Sequence and transcript expression of the super-kdr locus of the horn fly, Haematobia irritans.

In horn flies, Haematobia irritans irritans (Diptera: Muscidae) (Linnaeus, 1758), target site resistance to pyrethroids can be diagnosed by an allele-specific PCR that genotypes individual flies at both the super-kdr (skdr) and the knock down resistance (kdr) associated loci. When this technique uses genomic DNA as template, modifications, such as alternative RNA splicing and RNA editing are not specifically detected. Alternative splicing at the skdr locus has been reported in Dipterans; thus, the genomic DNA-based allele-specific PCR may not accurately reflect the frequency of the skdr mutation in horn fly field populations. To investigate if alternative splicing occurs at the skdr locus of horn flies, genomic DNA and cDNA sequences isolated from two wild populations and two laboratory-reared colonies with varying degrees of pyrethroid resistance were compared. There was no indication of alternative splicing at the super-kdr locus neither in the wild populations nor in the laboratory-reared colonies.

Resistance profile and molecular characterization of pyrethroid resistance in a Rhipicephalus microplus strain from Colombia.

Intensive use of chemical acaricides for the control of cattle ticks (Rhipicephalus microplus) has led to the development of multiple acaricide resistance in Colombia. The present study aimed to characterize, using toxicological bioassays and molecular biology techniques, the resistance profile of a tick strain isolated from the Arauca state, Northeast Colombia. Commercial acaricides were used in adult immersion tests to determine its in vitro efficacies. Deltamethrin showed very low activity (4-7.3%), a mixture of cypermethrin and chlorpyrifos had intermediate efficacy (64-75.2%), and ethion presented the highest activity (88.5-100%). A colony (Arauquita strain) was established and larval immersion tests confirmed high resistance level to deltamethrin (241-fold) and susceptibility to ivermectin. A quantitative polymerase chain reaction-high resolution melt technique was used to identify single nucleotide polymorphisms (SNPs) in the para-sodium channel gene. All of the genotyped individuals were mutant, presenting one (n = 7), two (n = 7) or three (n = 9) SNPs previously associated with pyrethroid resistance. Sequencing revealed a novel mutation (F712L), that was found for the first time in R. microplus ticks from South America. This is the first description of mutations associated with pyrethroid resistance in R. microplus from Colombia. The acaricide resistance pattern found in the Arauquita strain is similar to other parts of Colombia.

Seroepidemiology of Bovine Herpes Virus-1 Infection in Water Buffaloes from the state of Veracruz, Mexico.

Water buffaloes (Bubalus bubalis) were introduced to Mexico at the end of the last century. In Mexico, buffaloes are commonly pastured together with cattle; however few studies have been done on buffalo herd health in Mexico. We hypothesized that a better knowledge of the epidemiology of infections shared between cattle and buffaloes may improve herd profitability and promote buffalo production in areas unsuitable to cattle farming. This study aimed to determine the prevalence of antibodies against bovine herpes virus - 1 (BoHV1) in water buffaloes raised on six farms from the state of Veracruz, Mexico. Of 368 buffaloes sampled, 217 (59%) were seropositive for BoHV-1. Age was identified as a risk factor for BoHV-1 infection with buffaloes older than 5 years being the most likely to be infected. Animals more than 7 years old had the highest prevalence (86.0%). Females and males had similar seroprevalence rates. Females with history of abortion had higher prevalence of antiBoHV-1 antibodies than those with no record of abortion. Buffaloes and cattle were raised together in only one of the six farms under study. Interaction with cattle was not a risk factor for BoHV-1 seropositivity. This study showed that BoHV-1 is prevalent among buffalo herds in the state of Veracruz, Mexico. Buffaloes appear to play an important role in the epidemiology of BoHV-1 infection in parts of Mexico when there is no apparent risk of interaction with cattle. Animal health programs established to mitigate the burden caused by BoHV-1 must take into consideration buffaloes when this bovid species is part of the agroecosystem shared with cattle.

Spatial multi-criteria decision analysis for modelling suitable habitats of Ornithodoros soft ticks in the Western Palearctic region.

