Nilgai antelope display no signs of infection upon experimental challenge with a virulent Babesia bovis strain.

BACKGROUND: Bovine babesiosis is caused by infection with the protozoal parasite Babesia bovis, which is transmitted by Rhipicephalus (Boophilus) spp. It can cause mortality rates up to 90% in immunologically naive Bos taurus cattle. In south Texas, R. (B.) microplus is known to infest nilgai antelope (Boselaphus tragocamelus); however, their susceptibility to infection with B. bovis and their role in the transmission of the parasite remain unknown. In this study, we challenged nilgai antelope with B. bovis and evaluated their susceptibility to infection. METHODS: Nilgai were needle inoculated with ≈108 B. bovis-parasitized erythrocytes (merozoites) or a homogenate of B. bovis-infected larval ticks (sporozoite) delivered intravenously. Bos taurus beef calves were inoculated in parallel, as this strain of B. bovis is lethal to cattle. Temperature and hematocrit were monitored daily over the course of each study, and whole blood was collected for molecular [polymerase chain reaction (PCR)] and serological [indirect enzyme-linked immunosorbent assay (ELISA)] diagnostic evaluation. Histological sections of nilgai cerebral tissue were examined for evidence of infection. Recipient bovine calves were sub-inoculated with blood from nilgai challenged with either stage of the parasite, and they were monitored for clinical signs of infection and evaluated by a PCR diagnostic assay. Red blood cells (RBCs) from prechallenged nilgai and B. taurus beef cattle were cultured with an in vitro B. bovis merozoite culture to examine colonization of the RBCs by the parasite. RESULTS: Nilgai did not display clinical signs of infection upon inoculation with either the merozoite or sporozoite stage of B. bovis. All nilgai were PCR-negative for the parasite, and they did not develop antibodies to B. bovis. No evidence of infection was detected in histological sections of nilgai tissues, and in vitro culture analysis indicated that the nilgai RBCs were not colonized by B. bovis merozoites. Cattle subinoculated with blood from challenged nilgai did not display clinical signs of infection, and they were PCR-negative up to 45 days after transfer. CONCLUSIONS: Nilgai do not appear to be susceptible to infection with a strain of B. bovis that is lethal to cattle. Tick control on these alternative hosts remains a critical priority, especially given their potential to disseminate ticks over long distances.

Developmental stage and level of submersion in water impact the viability of lone star and winter tick eggs during flooding.

Female ticks deposit large egg clusters that range in size from hundreds to thousands. These clusters are restricted to a deposition site, usually under leaf litter and other debris. These sites can be exposed to periodic flooding, where the cluster of tick eggs can float to the surface or remain underneath organic debris entirely underwater. Here, we examined the viability of egg clusters from winter ticks, Dermacentor albipictus , and lone star ticks, Amblyomma americanum , when partially submerged or fully submerged in water in relation to the developmental stages of the eggs. In general, egg clusters that were older and partially submerged had a higher viability than fully submerged, young eggs in water. A. americanum was much more resistant to water exposure between the two species. These studies highlight that egg clusters for specific tick species can remain viable when exposed to water for at least two weeks, where eggs float on the surface. These studies also suggest that water-based distribution of egg clusters could occur for some species, and flooding will differentially impact tick egg survival based on the specific developmental stage of exposure and species.

Feeding and reproductive parameters of adult female Ixodes scapularis (Acari: Ixodidae) and Amblyomma americanum parasitizing white-tailed deer (Odocoileus virginianus).

