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Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus) microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas. Raw data from bTEFAP were screened and trimmed based upon quality scores and binned into individual sample collections. Bacteria identified to the species level include Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus sciuri, Serratia marcescens, Corynebacterium glutamicum, and Finegoldia magna. One hundred twenty-one bacterial genera were detected in all the life stages and tissues sampled. The total number of genera identified by tick sample comprised: 53 in adult males, 61 in adult females, 11 in gut tissue, 7 in ovarian tissue, and 54 in the eggs. Notable genera detected in the cattle tick include Wolbachia, Coxiella, and Borrelia. The molecular approach applied in this study allowed us to assess the relative abundance of the microbiota associated with R. microplus. This report represents the first survey of the bacteriome in the cattle tick using non-culture based molecular approaches. Comparisons of our results with previous bacterial surveys provide an indication of geographic variation in the assemblages of bacteria associated with R. microplus. Additional reports on the identification of new bacterial species maintained in nature by R. microplus that may be pathogenic to its vertebrate hosts are expected as our understanding of its microbiota expands. Increased awareness of the role R. microplus can play in the transmission of pathogenic bacteria will enhance our ability to mitigate its economic impact on animal agriculture globally. This recognition should be included as part of analyses to assess the risk for re-invasion of areas like the United States of America where R. microplus was eradicated.

Background The horn fly, Haematobia irritans irritans , causes significant production losses to the cattle industry. Horn fly control relies on insecticides; however, alternative control methods such as vaccines are needed due to the fly's capacity to quickly develop resistance to insecticides, and the pressure for eco-friendly options. Methods We used a reverse vaccinology approach comprising three vaccine prediction and 11 annotation tools to evaluate and rank 79,542 translated open reading frames (ORFs) from the horn fly's transcriptome, and selected 10 transcript ORFs as vaccine candidates for expression in Pichia pastoris . The expression of the 10 selected transcripts and the proteins that they encoded were investigated in adult flies by reverse transcription polymerase chain reaction (RT-PCR) and mass spectrometry, respectively. Then, we evaluated the immunogenicity of a vaccine candidate in an immunization trial and the antigen’s effects on horn fly mortality and fecundity in an in vitro feeding assay. Results Six of the ten vaccine candidate antigens were successfully expressed in P. pastoris . RT-PCR confirmed the expression of all six ORFs in adult fly RNA. One of the vaccine candidate antigens, BI-HS009, was expressed in sufficient quantity for immunogenicity and efficacy trials. The IgG titers of animals vaccinated with BI-HS009 plus adjuvant were significantly higher than those of animals vaccinated with buffer plus adjuvant only from days 42 to 112, with a peak on day 56. Progeny of horn flies feeding upon blood from animals vaccinated with BI-HS009 plus adjuvant collected on day 56 had 63% lower pupariation rate and 57% lower adult emergence than the control group (ANOVA: F (1, 6)  = 8.221, P  = 0.028 and F (1, 6)  = 8.299, P  = 0.028, respectively). Conclusions The reverse vaccinology approach streamlined the discovery process by prioritizing possible vaccine antigen candidates. Through a thoughtful process of selection and in vivo and in vitro evaluations, we were able to identify a promising antigen for an anti-horn fly vaccine. Graphical abstract

Background Vaccination as a control method against the cattle tick, Rhipicephalus ( Boophilus ) microplus has been practiced since the introduction of two products in the mid-1990s. There is a need for a vaccine that could provide effective control of R. microplus in a more consistent fashion than existing products. During our transcriptome studies of R. microplus , several gene coding regions were discovered to encode proteins with significant amino acid similarity to aquaporins. Methods A cDNA encoding an aquaporin from the cattle tick, Rhipicephalus microplus , was isolated from transcriptomic studies conducted on gut tissues dissected from fully engorged adult female R. microplus . Results Bioinformatic analysis indicates this aquaporin, designated RmAQP1, shows greatest amino acid similarity to the human aquaporin 7 family. Members of this family of water-conducting channels can also facilitate the transport of glycerol in addition to water. The efficacy of this aquaporin as an antigen against the cattle tick was explored in cattle vaccine trials conducted in Brazil. A cDNA encoding a significant portion of RmAQP1 was expressed as a recombinant protein in Pichia pastoris , purified under native conditions using a polyhistidine C-terminus tag and nickel affinity chromatography, emulsified with Montanide adjuvant, and cattle vaccinated intramuscularly. The recombinant protein provided 75% and 68% efficacy in two cattle pen trials conducted in Campo Grande, Brazil on groups of 6 one year old Holstein calves. Conclusion The effectiveness of this vaccine in reducing the numbers of adult female ticks shows this aquaporin antigen holds promise as an active ingredient in cattle vaccines targeted against infestations of R. microplus .