Ticks are economically and medically important ectoparasites due to the injuries inflicted through their bite, and their ability to transmit pathogens to humans, livestock, and wildlife. Whereas hard ticks have been intensively studied, little is known about soft ticks, even though they can also transmit pathogens, including African Swine Fever Virus (ASFV) affecting domestic and wild suids or Borrelia bacteria causing tick-borne relapsing fever (TBRF) in humans. We thus developed a regional model to identify suitable spatial areas for a community of nine Ornithodoros tick species (O. erraticus, O. sonrai, O. alactagalis, O. nereensis, O. tholozani, O. papillipes, O. tartakovskyi, O. asperus, O. verrucosus), which may be of medical and veterinary importance in the Western Palearctic region. Multi-Criteria Decision Analysis was used due to the relative scarcity of high-quality occurrence data. After an in-depth literature review on the ecological requirements of the selected tick community, five climate-related factors appeared critical for feeding activity and tick development: (i) a spring temperature exceeding 10°C to induce the end of winter soft tick quiescent period, (ii) a three-months summer temperature above 20°C to allow tick physiological activities, (iii) annual precipitation ranging from 60mm to 750mm and, in very arid areas, (iv) dry seasons interrupted by small rain showers to maintain minimum moisture inside their habitat along the year or (v) residual water provided by perennial rivers near habitats. We deliberately chose not to include biological factors such as host availability or vegetation patterns. A sensitivity analysis was done by performing multiple runs of the model altering the environmental variables, their suitability function, and their attributed weights. To validate the models, we used 355 occurrence data points, complemented by random points within sampled ecoregions. All models indicated suitable areas in the Mediterranean Basin and semi-desert areas in South-West and Central Asia. Most variability between models was observed along northern and southern edges of highly suitable areas. The predictions featured a relatively good accuracy with an average Area Under Curve (AUC) of 0.779. These first models provide a useful tool for estimating the global distribution of Ornithodoros ticks and targeting their surveillance in the Western Palearctic region.

Mutation in the Sodium Channel Gene Corresponds With Phenotypic Resistance of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) to Pyrethroids.

The brown dog tick, Rhipicephalus sanguineus sensu lato (Latreille), is a cosmopolitan ectoparasite and vector of pathogens that kill humans and animals. Pyrethroids represent a class of synthetic acaricides that have been used intensely to try to control the brown dog tick and mitigate the risk of tick-borne disease transmission. However, acaricide resistance is an emerging problem in the management of the brown dog tick. Understanding the mechanism of resistance to acaricides, including pyrethroids, is important to adapt brown dog tick control strategies. The main objective of this study was to determine if target-site mutations associated with pyrethroid resistance in other pests could be associated with phenotypic resistance detected in a brown dog tick population from Florida. We amplified segment 6 of the domain III of the voltage-sensitive sodium channel protein, using cDNAs synthesized from pyrethroid-susceptible and pyrethroid-resistant tick strains. A single nucleotide point mutation (SNP) identified in a highly conserved region of domain III S6 in the resistant ticks resulted in an amino acid change from phenylalanine to leucine. This mutation is characteristic of resistance phenotypes in other tick species, and is the first report of this mutation in R. sanguineus. Molecular assays based on this knowledge could be developed to diagnose the risk for pyrethroid resistance, and to inform decisions on integrated brown dog tick management practices.

Movement patterns of nilgai antelope in South Texas: Implications for cattle fever tick management.

Wildlife, both native and introduced, can harbor and spread diseases of importance to the livestock industry. Describing movement patterns of such wildlife is essential to formulate effective disease management strategies. Nilgai antelope (Boselaphus tragocamelus) are a free-ranging, introduced ungulate in southern Texas known to carry cattle fever ticks (CFT, Rhipicephalus (Boophilus) microplus, R. (B.) annulatus). CFT are the vector for the etiological agent of bovine babesiosis, a lethal disease causing high mortality in susceptible Bos taurus populations and severely affecting the beef cattle industry. Efforts to eradicate CFT from the United States have been successful. However, a permanent quarantine area is maintained between Texas and Mexico to check its entry from infested areas of neighboring Mexico states on wildlife and stray cattle. In recent years, there has been an increase in CFT infestations outside of the permanent quarantine area in Texas. Nilgai are of interest in understanding how CFT may be spread through the landscape. Thirty nilgai of both sexes were captured and fitted with satellite radio collars in South Texas to gain information about movement patterns, response to disturbances, and movement barriers. Median annual home range sizes were highly variable in males (4665ha, range=571-20,809) and females (1606ha, range=848-29,909). Female movement patterns appeared to be seasonal with peaks during June-August; these peaks appeared to be a function of break-ups in female social groups rather than environmental conditions. Nilgai, which reportedly are sensitive to disturbance, were more likely to relocate into new areas immediately after being captured versus four other types of helicopter activities. Nilgai did not cross 1.25m high cattle fences parallel to paved highways but did cross other fence types. Results indicate that females have a higher chance of spreading CFT through the landscape than males, but spread of CFT may be mitigated via maintenance of cattle fences running parallel with paved highways. Our results highlight the importance of documenting species-specific behavior in wildlife-livestock interfaces that can be used to develop effective disease management strategies in the United States and worldwide.