White-tailed deer Odocoileus virginianus (Zimmermann) (Artiodactyla: Cervidae) are the main host for adult Ixodes scapularis Say (Acari: Ixodidae) (blacklegged tick) and all stages of Amblyomma americanum Linnaeus (Acari: Ixodidae) (lone star tick). However, literature describing the feeding and reproductive parameters of these tick species when feeding on this host is limited. We experimentally infested white-tailed deer with adult pairs of either I. scapularis or A. americanum to improve our understanding of these tick-host relationships. Our study used tick-naïve white-tailed deer and restricted host grooming throughout the infestation. For I. scapularis, the days to repletion (mean ±â€…SE, 6.04 ±â€…0.07), engorgement weight of replete females (0.20 ±â€…0.0032 g), duration of oviposition (32 ±â€…0.45 d), egg mass weight (0.10 ±â€…0.0027 g), and number of eggs laid per tick (1,803.00 ±â€…49.00) were recorded. Data from A. americanum were also recorded, including days to repletion (11.00 ±â€…0.063), engorgement weight of replete females (0.63 ±â€…0.025 g), duration of oviposition (37.00 ±â€…1.30 d), egg mass weight (0.34 ±â€…0.017 g), and number of eggs laid per tick (5,873.00 ±â€…291.00). These biological parameter data could be used as variables in models (e.g., LYMESIM 2.0) to determine how white-tailed deer influence I. scapularis and A. americanum populations in nature, and to evaluate the protective efficacy of tick-antigen-based antitick vaccines.

Artificial infestation of white-tailed deer with ticks (Acari: Ixodidae) to study tick-host interactions.

White-tailed deer (Odocoileus virginianus) are a main host for the adult life stages of tick species of medical and veterinary importance. Since white-tailed deer play a vital role in tick ecology, research has been conducted to understand this tick-host relationship. To date, research involving captive white-tailed deer and artificial infestation of these animals with ticks has focused on host suitability, the role of white-tailed deer in tick-borne diseases, and anti-tick vaccine research. The methodology reported for these studies was at times not descriptive and inconsistent regarding how and what region of the white-tailed deer was infested with ticks. Here, we propose a standardized method to artificially infest captive white-tailed deer with ticks for research purposes. The protocol describes a method proven effective to experimentally infest captive white-tailed deer with blacklegged ticks (Ixodes scapularis) to study tick-host interactions. The methods can be reliably transferred for experimental infestation of white-tailed deer by other multi-host and one-host tick species.

Patterns of deer ked (Diptera: Hippoboscidae) and tick (Ixodida: Ixodidae) infestation on white-tailed deer (Odocoileus virginianus) in the eastern United States.

BACKGROUND: White-tailed deer (Odocoileus virginianus) host numerous ectoparasitic species in the eastern USA, most notably various species of ticks and two species of deer keds. Several pathogens transmitted by ticks to humans and other animal hosts have also been found in deer keds. Little is known about the acquisition and potential for transmission of these pathogens by deer keds; however, tick-deer ked co-feeding transmission is one possible scenario. On-host localization of ticks and deer keds on white-tailed deer was evaluated across several geographical regions of the eastern US to define tick-deer ked spatial relationships on host deer, which may impact the vector-borne disease ecology of these ectoparasites. METHODS: Ticks and deer keds were collected from hunter-harvested white-tailed deer from six states in the eastern US. Each deer was divided into three body sections, and each section was checked for 4 person-minutes. Differences in ectoparasite counts across body sections and/or states were evaluated using a Bayesian generalized mixed model. RESULTS: A total of 168 white-tailed deer were inspected for ticks and deer keds across the study sites. Ticks (n = 1636) were collected from all surveyed states, with Ixodes scapularis (n = 1427) being the predominant species. Counts of I. scapularis from the head and front sections were greater than from the rear section. Neotropical deer keds (Lipoptena mazamae) from Alabama and Tennessee (n = 247) were more often found on the rear body section. European deer keds from Pennsylvania (all Lipoptena cervi, n = 314) were found on all body sections of deer. CONCLUSIONS: The distributions of ticks and deer keds on white-tailed deer were significantly different from each other, providing the first evidence of possible on-host niche partitioning of ticks and two geographically distinct deer ked species (L. cervi in the northeast and L. mazamae in the southeast). These differences in spatial distributions may have implications for acquisition and/or transmission of vector-borne pathogens and therefore warrant further study over a wider geographic range and longer time frame.

The genome of the stable fly, Stomoxys calcitrans, reveals potential mechanisms underlying reproduction, host interactions, and novel targets for pest control.