This study was conducted to enhance the identification of novel targets to develop acaricides that can be used to advance integrated tick-borne disease management. Drivers for the emergence and re-emergence of tick-borne diseases affecting humans, livestock, and other domestic animals in many parts of the world include the increased abundance and expanded geographic distribution of tick species that vector pathogens. The evolution of resistance to acaricides among some of the most important tick vector species highlights the vulnerability of relying on chemical treatments for tick control to mitigate the health burden of tick-borne diseases. The involvement of inward rectifier potassium (Kir) channels in homeostasis, diuresis, and salivary gland secretion in ticks and other pests identified them as attractive targets to develop novel acaricides. However, few studies exist on the molecular characteristics of Kir channels in ticks. This bioinformatic analysis described Kir channels in 20 species of hard and soft ticks. Summarizing relevant investigations on Kir channel function in invertebrate pests allowed the phylogenomic study of this class of ion channels in ticks. How this information can be adapted to innovate tick control technologies is discussed.

Background Cattle babesiosis is a tick-borne disease of cattle that has severe economic impact on cattle producers throughout the world’s tropical and subtropical countries. The most severe form of the disease is caused by the apicomplexan, Babesia bovis, and transmitted to cattle through the bite of infected cattle ticks of the genus Rhipicephalus , with the most prevalent species being Rhipicephalus (Boophilus) microplus . We studied the reaction of the R. microplus larval transcriptome in response to infection by B. bovis . Methods Total RNA was isolated for both uninfected and Babesia bovis-infected larval samples. Subtracted libraries were prepared by subtracting the B. bovis-infected material with the uninfected material, thus enriching for expressed genes in the B. bovis-infected sample. Expressed sequence tags from the subtracted library were generated, assembled, and sequenced. To complement the subtracted library method, differential transcript expression between samples was also measured using custom high-density microarrays. The microarray probes were fabricated using oligonucleotides derived from the Bmi Gene Index database (Version 2). Array results were verified for three target genes by real-time PCR. Results Ticks were allowed to feed on a B. bovis -infected splenectomized calf and on an uninfected control calf. RNA was purified in duplicate from whole larvae and subtracted cDNA libraries were synthesized from Babesia -infected larval RNA, subtracting with the corresponding uninfected larval RNA. One thousand ESTs were sequenced from the larval library and the transcripts were annotated. We used a R. microplus microarray designed from a R. microplus gene index, BmiGI Version 2, to look for changes in gene expression that were associated with infection of R. microplus larvae. We found 24 transcripts were expressed at a statistically significant higher level in ticks feeding upon a B. bovis -infected calf contrasted to ticks feeding on an uninfected calf. Six transcripts were expressed at a statistically significant lower level in ticks feeding upon a B. bovis -infected calf contrasted to ticks feeding on an uninfected calf. Conclusion Our experimental approaches yielded specific differential gene expression associated with the infection of R. microplus by B. bovis . Overall, an unexpectedly low number of transcripts were found to be differentially expressed in response to B. bovis infection. Although the BmiGI Version 2 gene index ( http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=b_microplus ) was a useful database to help assign putative function to some transcripts, a majority of the differentially expressed transcripts did not have annotation that was useful for assignment of function and specialized bioinformatic approaches were necessary to increase the information from these transcriptome experiments.

Rhipicephalus (Boophilus) microplus is globally one of the most economically important ectoparasites of cattle costing the agriculture industry billions of dollars annually. Resistance to chemical control measures has prompted the development of novel methods of control. Recent advancements in genetic control measures for human and other animal vectors have utilized sex determination research to manipulate sex ratios, which have shown promising results in mosquitoes, namely Aedes aegypti and Anopheles stephensi. Here, we use R. (B.) microplus as a model to provide foundational research to allow similar avenues of investigation in ticks using R. (B.) microplus as a model. Karyotypes for R. (B.) microplus show an XX:XO sex determining system with the largest chromosome being the sex chromosome. Using flow cytometric methods, the size of the sex chromosome was estimated at 526.91 Mb. All measures to identify the sex chromosome within the cattle tick genome assembly associated sex chromosomal characteristics to 2 chromosomes. This discrepancy between the assembly and karyotypes of the tick led to generating a new genome assembly with a single adult male specimen. The 2 chromosomes in question aligned with a single scaffold within the new genome that had a length of 513.29 Mb and was the first time the sex chromosome was identified in an Ixodid genome assembly.

The cattle tick, Rhipicephalus microplus, is a serious pest of cattle, with significant economic consequences to the livestock industries of tropical and semitropical countries. Rhipicephalus microplus belongs to the Metastriata group of the Ixodidae family known as hard ticks. When adult hard ticks feed, mating has not yet occurred and an initial host attachment phase of 1–2 days is followed by a slow feeding phase that can last several days. Once mating occurs, feeding concludes with a rapid engorgement phase that is completed in 12–36 h. Our group’s interest in mining the genome and transcriptome of R. microplus for novel targets for development of tick control technologies led us to investigate the early transcriptional events occurring upon tick attachment and subsequent feeding. We placed newly molted unfed adult R. microplus females upon a bovine host and harvested the attached ticks after 3, 6, 12, and 24 h. We also placed a group of these ticks in a gas-permeable tube taped onto the side of the bovine host. These ticks were able to sense the host but unable to penetrate the tube to begin attachment and were ultimately harvested after 3 h. This study produced a comprehensive transcriptome from newly molted adult ticks and will provide a useful resource for studies of tick feeding and host perception and also assist genome annotation refinements.

