Isolation and Molecular Characterization of Tick-Borne Relapsing Fever Borrelia Infecting Ornithodoros (Pavlovskyella) verrucosus Ticks Collected in Ukraine.

BACKGROUND: Tick-borne relapsing fever (TBRF) is a neglected zoonotic bacterial disease known to occur on 5 continents. We report a laboratory-acquired case of TBRF caused by Borrelia caucasica, which is endemic in Ukraine and transmitted by Ornithodoros verrucosus ticks. METHODS: We isolated spirochetes and characterized them by partially sequencing the 16s ribosomal ribonucleic acid (rrs), flagellin (flaB), and deoxyribonucleic acid gyrase (gyrB) genes and conducting a phylogenetic analysis. RESULTS: These analyses revealed a close relationship of Ukrainian spirochetes with the Asian TBRF species, Borrelia persica. The taxonomic and nomenclature problems related to insufficient knowledge on the spirochetes and their vectors in the region are discussed. CONCLUSIONS: Although these findings enhance our understanding of species identities for TBRF Borrelia in Eurasia, further work is required to address the neglected status of TBRF in this part of the world. Public health practitioners should consider TBRF and include the disease into differential diagnosis of febrile illnesses with unknown etiology.

Integrative taxonomic description of the chewing louse Tricholipeurus lipeuroides infesting Odocoileus virginianus veraecrucis white-tailed deer in Veracruz, Mexico.

Chewing lice (Phthiraptera: Amblycera, Ischnocera) represent a component of the ectoparasite fauna associated with large sized mammals as deers. However, the diversity of chewing louse species infesting deer remains to be fully characterized in the Neotropics. Little is known about the chewing lice infesting the extant fourteen subspecies of white-tailed deer (Odocoileus virginianus) in Mexico. Known to infest white-tailed deer (WTD) in Canada and the United States (U.S.), Tricholipeurus lipeuroides is a chewing louse species that was originally described in the nineteenth century infesting O. v. mexicanus in Mexico. For the first time, infestation of O. v. veraecrucis, a Neotropical WTD subspecies in Mexico, with T. lipeuroides is reported herein. An integrative taxonomic approach was taken by combining morphological and molecular analyses to describe the T. lipeuroides infestion of O. v. veraecrucis. Ecological parameters of the T. lipeuroides infestations were also calculated. The prevalence was 91.7% of the 56 O. v. veraecrucis (29 females and 27 males) inspected while under chemical restraint that were sampled at 3 sites in the central region of Veracruz state in Mexico. The amplification and sequencing of previously reported T. lipeuroides Cytochrome Oxidase Subunit I gene confirmed the identity of all the chewing louse life stages. These results are discussed in the context of comparative analyses on the emergence of novel chewing lice-deer associations.

Multistate Infestation with the Exotic Disease-Vector Tick Haemaphysalis longicornis - United States, August 2017-September 2018.

Haemaphysalis longicornis is a tick indigenous to eastern Asia and an important vector of human and animal disease agents, resulting in such outcomes as human hemorrhagic fever and reduction of production in dairy cattle by 25%. H. longicornis was discovered on a sheep in New Jersey in August 2017 (1). This was the first detection in the United States outside of quarantine. In the spring of 2018, the tick was again detected at the index site, and later, in other counties in New Jersey, in seven other states in the eastern United States, and in Arkansas. The hosts included six species of domestic animals, six species of wildlife, and humans. To forestall adverse consequences in humans, pets, livestock, and wildlife, several critical actions are indicated, including expanded surveillance to determine the evolving distribution of H. longicornis, detection of pathogens that H. longicornis currently harbors, determination of the capacity of H. longicornis to serve as a vector for a range of potential pathogens, and evaluation of effective agents and methods for the control of H. longicornis.

Imaging of Borrelia turicatae Producing the Green Fluorescent Protein Reveals Persistent Colonization of the Ornithodoros turicata Midgut and Salivary Glands from Nymphal Acquisition through Transmission.

