus (Boophilus) microplus candidate protective antigens for the control of cattle tick infestations.

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.

Failed Disruption of Tick Feeding, Viability, and Molting after Immunization of Mice and Sheep with Recombinant Ixodes ricinus Salivary Proteins IrSPI and IrLip1.

To identify potential vaccine candidates against Ixodes ricinus and tick-borne pathogen transmission, we have previously sequenced the salivary gland transcriptomes of female ticks infected or not with Bartonella henselae. The hypothesized potential of both IrSPI (I. ricinus serine protease inhibitor) and IrLip1 (I. ricinus lipocalin 1) as protective antigens decreasing tick feeding and/or the transmission of tick-borne pathogens was based on their presumed involvement in dampening the host immune response to tick feeding. Vaccine endpoints included tick larval and nymphal mortality, feeding, and molting in mice and sheep. Whether the antigens were administered individually or in combination, the vaccination of mice or sheep elicited a potent antigen-specific antibody response. However, and contrary to our expectations, vaccination failed to afford protection against the infestation of mice and sheep by I. ricinus nymphs and larvae, respectively. Rather, vaccination with IrSPI and IrLip1 appeared to enhance tick engorgement and molting and decrease tick mortality. To the best of our knowledge, these observations represent the first report of induction of vaccine-mediated enhancement in relation to anti-tick vaccination.

Multiple Antigenic Peptide-Based Vaccines Targeting Ixodes ricinus Neuropeptides Induce a Specific Antibody Response but Do Not Impact Tick Infestation.

Synthetic peptide vaccines were designed to target the neuropeptides innervating Ixodes ricinus salivary glands and hindgut and they were tested for their capacity to afford protective immunity against nymphs or larvae and Anaplasma phagocytophilum-infected nymph infestation, in mice and sheep, respectively. In both models, the assembly of SIFamide (SIFa) or myoinhibitory peptide (MIP) neuropeptides into multiple antigenic peptide constructs (MAPs) elicited a robust IgG antibody response following immunization. Nevertheless, no observable detrimental impact on nymphs was evidenced in mice, and, unfortunately, the number of engorged nymphs on sheep was insufficient for firm conclusions to be drawn, including for bacterial transmission. Regarding larvae, while vaccination of the sheep did not globally diminish tick feeding success or development, analyses of animals at the individual level revealed a negative correlation between anti-SIFa and MIP antibody levels and larva-to-nymph molting success for both antigens. Our results provide a proof of principle and precedent for the use of MAPs for the induction of immunity against tick peptide molecules. Although the present study did not provide the expected level of protection, it inaugurates a new strategy for protection against ticks based on the immunological targeting of key components of their nervous system.

Experimental Ixodes ricinus-Sheep Cycle of Anaplasma phagocytophilum NV2Os Propagated in Tick Cell Cultures.

The causative agent of tick-borne fever and human granulocytic anaplasmosis, Anaplasma phagocytophilum, is transmitted by Ixodes ricinus, and is currently considered an emerging disease throughout Europe. In this study, we established a model of A. phagocytophilum sheep infection and I. ricinus transmission using the European Norway variant 2 ovine strain (NV2Os) propagated in both IDE8 and ISE6 tick cells. Two sheep were inoculated with IDE8 tick cells infected with NV2Os. Both sheep developed A. phagocytophilum infection as determined by qPCR and PCR, the presence of fever 4 days post inoculation (dpi), the observation of morulae in granulocytes at 6 dpi, and the detection of A. phagocytophilum antibodies at 14 dpi. A. phagocytophilum was detected by PCR in skin, lung, small intestine, liver, spleen, uterus, bone marrow, and mesenteric lymph node from necropsies performed at 14 and 15 dpi. One sheep was infested during the acute phase of infection with I. ricinus nymphs from a pathogen-free colony. After molting, A. phagocytophilum transstadial transmission in ticks was validated with qPCR positive bacterial detection in 80% of salivary glands and 90% of midguts from female adults. Infected sheep blood collected at 14 dpi was demonstrated to be able to infect ISE6 tick cells, thus enabling the infection of two additional naive sheep, which then went on to develop similar clinical signs to the sheep infected previously. One of the sheep remained persistently infected until 115 dpi when it was euthanized, and transmitted bacteria to 70 and 2.7% of nymphs engorged as larvae during the acute and persistent infection stages, respectively. We then demonstrated that these infected nymphs were able to transmit the bacteria to one of two other naive infested sheep. As expected, when I. ricinus females were engorged during the acute phase of infection, no A. phagocytophilum transovarial transmission was detected. The development of this new experimental model will facilitate future research on this tick-borne bacterium of increasing importance, and enable the evaluation of any new tick/transmission control strategies.