BACKGROUND: Cattle babesiosis is a tick-borne disease of cattle with the most severe form of the disease caused by the apicomplexan, Babesia bovis. Babesiosis is transmitted to cattle through the bite of infected cattle ticks of the genus Rhipicephalus. The most prevalent species is Rhipicephalus (Boophilus) microplus, which is distributed throughout the tropical and subtropical countries of the world. The transmission of B. bovis is transovarian and a previous study of the R. microplus ovarian proteome identified several R. microplus proteins that were differentially expressed in response to infection. Through various approaches, we studied the reaction of the R. microplus ovarian transcriptome in response to infection by B. bovis. METHODS: A group of ticks were allowed to feed on a B. bovis-infected splenectomized calf while a second group fed on an uninfected splenectomized control calf. RNA was purified from dissected adult female ovaries of both infected and uninfected ticks and a subtracted B. bovis-infected cDNA library was synthesized, subtracting with the uninfected ovarian RNA. Four thousand ESTs were sequenced from the ovary subtracted library and annotated. RESULTS: The subtracted library dataset assembled into 727 unique contigs and 2,161 singletons for a total of 2,888 unigenes, Microarray experiments designed to detect B. bovis-induced gene expression changes indicated at least 15 transcripts were expressed at a higher level in ovaries from ticks feeding upon the B. bovis-infected calf as compared with ovaries from ticks feeding on an uninfected calf. We did not detect any transcripts from these microarray experiments that were expressed at a lower level in the infected ovaries compared with the uninfected ovaries. Using the technique called serial analysis of gene expression, 41 ovarian transcripts from infected ticks were differentially expressed when compared with transcripts of controls. CONCLUSION: Collectively, our experimental approaches provide the first comprehensive profile of the R. microplus ovarian transcriptome responding to infection by B. bovis. This dataset should prove useful in molecular studies of host-pathogen interactions between this tick and its apicomplexan parasite.

Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus) microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas. Raw data from bTEFAP were screened and trimmed based upon quality scores and binned into individual sample collections. Bacteria identified to the species level include Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus sciuri, Serratia marcescens, Corynebacterium glutamicum, and Finegoldia magna. One hundred twenty-one bacterial genera were detected in all the life stages and tissues sampled. The total number of genera identified by tick sample comprised: 53 in adult males, 61 in adult females, 11 in gut tissue, 7 in ovarian tissue, and 54 in the eggs. Notable genera detected in the cattle tick include Wolbachia, Coxiella, and Borrelia. The molecular approach applied in this study allowed us to assess the relative abundance of the microbiota associated with R. microplus. This report represents the first survey of the bacteriome in the cattle tick using non-culture based molecular approaches. Comparisons of our results with previous bacterial surveys provide an indication of geographic variation in the assemblages of bacteria associated with R. microplus. Additional reports on the identification of new bacterial species maintained in nature by R. microplus that may be pathogenic to its vertebrate hosts are expected as our understanding of its microbiota expands. Increased awareness of the role R. microplus can play in the transmission of pathogenic bacteria will enhance our ability to mitigate its economic impact on animal agriculture globally. This recognition should be included as part of analyses to assess the risk for re-invasion of areas like the United States of America where R. microplus was eradicated.

Background Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus ( Boophilus ) microplus , hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas. Results Raw data from bTEFAP were screened and trimmed based upon quality scores and binned into individual sample collections. Bacteria identified to the species level include Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus sciuri, Serratia marcescens, Corynebacterium glutamicum , and Finegoldia magna . One hundred twenty-one bacterial genera were detected in all the life stages and tissues sampled. The total number of genera identified by tick sample comprised: 53 in adult males, 61 in adult females, 11 in gut tissue, 7 in ovarian tissue, and 54 in the eggs. Notable genera detected in the cattle tick include Wolbachia , Coxiella , and Borrelia . The molecular approach applied in this study allowed us to assess the relative abundance of the microbiota associated with R. microplus . Conclusions This report represents the first survey of the bacteriome in the cattle tick using non-culture based molecular approaches. Comparisons of our results with previous bacterial surveys provide an indication of geographic variation in the assemblages of bacteria associated with R. microplus . Additional reports on the identification of new bacterial species maintained in nature by R. microplus that may be pathogenic to its vertebrate hosts are expected as our understanding of its microbiota expands. Increased awareness of the role R. microplus can play in the transmission of pathogenic bacteria will enhance our ability to mitigate its economic impact on animal agriculture globally. This recognition should be included as part of analyses to assess the risk for re-invasion of areas like the United States of America where R. microplus was eradicated.