Relapsing fever (RF) spirochetes colonize and are transmitted to mammals primarily by Ornithodoros ticks, and little is known regarding the pathogen's life cycle in the vector. To further understand vector colonization and transmission of RF spirochetes, Borrelia turicatae expressing a green fluorescent protein (GFP) marker (B. turicatae-gfp) was generated. The transformants were evaluated during the tick-mammal infectious cycle, from the third nymphal instar to adult stage. B. turicatae-gfp remained viable for at least 18 months in starved fourth-stage nymphal ticks, and the studies indicated that spirochete populations persistently colonized the tick midgut and salivary glands. Our generation of B. turicatae-gfp also revealed that within the salivary glands, spirochetes are localized in the ducts and lumen of acini, and after tick feeding, the tissues remained populated with spirochetes. The B. turicatae-gfp generated in this study is an important tool to further understand and define the mechanisms of vector colonization and transmission.IMPORTANCE In order to interrupt the infectious cycle of tick-borne relapsing fever spirochetes, it is important to enhance our understanding of vector colonization and transmission. Toward this, we generated a strain of Borrelia turicatae that constitutively produced the green fluorescent protein, and we evaluated fluorescing spirochetes during the entire infectious cycle. We determined that the midgut and salivary glands of Ornithodoros turicata ticks maintain the pathogens throughout the vector's life cycle and remain colonized with the spirochetes for at least 18 months. We also determined that the tick's salivary glands were not depleted after a transmission blood feeding. These findings set the framework to further understand the mechanisms of midgut and salivary gland colonization.

Evolutionary changes in symbiont community structure in ticks.

Ecological specialization to restricted diet niches is driven by obligate, and often maternally inherited, symbionts in many arthropod lineages. These heritable symbionts typically form evolutionarily stable associations with arthropods that can last for millions of years. Ticks were recently found to harbour such an obligate symbiont, Coxiella-LE, that synthesizes B vitamins and cofactors not obtained in sufficient quantities from blood diet. In this study, the examination of 81 tick species shows that some Coxiella-LE symbioses are evolutionarily stable with an ancient acquisition followed by codiversification as observed in ticks belonging to the Rhipicephalus genus. However, many other Coxiella-LE symbioses are characterized by low evolutionary stability with frequent host shifts and extinction events. Further examination revealed the presence of nine other genera of maternally inherited bacteria in ticks. Although these nine symbionts were primarily thought to be facultative, their distribution among tick species rather suggests that at least four may have independently replaced Coxiella-LE and likely represent alternative obligate symbionts. Phylogenetic evidence otherwise indicates that cocladogenesis is globally rare in these symbioses as most originate via horizontal transfer of an existing symbiont between unrelated tick species. As a result, the structure of these symbiont communities is not fixed and stable across the tick phylogeny. Most importantly, the symbiont communities commonly reach high levels of diversity with up to six unrelated maternally inherited bacteria coexisting within host species. We further conjecture that interactions among coexisting symbionts are pivotal drivers of community structure both among and within tick species.

Role of inward rectifier potassium channels in salivary gland function and sugar feeding of the fruit fly, Drosophila melanogaster.

The arthropod salivary gland is of critical importance for horizontal transmission of pathogens, yet a detailed understanding of the ion conductance pathways responsible for saliva production and excretion is lacking. A superfamily of potassium ion channels, known as inward rectifying potassium (Kir) channels, is overexpressed in the Drosophila salivary gland by 32-fold when compared to the whole body mRNA transcripts. Therefore, we aimed to test the hypothesis that pharmacological and genetic depletion of salivary gland specific Kir channels alters the efficiency of the gland and reduced feeding capabilities using the fruit fly Drosophila melanogaster as a model organism that could predict similar effects in arthropod disease vectors. Exposure to VU041, a selective Kir channel blocker, reduced the volume of sucrose consumption by up to 3.2-fold and was found to be concentration-dependent with an EC50 of 68µM. Importantly, the inactive analog, VU937, was shown to not influence feeding, suggesting the reduction in feeding observed with VU041 is due to Kir channel inhibition. Next, we performed a salivary gland specific knockdown of Kir1 to assess the role of these channels specifically in the salivary gland. The genetically depleted fruit flies had a reduction in total volume ingested and an increase in the time spent feeding, both suggestive of a reduction in salivary gland function. Furthermore, a compensatory mechanism appears to be present at day 1 of RNAi-treated fruit flies, and is likely to be the Na+-K+-2Cl- cotransporter and/or Na+-K+-ATPase pumps that serve to supplement the inward flow of K+ ions, which highlights the functional redundancy in control of ion flux in the salivary glands. These findings suggest that Kir channels likely provide, at least in part, a principal potassium conductance pathway in the Drosophila salivary gland that is required for sucrose feeding.

Prevalence of Cryptosporidium in small ruminants from Veracruz, Mexico.