Silencing of genes involved in Anaplasma marginale-tick interactions affects the pathogen developmental cycle in Dermacentor variabilis.

BACKGROUND: The cattle pathogen, Anaplasma marginale, undergoes a developmental cycle in ticks that begins in gut cells. Transmission to cattle occurs from salivary glands during a second tick feeding. At each site of development two forms of A. marginale (reticulated and dense) occur within a parasitophorous vacuole in the host cell cytoplasm. However, the role of tick genes in pathogen development is unknown. Four genes, found in previous studies to be differentially expressed in Dermacentor variabilis ticks in response to infection with A. marginale, were silenced by RNA interference (RNAi) to determine the effect of silencing on the A. marginale developmental cycle. These four genes encoded for putative glutathione S-transferase (GST), salivary selenoprotein M (SelM), H+ transporting lysosomal vacuolar proton pump (vATPase) and subolesin. RESULTS: The impact of gene knockdown on A. marginale tick infections, both after acquiring infection and after a second transmission feeding, was determined and studied by light microscopy. Silencing of these genes had a different impact on A. marginale development in different tick tissues by affecting infection levels, the densities of colonies containing reticulated or dense forms and tissue morphology. Salivary gland infections were not seen in any of the gene-silenced ticks, raising the question of whether these ticks were able to transmit the pathogen. CONCLUSION: The results of this RNAi and light microscopic analyses of tick tissues infected with A. marginale after the silencing of genes functionally important for pathogen development suggest a role for these molecules during pathogen life cycle in ticks.

Vaccination with recombinant Boophilus annulatus Bm86 ortholog protein, Ba86, protects cattle against B. annulatus and B. microplus infestations.

BACKGROUND: The cattle ticks, 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 B. microplus Bm86 protective antigen has been shown to protect cattle against tick infestations. Recently, the gene coding for B. annulatus Bm86 ortholog, Ba86, was cloned and the recombinant protein was secreted and purified from the yeast Pichia pastoris. RESULTS: Recombinant Ba86 (Israel strain) was used to immunize cattle to test its efficacy for the control of B. annulatus (Mercedes, Texas, USA strain) and B. microplus (Susceptible, Mexico strain) infestations. Bm86 (Gavac and Mozambique strain) and adjuvant/saline were used as positive and negative controls, respectively. Vaccination with Ba86 reduced tick infestations (71% and 40%), weight (8% and 15%), oviposition (22% and 5%) and egg fertility (25% and 50%) for B. annulatus and B. microplus, respectively. The efficacy of both Ba86 and Bm86 was higher for B. annulatus than for B. microplus. The efficacy of Ba86 was higher for B. annulatus (83.0%) than for B. microplus (71.5%). The efficacy of Bm86 (Gavac; 85.2%) but not Bm86 (Mozambique strain; 70.4%) was higher than that of Ba86 (71.5%) on B. microplus. However, the efficacy of Bm86 (both Gavac and Mozambique strain; 99.6%) was higher than that of Ba86 (83.0%) on B. annulatus. CONCLUSION: These experiments showed the efficacy of recombinant Ba86 for the control of B. annulatus and B. microplus infestations in cattle and suggested that physiological differences between B. microplus and B. annulatus and those encoded in the sequence of Bm86 orthologs may be responsible for the differences in susceptibility of these tick species to Bm86 vaccines.

Conservation and immunogenicity of the mosquito ortholog of the tick-protective antigen, subolesin.