BACKGROUND: The stable fly, Stomoxys calcitrans, is a major blood-feeding pest of livestock that has near worldwide distribution, causing an annual cost of over $2 billion for control and product loss in the USA alone. Control of these flies has been limited to increased sanitary management practices and insecticide application for suppressing larval stages. Few genetic and molecular resources are available to help in developing novel methods for controlling stable flies. RESULTS: This study examines stable fly biology by utilizing a combination of high-quality genome sequencing and RNA-Seq analyses targeting multiple developmental stages and tissues. In conjunction, 1600 genes were manually curated to characterize genetic features related to stable fly reproduction, vector host interactions, host-microbe dynamics, and putative targets for control. Most notable was characterization of genes associated with reproduction and identification of expanded gene families with functional associations to vision, chemosensation, immunity, and metabolic detoxification pathways. CONCLUSIONS: The combined sequencing, assembly, and curation of the male stable fly genome followed by RNA-Seq and downstream analyses provide insights necessary to understand the biology of this important pest. These resources and new data will provide the groundwork for expanding the tools available to control stable fly infestations. The close relationship of Stomoxys to other blood-feeding (horn flies and Glossina) and non-blood-feeding flies (house flies, medflies, Drosophila) will facilitate understanding of the evolutionary processes associated with development of blood feeding among the Cyclorrhapha.

Sudden Mortality in Captive White-Tailed Deer With Atypical Infestation of Winter Tick.

In October 2020, three captive male white-tailed deer, Odocoileus virginianus [Zimmermann] (artiodactyla: Cervidae), were found dead in central Pennsylvania and a fourth was euthanized due to extreme lethargy. The deer presented with high burdens of Dermacentor albipictus (Packard) (Ixoda: Ixodidae) (winter tick). There were no other clinical symptoms and deer were in otherwise good physical condition with no observed alopecia. Winter tick epizootics have been associated with mortalities of moose, Alces alces [Linnaeus] (artiodactyla: cervidae), and more recently elk, Cervus canadensis [Erxleben] (artiodactyla: cervidae), in Pennsylvania, but have not been reported in white-tailed deer. Mild winters are favorable to winter ticks and deer producers and managers should be aware of possible infestations as a result.

Molecular screening for rickettsial bacteria and piroplasms in ixodid ticks surveyed from white-tailed deer (Odocoileus virginianus) and nilgai antelope (Boselaphus tragocamelus) in southern Texas.

A survey of ixodid ticks parasitizing white-tailed deer (Odocoileus virginianus) and nilgai antelope (Boselaphus tragocamelus) was completed during the 2018-2019 public hunt season on the Laguna Atascosa National Wildlife Refuge (Cameron County, Texas) and the East Foundation's El Sauz Ranch in nearby Willacy County (Texas). Anocenter nitens was the predominant tick species identified with 5% of these ticks collected from nilgai. All life stages were encountered in high numbers on white-tailed deer, indicating that deer may be a primary host in this region. Amblyomma maculatum and Amblyomma inornatum were identified from both hosts, while Ixodes scapularis was encountered only on white-tailed deer. This is the first published record of A. inornatum on nilgai. A subset of ticks was used in PCR assays to detect Rickettsia spp., family Anaplasmataceae, Borrelia spp., and Theileria-Babesia spp. Borrelia spp. were not detected in any of the ticks analyzed. Rickettsia parkeri was detected in three A. maculatum adult ticks from deer, Rickettsia sp. endosymbiont sequences were present in all I. scapularis ticks, and Rickettsia amblyommatis was detected in three A. inornatum adult ticks from deer. Sequence analysis of Anaplasmataceae-positive amplicons from A. nitens and A. maculatum had low percent identity to published Anaplasma spp. sequences, suggesting a unique Anaplasma sp. may be circulating in the population. Anaplasma platys was detected from A. nitens larvae and an Ehrlichia sp. Delta strain was present in A. maculatum, both of unknown pathogenicity towards deer. Theileria cervi was detected in all stages of A. nitens ticks, and positive ticks originated from 27 of 31 deer and a single nilgai sampled from throughout the survey site. The primary vector for T. cervi is absent from this region, suggesting T. cervi is possibly maintained by a different tick species.

Association of Salivary Cholinesterase With Arthropod Vectors of Disease.