BACKGROUND: Cryptosporidiosis is a zoonotic disease caused by the protozoan parasite Cryptosporidium spp. that can affect domestic animal and human populations. In newborn ruminants, cryptosporidiosis is characterized by outbreaks of diarrhea, which can result in high morbidity and economic impact. The aim of the present study was to determine the prevalence of Cryptosporidium spp. in small ruminants from the Perote municipality in Veracruz State, Mexico. One hundred and sixty small ruminants (80 sheep and 80 goats) from eight farms located in four towns of the Perote municipality were examined following a cross-sectional study design. Stool samples were analyzed by a modification of the Faust centrifugation method, and the presence of Cryptosporidium spp. oocysts was examined using a modification of the Ziehl-Neelsen staining procedure. Bivariate and multivariate analyses were used to assess the association of Cryptosporidium infection and the general characteristics of the animals studied. RESULTS: Overall, 112 (70%, 95% CI: 62.3-76.9) of the 160 small ruminants sampled were infected with Cryptosporidium spp. The prevalence of Cryptosporidium spp. infection in goats was 72.5% (95% CI: 61.4-81.9) and in sheep 67.5% (95% CI: 56.1-77.6). Small ruminants aged 1 month old had the highest (88.2%; 95% CI: 63.6-98.5) prevalence of infection. Prevalence varied from 60% to 85% among herds. Animal species, age, sex, breed, farm, town or cohabitation with cattle did not influence the prevalence of Cryptosporidium infection. CONCLUSIONS: A high prevalence of infection with Cryptosporidium spp. was observed in small ruminants from the Perote municipality in Veracruz, Mexico. Infection was widely distributed among sheep and goats regardless of their age, breed or farm location. Further research is required to identify risk factors for, and to assess the veterinary public health significance of Cryptosporidium infection among sheep and goats in the Mexican state of Veracruz.

Surface Polar Lipids Differ in Male and Female Phlebotomus papatasi (Diptera: Psychodidae).

The polar lipids on the surface of the Old World sand fly, Phlebotomus papatasi (Scopoli), were analyzed by high-resolution mass spectrometry. Blood-fed females and nonblood-fed females and males were separately analyzed and compared. The major polar lipids were found to be long-chain diols and fatty acids. Relatively high levels of diacylglycerols were found in blood-fed females and in males. A wide variety of lipids were found at low levels, including esters, sterols, monoacylglycerols, and hydroxy fatty acids. Blood-fed females had several lyso lipids and N-acyl amino acids that were not found on unfed females or males. These substances may be surfactants used in blood feeding. Heneicosenoic acid was found on females at more than twice the level of males, suggesting it could be a component of a female pheromone. Four substances were identified on males at twofold higher levels than on females: tetradienoic acid, methoxyhexadecasphinganine, butyl octadecanoate, and diacylglycerol(14:1/12:0/0:0). These could be short-range pheromones involved in courtship, and they will be further analyzed in future behavioral bioassays.

Seroprevalence of Toxoplasma gondii infection in water buffaloes (Bubalus bubalis) in Veracruz State, Mexico and its association with climatic factors.

BACKGROUND: Infection with Toxoplasma gondii in water buffaloes (Bubalus bubalis) is of epidemiological importance because of the risk for transmission to humans. We sought to determine the seroprevalence of T. gondii infection in 339 water buffaloes in Veracruz State, Mexico using the modified agglutination test (MAT, cut off 1:25). Seroprevalence association with general characteristics of buffaloes and their environment was also investigated. RESULTS: Antibodies to T. gondii were found in 165 (48.7%) of the 339 buffaloes with MAT titers of 1:25 in 104, 1:50 in 52, and 1:100 in 9. Bivariate analysis showed that seroprevalence of T. gondii infection was similar in buffaloes regardless of their general characteristics i.e., age, sex, and breed. In contrast, the seroprevalence in buffaloes varied significantly with environmental characteristics including altitude, mean annual temperature, and mean annual rainfall of the municipalities studied. Multivariate analysis showed that T. gondii seropositivity in buffaloes was associated with a mean annual rainfall between 1266-1650 mm (OR = 1.84; 95% CI: 1.15-2.94; P = 0.01). CONCLUSIONS: Results indicate that environmental characteristics may influence the seroprevalence of T. gondii infection in buffaloes. This is the first report on the seroprevalence of T. gondii infection in buffaloes in Mexico. Further research is needed to assess the risk for infection in humans associated with the ingestion of raw or undercooked meat from buffaloes infected with T. gondii.