The control of arthropod vectors of pathogens that affect human and animal health is important for the eradication of vector-borne diseases. The ortholog of the tick-protective antigen, subolesin, was identified in Aedes albopictus and found to have conserved epitopes in ticks and mosquitoes. RNA interference with the tick and mosquito double-stranded RNA in three tick species resulted in significant gene knockdown and decreased tick weight and/or survival. Feeding Anopheles atroparvus, Aedes caspius, and Culex pipiens female mosquitoes on an A. albopictus subolesin hyperimmune serum resulted in 11 +/- 5% to 29 +/- 6% survival inhibition when compared to controls fed on preimmune serum. Feeding sand flies, Phlebotomus perniciosus, on antimosquito subolesin ortholog protein antibodies inhibited female survival and the number of larvae and adults obtained after hatching by 28 +/- 22% and 16 +/- 3%, respectively, when compared to controls. Vaccination with tick and mosquito subolesin ortholog proteins significantly reduced Ixodes scapularis tick infestation and weight in a similar way. However, vaccination with the recombinant mosquito subolesin ortholog antigen did not protect against Amblyomma americanum and Rhipicephalus sanguineus tick infestations. Collectively, these preliminary results provided the first evidence that development of vaccines may be possible for control of multiple arthropod vectors using subolesin orthologs but suggested that multiple antigens may be required to produce an effective vaccine.

The leucokinin-like peptide receptor from the cattle fever tick, Rhipicephalus microplus, is localized in the midgut periphery and receptor silencing with validated double-stranded RNAs causes a reproductive fitness cost.

The cattle fever tick, Rhipicephalus microplus (Canestrini) (Acari: Ixodidae), is a one-host tick that infests primarily cattle in tropical and sub-tropical regions of the world. This species transmits deadly cattle pathogens, especially Babesia spp., for which a recombinant vaccine is not available. Therefore, disease control depends on tick vector control. Although R. microplus was eradicated in the USA, tick populations in Mexico and South America have acquired resistance to many of the applied acaricides. Recent acaricide-resistant tick reintroductions detected in the U.S. underscore the need for novel tick control methods. The octopamine and tyramine/octopamine receptors, both G protein-coupled receptors (GPCR), are believed to be the main molecular targets of the acaricide amitraz. This provides the proof of principle that investigating tick GPCRs, especially those that are invertebrate-specific, may be a feasible strategy for discovering novel targets and subsequently new anti-tick compounds. The R. microplus leucokinin-like peptide receptor (LKR), also known as the myokinin- or kinin receptor, is such a GPCR. While the receptor was previously characterized in vitro, the function of the leucokinin signaling system in ticks remains unknown. In this work, the LKR was immunolocalized to the periphery of the female midgut and silenced through RNA interference (RNAi) in females. To optimize RNAi experiments, a dual-luciferase system was developed to determine the silencing efficiency of LKR-double stranded RNA (dsRNA) constructs prior to testing those in ticks placed on cattle. This assay identified two effective dsRNAs. Silencing of the LKR with these two validated dsRNA constructs was verified by quantitative real time PCR (qRT-PCR) of female tick dissected tissues. Silencing was significant in midguts and carcasses. Silencing caused decreases in weights of egg masses and in the percentages of eggs hatched per egg mass, as well as delays in time to oviposition and egg hatching. A role of the kinin receptor in tick reproduction is apparent.

Detection of Rickettsia spp. in Rhipicephalus sanguineus (sensu lato) collected from free-roaming dogs in Coahuila state, northern Mexico.

BACKGROUND: The aim of this study was to detect and molecularly identify Rickettsia spp. in Rhipicephalus sanguineus (sensu lato) collected from free-roaming dogs in 30 communities from five municipalities in the south of Coahuila State, northern Mexico, where Rocky Mountain spotted fever is endemic. METHODS: In total, 60 dogs from each municipality were examined for engorged ticks. DNA was isolated from tick pools and conventional PCR assays targeting the 23S-5S ribosomal RNA intergenic spacer and outer membrane protein (ompA) gene of Rickettsia spp. were performed. RESULTS: All ticks (n = 1238) were morphologically identified as R. sanguineus (s.l.). Six pools (each with six engorged females) from four municipalities were positive to Rickettsia spp. DNA sequencing and phylogenetic analyses confirmed the presence of R. rickettsii and R. rhipicephali in R. sanguineus (s.l.) in these ticks. CONCLUSIONS: This study confirms the presence of R. rickettsii and R. rhipicephali in R. sanguineus (s.l.) from stray dogs in the south of Coahuila. This suggests that stray dogs may play a role in the inter-municipal dissemination of infected ticks in this region. Further research is required to assess whether ticks from stray dogs could serve as good indicators for the molecular xenomonitoring of R. rickettsii in this region. Considering that R. sanguineus (s.l.) is a proven vector of R. rickettsii in Mexico, increased awareness regarding permanent tick control in dogs is warranted.