Acetylcholinesterase (AChE) was previously reported to be present in saliva of the southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini), with proposed potential functions to 1) reduce acetylcholine toxicity during rapid engorgement, 2) modulate host immune responses, and 3) to influence pathogen transmission and establishment in the host. Potential modulation of host immune responses might include participation in salivary-assisted transmission and establishment of pathogens in the host as has been reported for a number of arthropod vector-borne diseases. If the hypothesis that tick salivary AChE may alter host immune responses is correct, we reasoned that similar cholinesterase activities might be present in saliva of additional arthropod vectors. Here, we report the presence of AChE-like activity in the saliva of southern cattle ticks, Rhipicephalus (Boophilus) microplus; the lone star tick, Amblyomma americanum (Linnaeus); Asian tiger mosquitoes, Aedes albopictus (Skuse); sand flies, Phlebotomus papatasi (Scopoli); and biting midges, Culicoides sonorensis Wirth and Jones. Salivary AChE-like activity was not detected for horn flies Haematobia irritans (L.), stable flies Stomoxys calcitrans (L.), and house flies Musca domestica L. Salivary cholinesterase (ChE) activities of arthropod vectors of disease-causing agents exhibited various Michaelis-Menten KM values that were each lower than the KM value of bovine serum AChE. A lower KM value is indicative of higher affinity for substrate and is consistent with a hypothesized role in localized depletion of host tissue acetylcholine potentially modulating host immune responses at the arthropod bite site that may favor ectoparasite blood-feeding and alter host defensive responses against pathogen transmission and establishment.

Sex Chromosome Evolution in Muscid Flies.

Sex chromosomes and sex determining genes can evolve fast, with the sex-linked chromosomes often differing between closely related species. Population genetics theory has been developed and tested to explain the rapid evolution of sex chromosomes and sex determination. However, we do not know why the sex chromosomes are divergent in some taxa and conserved in others. Addressing this question requires comparing closely related taxa with conserved and divergent sex chromosomes to identify biological features that could explain these differences. Cytological karyotypes suggest that muscid flies (e.g., house fly) and blow flies are such a taxonomic pair. The sex chromosomes appear to differ across muscid species, whereas they are conserved across blow flies. Despite the cytological evidence, we do not know the extent to which muscid sex chromosomes are independently derived along different evolutionary lineages. To address that question, we used genomic and transcriptomic sequence data to identify young sex chromosomes in two closely related muscid species, horn fly (Haematobia irritans) and stable fly (Stomoxys calcitrans). We provide evidence that the nascent sex chromosomes of horn fly and stable fly were derived independently from each other and from the young sex chromosomes of the closely related house fly (Musca domestica). We present three different scenarios that could have given rise to the sex chromosomes of horn fly and stable fly, and we describe how the scenarios could be distinguished. Distinguishing between these scenarios in future work could identify features of muscid genomes that promote sex chromosome divergence.

TICK VECTOR AND DISEASE PATHOGEN SURVEILLANCE OF NILGAI ANTELOPE ( BOSELAPHUS TRAGOCAMELUS) IN SOUTHEASTERN TEXAS, USA.

Nilgai ( Boselaphus tragocamelus) are nonnative bovines that were originally introduced as game animals to one large, south Texas, US ranch but that are now present throughout southeastern Texas from Baffin Bay to Harlingen and in northern Mexico at least as far west as Durango. Between October 2014 and January 2017, nilgai ( n=517) were examined for the presence of tick ectoparasites, with particular interest in the cattle fever tick, Rhipicephalus ( Boophilus) microplus. These animals were either hunter killed or they were culled as part of federal cooperative harvesting from Cameron and Willacy counties in southeastern Texas. The proportion of fever tick-infested animals differed in a N-to-S pattern, and this was at least partly attributed to differences in habitat. The southern area is a lowland floodplain predominated by halophytes, whereas the northerly area is upland thorn scrub, the latter of which provides a vegetative canopy that is more conducive to tick survival and persistence. A subset of nilgai, all from the Texas-Mexico border area, were screened for livestock pathogens using molecular and serological assays. All nilgai were seronegative for Babesia ( Theileria) equi and Babesia cabalii. Although 11 animals were seropositive for Anaplasma marginale by competitive enzyme-linked immunosorbent assay (cELISA), these were interpreted with caution because of the lack of concordance between cELISA and molecular detection assays. All animals were PCR negative for presence of Babesia spp. DNA, and a single nilgai was seropositive for Babesia bovis and Babesia bigemina by complement fixation. It remains unknown whether cattle Babesia spp. can establish an infection in nilgai.