High prevalence of Toxoplasma gondii antibodies in dogs in Veracruz, Mexico.

BACKGROUND: Little is known concerning the prevalence of Toxoplasma gondii infection in dogs in Mexico. Here, we investigated antibodies to T. gondii and associated risk factors in 101 dogs from an animal shelter in Veracruz State, Mexico. Canine sera were assayed for T. gondii IgG antibodies by using the modified agglutination test (MAT, cut off 1:25). RESULTS: Sixty eight (67.3%) of 101 dogs were seropositive with titers of 1:25 in 16, 1:50 in 8, 1:100 in 9, 1:200 in 10, 1:400 in 10, 1:800 in 10, 1:1600 in 3, and 1:3200 or higher in 2. None of the dogs' characteristics studied including age, sex, breed, and history of deworming, rabies vaccination and contact with cats was associated with seroprevalence of T. gondii infection. CONCLUSION: Using the dogs as sentinel animals, the results indicate high contamination with T. gondii of the environment in Veracruz, Mexico. Results have public health implications, and further studies in Veracruz should be conducted to establish the sources of environmental contamination with T. gondii and to determine optimal preventive measures against T. gondii infection in humans.

In Vitro Evaluation of Essential Oils and Saturated Fatty Acids for Repellency against the Old-World Sand Fly, Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae).

The sand fly, Phlebotomus papatasi (Scopoli, 1786), is a major vector for Leishmania major in the Middle East, which has impacted human health and US military operations in the area, demonstrating the need to develop effective sand fly control and repellent options. Here, we report the results of spatial repellency and avoidance experiments in a static air olfactometer using the female P. papatasi testing essential oils of Lippia graveolens (Mexican oregano), Pimenta dioica (allspice), Amyris balsamifera (amyris), Nepeta cataria (catnip), Mentha piperita (peppermint), and Melaleuca alternifolia (tea tree); the 9-12 carbon saturated fatty acids (nonanoic acid, decanoic acid, undecanoic acid, and dodecanoic acid); and the synthetic repellents DEET and IR3535. The materials applied at 1% exhibited varying activity levels but were not significantly different in mean repellency and avoidance from DEET and IR3535, except in regards to nonanoic acid. Some materials, particularly nonanoic and undecanoic acids, produced sand fly mortality. The observed trends in mean repellency over exposure time included the following: (1) P. dioica oil, M. alternifolia oil, decanoic acid, undecanoic acid, DEET, and IR3535 exhibited increasing mean repellency over time; (2) oils of N. cataria, A. balsamifera, M. piperita, and dodecanoic acid exhibited relatively constant mean repellency over time; and (3) L. graveolens oil and nonanoic acid exhibited a general decrease in mean repellent activity over time. These studies identified the essential oils of N. cataria and A. balsamifera as effective spatial repellents at reduced concentrations compared to those of DEET. Additional research is required to elucidate the modes of action and potential synergism of repellents and essential oil components for enhanced repellency activity.

Discovery of microRNAs of the stable fly (Diptera: Muscidae) by High-throughput sequencing.

The stable fly, Stomoxys calcitrans (L.), is a serious ectoparasite affecting animal production and health of both animals and humans. Stable fly control relies largely on chemical insecticides; however, the development of insecticide resistance as well as environmental considerations requires continued discovery research to develop novel control technologies. MicroRNAs (miRNAs) are a class of short noncoding RNAs that have been shown to be important regulators of gene expression across a wide variety of organisms, and may provide an innovative approach with regard to development of safer more targeted control technologies. The current study reports discovery ad initial comparative analysis of 88 presumptive miRNA sequences from the stable fly, obtained using high-throughput sequencing of small RNAs. The majority of stable fly miRNAs were 22-23 nt in length. Many miRNAs were arthropod specific, and several mature miRNA sequences showed greater sequence identity to miRNAs from other blood-feeding dipterans such as mosquitoes rather than to Drosophilids. This initial step in characterizing the stable fly microRNAome provides a basis for further analyses of life stage-specific and tissue-specific expression to elucidate their functional roles in stable fly biology.

Inhibitor profile of bis(n)-tacrines and N-methylcarbamates on acetylcholinesterase from Rhipicephalus (Boophilus) microplus and Phlebotomus papatasi.