Fungi Isolated From House Flies (Diptera: Muscidae) on Penned Cattle in South Texas.

Musca domestica L. were collected from cattle diagnosed with bovine ringworm to evaluate the potential of the house fly to disseminate Trichophyton verrucosum E. Bodin, a fungal dermatophyte that is the causative agent for ringworm in cattle. Fungal isolates were cultured from 45 individual flies on supplemented Sabouraud dextrose agar, and isolates were identified using morphological and microscopic approaches. Each isolate was identified further by PCR amplification of the ribosomal DNA locus with fungal-specific primers and subsequent amplicon sequencing. Trichophyton verrucosum was not identified using these approaches. However, 35 different fungal species representing 17 genera were cultured from collected flies, including several species that are allergenic and pathogenic to humans and animals. Several species within the fungal orders Hypocreales, Microascales, Onygenales, Saccharomycetales, Xylaniales, and Agaricales were observed for the first time on house flies. The most frequent fungus recovered was Cladosporium cladosporoides Fresen, which is known to be a ubiquitous, airborne allergen to humans.

A Virulent Babesia bovis Strain Failed to Infect White-Tailed Deer (Odocoileus virginianus).

Wildlife are an important component in the vector-host-pathogen triangle of livestock diseases, as they maintain biological vectors that transmit pathogens and can serve as reservoirs for such infectious pathogens. Babesia bovis is a tick-borne pathogen, vectored by cattle fever ticks, Rhipicephalus spp., that can cause up to 90% mortality in naive adult cattle. While cattle are the primary host for cattle fever ticks, wild and exotic ungulates, including white-tailed deer (WTD), are known to be viable alternative hosts. The presence of cattle fever tick populations resistant to acaricides raises concerns regarding the possibility of these alternative hosts introducing tick-borne babesial parasites into areas free of infection. Understanding the B. bovis reservoir competence of these alternative hosts is critical to mitigating the risk of introduction. In this study, we tested the hypothesis that WTD are susceptible to infection with a B. bovis strain lethal to cattle. Two groups of deer were inoculated intravenously with either B. bovis blood stabilate or a larval extract supernatant containing sporozoites from infected R. microplus larvae. The collective data demonstrated that WTD are neither a transient host nor reservoir of B. bovis. This conclusion is supported by the failure of B. bovis to establish an infection in deer regardless of inoculum. Although specific antibody was detected for a short period in the WTD, the PCR results were consistently negative at multiple time points throughout the experiment and blood from WTD that had been exposed to parasite, transferred into naïve recipient susceptible calves, failed to establish infection. In contrast, naïve steers inoculated intravenously with either B. bovis blood stabilate or the larval extract supernatant containing sporozoites rapidly succumbed to disease. These findings provide evidence that WTD are not an epidemiological component in the maintenance of B. bovis infectivity to livestock.

Multiple mutations in the para-sodium channel gene are associated with pyrethroid resistance in Rhipicephalus microplus from the United States and Mexico.

BACKGROUND: Acaricide resistant Rhipicephalus microplus populations have become a major problem for many cattle producing areas of the world. Pyrethroid resistance in arthropods is typically associated with mutations in domains I, II, III, and IV of voltage-gated sodium channel genes. In R. microplus, known resistance mutations include a domain II change (C190A) in populations from Australia, Africa, and South America and a domain III mutation (T2134A) that only occurs in Mexico and the U.S. METHODS: We investigated pyrethroid resistance in cattle fever ticks from Texas and Mexico by estimating resistance levels in field-collected ticks using larval packet discriminating dose (DD) assays and identifying single nucleotide polymorphisms (SNPs) in the para-sodium channel gene that associated with resistance. We then developed qPCR assays for three SNPs and screened a larger set of 1,488 R. microplus ticks, representing 77 field collections and four laboratory strains, for SNP frequency. RESULTS: We detected resistance SNPs in 21 of 68 U.S. field collections and six of nine Mexico field collections. We expected to identify the domain III SNP (T2134A) at a high frequency; however, we only found it in three U.S. collections. A much more common SNP in the U.S. (detected in 19 of 21 field collections) was the C190A domain II mutation, which has never before been reported from North America. We also discovered a novel domain II SNP (T170C) in ten U.S. and two Mexico field collections. The T170C transition mutation has previously been associated with extreme levels of resistance (super-knockdown resistance) in insects. We found a significant correlation (r = 0.81) between the proportion of individuals in field collections that carried any two resistance SNPs and the percent survivorship of F1 larvae from these collections in DD assays. This relationship is accurately predicted by a simple linear regression model (R2 = 0.6635). CONCLUSIONS: These findings demonstrate that multiple mutations in the para-sodium channel gene independently associate with pyrethroid resistance in R. microplus ticks, which is likely a consequence of human-induced selection.

Expression of bovine genes associated with local and systemic immune response to infestation with the Lone Star tick, Amblyomma americanum.

The Lone Star tick, Amblyomma americanum Linnaeus 1758 (Acari; Ixodidae), causes considerable production losses to the southern U.S. cattle industry due to reduced weight, infertility, secondary infections at bite wound sites, damaged hides, and potentially death, as these ticks tend to infest livestock in large numbers. Increasing environmental concerns, along with the potential for chemical residue in food products, have led to more emphasis on alternative tick control strategies, such as selective breeding practices and anti-tick vaccines. To enable progress toward these goals, a better understanding of bovine host immune mechanisms elicited by ticks is needed. In this study, 7 calves were phenotyped as susceptible, moderately resistant, or highly resistant to adult A. americanum ticks. Tick bite-site biopsies and blood leukocytes were collected at multiple time points throughout 3 successive tick infestations. Gene expression at tick bite-site biopsies was assessed by microarray analysis over 3 time points for each phenotype group. Quantitative reverse transcriptase-PCR expression analysis evaluated 11 candidate genes in tick bite-site biopsies, and 6 in blood leukocytes. Regression curve estimates calculated from the expression values generated by qRT-PCR in tick bite-sites identified correlations between several candidate genes. Increased expression of IGHG1, IL6, IL1α, and IL1RN in bovine tick bite-site biopsies suggests that Th2 differentiation may be important for the local bovine response to A. americanum ticks. Strong correlations in expression for IL1α and IL1ß, for IL1α and IL1RN, and for IL1α and TLR4 were found in biopsies from the tick-resistant phenotypes. The up-regulation of IL12 and IL23 in blood leukocytes from Lone Star tick-infested calves of all phenotypes suggests a possible systemic recruitment of memory T cells. This study provides novel insight concerning the bovine immune response to Lone Star ticks and a basis for future investigations to characterize the importance of these factors for tick-resistance in cattle.

Widespread movement of invasive cattle fever ticks (Rhipicephalus microplus) in southern Texas leads to shared local infestations on cattle and deer.

BACKGROUND: Rhipicephalus (Boophilus) microplus is a highly-invasive tick that transmits the cattle parasites (Babesia bovis and B. bigemina) that cause cattle fever. R. microplus and Babesia are endemic in Mexico and ticks persist in the United States inside a narrow tick eradication quarantine area (TEQA) along the Rio Grande. This containment area is threatened by unregulated movements of illegal cattle and wildlife like white-tailed deer (WTD; Odocoileus virginianus). METHODS: Using 11 microsatellite loci we genotyped 1,247 R. microplus from 63 Texas collections, including outbreak infestations from outside the TEQA. We used population genetic analyses to test hypotheses about ecological persistence, tick movement, and impacts of the eradication program in southern Texas. We tested acaricide resistance with larval packet tests (LPTs) on 47 collections. RESULTS: LPTs revealed acaricide resistance in 15/47 collections (32%); 11 were outside the TEQA and three were resistant to multiple acaricides. Some collections highly resistant to permethrin were found on cattle and WTD. Analysis of genetic differentiation over time at seven properties revealed local gene pools with very low levels of differentiation (FST 0.00-0.05), indicating persistence over timespans of up to 29 months. However, in one neighborhood differentiation varied greatly over a 12-month period (FST 0.03-0.13), suggesting recurring immigration from distinct sources as another persistence mechanism. Ticks collected from cattle and WTD at the same location are not differentiated (FST = 0), implicating ticks from WTD as a source of ticks on cattle (and vice versa) and emphasizing the importance of WTD to tick control strategies. We identified four major genetic groups (K = 4) using Bayesian population assignment, suggesting multiple introductions to Texas. CONCLUSIONS: Two dispersal mechanisms give rise to new tick infestations: 1) frequent short-distance dispersal from the TEQA; and 2) rare long-distance, human-mediated dispersal from populations outside our study area, probably Mexico. The threat of cattle fever tick transport into Texas is increased by acaricide resistance and the ability of R. microplus to utilize WTD as an alternate host. Population genetic analyses may provide a powerful tool for tracking invasions in other parts of the world where these ticks are established.