The cattle tick, Rhipicephalus (Boophilus) microplus (Bm), and the sand fly, Phlebotomus papatasi (Pp), are disease vectors to cattle and humans, respectively. The purpose of this study was to characterize the inhibitor profile of acetylcholinesterases from Bm (BmAChE1) and Pp (PpAChE) compared to human and bovine AChE, in order to identify divergent pharmacology that might lead to selective inhibitors. Results indicate that BmAChE has low sensitivity (IC50 = 200 µM) toward tacrine, a monovalent catalytic site inhibitor with sub micromolar blocking potency in all previous species tested. Similarly, a series of bis(n)-tacrine dimer series, bivalent inhibitors and peripheral site AChE inhibitors possess poor potency toward BmAChE. Molecular homology models suggest the rBmAChE enzyme possesses a W384F orthologous substitution near the catalytic site, where the larger tryptophan side chain obstructs the access of larger ligands to the active site, but functional analysis of this mutation suggests it only partially explains the low sensitivity to tacrine. In addition, BmAChE1 and PpAChE have low nanomolar sensitivity to some experimental carbamate anticholinesterases originally designed for control of the malaria mosquito, Anopheles gambiae. One experimental compound, 2-((2-ethylbutyl)thio)phenyl methylcarbamate, possesses >300-fold selectivity for BmAChE1 and PpAChE over human AChE, and a mouse oral LD50 of >1500 mg/kg, thus providing an excellent new lead for vector control.

First Report of Eratyrus cuspidatus (Hemiptera: Reduviidae) Infected with Trypanosoma cruzi in Peridomestic Environment in Chiapas, Mexico.

Background: Triatomine bugs are natural vectors of Trypanosoma cruzi, which causes Chagas disease or American trypanosomiasis. The role of sylvatic triatomine species as vectors of T. cruzi in Mexico remains to be fully understood. Our research on the epidemiology of Chagas disease in Southeastern Mexico involved sampling triatomines in rural settings. Materials and Methods: A triatomine was collected in a peridomestic environment of a rural dwelling in the state of Chiapas. The triatomine was identified morphologically as an adult female Eratyrus cuspidatus Stal. Results: Microscopic analysis revealed flagellate forms of T. cruzi in the feces of the E. cuspidatus collected. This was confirmed by quantitative polymerase chain reaction. Amplification of the mini-exon gene showed that the T. cruzi infecting E. cuspidatus corresponded to lineage I. Conclusions: This is the first report from Mexico of E. cuspidatus found infected in a human dwelling, which represents an important adaptation process to inhabit human environments.

Odorant-binding protein from the stable fly (Stomoxys calcitrans) has a high-histidine N-terminal extension that binds transition metals.

The role of odorant- and pheromone-binding proteins (OBPs) in olfactory function is not fully understood. We found an OBP sequence from the stable fly, Stomoxys calcitrans, ScalOBP60, that has a 25 amino acid N-terminal extension with a high content of histidine and acidic amino acids, suggesting a possible metal binding activity. A search of public databases revealed a large number of other fly OBPs with histidine-rich N-terminal extensions, as well as beetle, wasp and ant OBPs with histidine-rich C-terminal extensions. We recombinantly expressed ScalOBP60, as well as a truncated sequence which lacks the histidine-rich N-terminal region, tScalOBP60. Using fluorescence quenching and electrospray quadrupole time-of-flight mass spectrometry (ESI-QTOF), we detected two different types of metal-binding sites. Divalent copper, nickel and zinc bind to the N-terminal histidine-rich region, and divalent copper binds to an internal sequence position. Comparison of the ESI-QTOF spectra of ScalOBP60 and tScalOBP60 showed that the histidine-rich sequence is structurally disordered, but it becomes more ordered in the presence of divalent metal. When copper is bound to the internal site, binding of a hydrophobic ligand to ScalOBP60 is inhibited. The internal and N-terminal metal sites interact allosterically, possibly through a conformational equilibrium, suggesting a mechanism for metal regulation of ligand binding to ScalOBP60. Based on our studies of ScalOBP60, we propose several possible olfactory and non-olfactory functions for this OBP.

Machine learning and analysis of genomic diversity of "Candidatus Liberibacter asiaticus" strains from 20 citrus production states in Mexico.