Invasive potential of cattle fever ticks in the southern United States.

ABSTRACT' BACKGROUND: For >100 years cattle production in the southern United States has been threatened by cattle fever. It is caused by an invasive parasite-vector complex that includes the protozoan hemoparasites Babesia bovis and B. bigemina, which are transmitted among domestic cattle via Rhipicephalus tick vectors of the subgenus Boophilus. In 1906 an eradication effort was started and by 1943 Boophilus ticks had been confined to a narrow tick eradication quarantine area (TEQA) along the Texas-Mexico border. However, a dramatic increase in tick infestations in areas outside the TEQA over the last decade suggests these tick vectors may be poised to re-invade the southern United States. We investigated historical and potential future distributions of climatic habitats of cattle fever ticks to assess the potential for a range expansion. METHODS: We built robust spatial predictions of habitat suitability for the vector species Rhipicephalus (Boophilus) microplus and R. (B.) annulatus across the southern United States for three time periods: 1906, present day (2012), and 2050. We used analysis of molecular variance (AMOVA) to identify persistent tick occurrences and analysis of bias in the climate proximate to these occurrences to identify key environmental parameters associated with the ecology of both species. We then used ecological niche modeling algorithms GARP and Maxent to construct models that related known occurrences of ticks in the TEQA during 2001-2011 with geospatial data layers that summarized important climate parameters at all three time periods. RESULTS: We identified persistent tick infestations and specific climate parameters that appear to be drivers of ecological niches of the two tick species. Spatial models projected onto climate data representative of climate in 1906 reproduced historical pre-eradication tick distributions. Present-day predictions, although constrained to areas near the TEQA, extrapolated well onto climate projections for 2050. CONCLUSIONS: Our models indicate the potential for range expansion of climate suitable for survival of R. microplus and R. annulatus in the southern United States by mid-century, which increases the risk of reintroduction of these ticks and cattle tick fever into major cattle producing areas.

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.

Evaluation of methods for the isolation of high quality RNA from bovine and cervine hide biopsies.

Molecular investigations of the ruminant response to ectoparasites at the parasite-host interface are critically dependent upon the quality of RNA. The complexity of ruminant skin decreases the capacity to obtain high quality RNA from biopsy samples, which directly affects the reliability of data produced by gene expression experiments. Two methods for isolating total RNA from skin were compared and the use of 4M guanidinium isothiocyanate (GITC) during frozen storage of the specimens was evaluated. In addition, the best procedure for RNA isolation from bovine skin punch biopsies was also tested on white-tailed deer skin biopsies. Skin biopsy punches were collected and frozen prior to pulverization for RNA isolation. Total RNA quantity and integrity were determined by spectrophotometry and capillary electrophoresis technology, respectively. Significantly increased total RNA yield (P < 0.05) and higher integrity (P < 0.05) were obtained with a TRI Reagent® isolation method. Freezing and subsequent storage of bovine skin punch biopsies in 4 M GITC did not affect the amount or integrity of total RNA recovered by either RNA isolation method. However, quantity and integrity of total RNA extracted with the TRI Reagent method were again significantly higher than with the alternate technique, confirming it as the superior method. The TRI Reagent isolation method also yielded high quality total RNA from white-tailed deer skin punch biopsies, suggesting the usefulness of this method for obtaining RNA of a quality suitable for gene expression studies in other ruminant species.