Background: Huanglongbing (HLB, yellow shoot disease) is a highly destructive citrus disease associated with a nonculturable bacterium, "Candidatus Liberibacter asiaticus" (CLas), which is transmitted by Asian citrus psyllid (ACP, Diaphorina citri). In Mexico, HLB was first reported in Tizimin, Yucatán, in 2009 and is now endemic in 351 municipalities of 25 states. Understanding the population diversity of CLas is critical for HLB management. Current CLas diversity research is exclusively based on analysis of the bacterial genome, which composed two regions, chromosome (> 1,000 genes) and prophage (about 40 genes). Methods and results: In this study, 40 CLas-infected ACP samples from 20 states in Mexico were collected. CLas was detected and confirmed by PCR assays. A prophage gene(terL)-based typing system (TTS) divided the Mexican CLas strains into two groups: Term-G including four strains from Yucatán and Chiapas, as well as strain psy62 from Florida, USA, and Term-A included all other 36 Mexican strains, as well as strain AHCA1 from California, USA. CLas diversity was further evaluated to include all chromosomal and prophage genes assisted by using machine learning (ML) tools to resolve multidimensional data handling issues. A Term-G strain (YTMX) and a Term-A strain (BCSMX) were sequenced and analyzed. The two Mexican genome sequences along with the CLas genome sequences available in GenBank were studied. An unsupervised ML was implemented through principal component analysis (PCA) on average nucleotide identities (ANIs) of CLas whole genome sequences; And a supervised ML was implemented through sparse partial least squares discriminant analysis (sPLS-DA) on single nucleotide polymorphisms (SNPs) of coding genes of CLas guided by the TTS. Two CLas Geno-groups, Geno-group 1 that extended Term-A and Geno-group 2 that extended Term-G, were established. Conclusions: This study concluded that: 1) there were at least two different introductions of CLas into Mexico; 2) CLas strains between Mexico and USA are closely related; and 3) The two Geno-groups provide the basis for future CLas subspecies research.

Geographic Population Genetic Structure of the New World Screwworm, Cochliomyia hominivorax (Diptera: Calliphoridae), Using SNPs.

The New World screwworm, Cochliomyia hominivorax (Coquerel 1858) (Diptera: Calliphoridae), is a serious parasite of livestock, humans, and other warm-blooded animals. It has been eradicated from the northern parts of its historical range down to the Panama-Colombian border where a permanent barrier zone is maintained. This eradication was accomplished through using the sterile insect technique (SIT). In 2016 there was an outbreak of C. hominivorax in the Florida Keys. In only six months, this pest was successfully re-eradicated using SIT, but the geographic origin of the invasion has yet to be resolved. It was previously determined that the Florida flies most likely represented a single invasion, and it was recommended that a finer-scale genetic assessment should be completed. Thus, this current proof-of-concept study aimed to develop a population genetic database using single nucleotide polymorphisms (SNPs) to reference outbreaks and potentially identify the origin of the Florida outbreak. This initial database consists of wild-caught samples from 4 geographic locations as well as laboratory colony samples that originated from 7 additional locations using a genotyping by sequencing (GBS) approach. Geographic population structuring was identified for twelve populations that clustered according to geographic location. The Florida outbreak samples appeared similar to samples from the outer Caribbean cluster which included samples from Dominican Republic and Trinidad and Tobago, however, these results will be further clarified with the replacement of laboratory colony samples with future wild-caught samples.

Interaction between anti-tick vaccine and a macrocyclic lactone improves acaricidal efficacy against Rhipicephalus (Boophilus) microplus (Canestrini) (Acari: Ixodidae) in experimentally infested cattle.

The southern cattle fever tick (SCFT) Rhipicephalus (Boophilus) microplus, is considered the most important ectoparasite of livestock in the world because of high financial losses associated with direct feeding and transmission of the hemoparasites Babesia bovis, B. bigemina, and Anaplasma marginale. Unfortunately, SCFT in many parts of the world have evolved resistance to all market-available pesticides thus driving development of new control technologies. Vaccination against ticks using the tick gut protein Bm86 has been shown to be effective against acaricide-resistant ticks. This technique has been successfully implemented in Puerto Rico for the control of acaricide-resistant R. microplus on dairy and beef cattle. Observations from Puerto Rico indicate a potentially positive interaction between anti-tick vaccination when used in conjunction with systemic acaricide treatment. In this project, controlled animal studies were completed directly comparing efficacy of anti-tick vaccination with and without systemic acaricide. Results show that the Bm86 anti-tick vaccine in combination with the macrocyclic lactone, Moxidectin, expressed a synergistic interaction, providing greater and longer efficacy than either treatment alone.

Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing.

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.