Blood feeding of Ornithodoros turicata larvae using an artificial membrane system.

An artificial membrane system was adapted to feed Ornithodoros turicata (Ixodida: Argasidae) larvae from a laboratory colony using defibrinated swine blood. Aspects related to larval feeding and moulting to the first nymphal instar were evaluated. A total of 55.6% of all larvae exposed to the artificial membrane in two experimental groups fed to repletion and 98.0% of all fed larvae moulted. Mortality rates of first instar nymphs differed significantly depending on the sorting tools used to handle engorged larvae (χ2 = 35.578, P < 0.0001): engorged larvae handled with featherweight forceps showed significantly higher mortality (odds ratio = 4.441) than those handled with a camel-hair brush. Differences in the physical properties of the forceps and camel-hair brush may affect the viability of fragile soft tick larvae even when care and the same technique are used to sort them during experimental manipulations. The current results represent those of the first study to quantify successful feeding to repletion, moulting and post-moulting mortality rates in O. turicata larvae using an artificial membrane feeding system. Applications of the artificial membrane feeding system to fill gaps in current knowledge of soft tick biology and the study of soft tick-pathogen interactions are discussed.

First documentation of ivermectin resistance in Rhipicephalus sanguineus sensu lato (Acari: Ixodidae).

The brown dog tick, Rhipicephalus sanguineus sensu lato (Latreille, 1806), is an ectoparasite and disease vector of significant veterinary and public health importance that is distributed widely around the world. The intensive use of synthetic acaricides for tick control exerts a strong selective pressure for brown dog ticks to become resistant to them. Here, we investigated claims from the field regarding treatment failure associated with the use of veterinary products containing ivermectin (IVM) to control brown dog ticks infesting dogs in Yucatan state, Mexico. Dogs in six state municipalities were inspected to sample 15 R. sanguineus s.l. POPULATIONS: Interviews were conducted with dog owners to gather information on the history of dog treatment with conventional acaricides and IVM. The larval immersion test was used on the progeny of adult female ticks infesting dogs to test for susceptibility to IVM. Dose-mortality regressions, lethal concentrations (LC), their confidence intervals, and slope were estimated by probit analysis. Ten tick populations (66.7%) were classified as resistant compared with the most susceptible population. A high inter-population variation in the phenotypic level of IVM resistance was evident (resistance ratio at LC50% and LC99% ranged from 1.0 to 30.5, and from 1.0 to 458.8, respectively). Tick populations classified as resistant were collected from dogs known to have been treated with IVM. To our knowledge, this is the first report of IVM resistance in R. sanguineus s.l. worldwide. Veterinary and pet owner education on integrated tick management practices is required to avoid widespread resistance and increased treatment failure with products containing IVM and other macrocyclic lactones used to control endo- and ectoparasites affecting dogs. Integrated tick management will also help mitigate the burden of brown dog tick-borne diseases on human and animal populations.

Ticks collected from humans, domestic animals, and wildlife in Yucatan, Mexico.

Domestic animals and wildlife play important roles as reservoirs of zoonotic pathogens that are transmitted to humans by ticks. Besides their role as vectors of several classes of microorganisms of veterinary and public health relevance, ticks also burden human and animal populations through their obligate blood-feeding habit. It is estimated that in Mexico there are around 100 tick species belonging to the Ixodidae and Argasidae families. Information is lacking on tick species that affect humans, domestic animals, and wildlife through their life cycle. This study was conducted to bridge that knowledge gap by inventorying tick species that infest humans, domestic animals and wildlife in the State of Yucatan, Mexico. Amblyomma ticks were observed as euryxenous vertebrate parasites because they were found parasitizing 17 animal species and human. Amblyomma mixtum was the most eryxenous species found in 11 different animal species and humans. Both A. mixtum and A. parvum were found parasitizing humans. Ixodes near affinis was the second most abundant species parasitizing six animal species (dogs, cats, horses, white-nosed coati, white-tail deer and black vulture) and was found widely across the State of Yucatan. Ixodid tick populations may increase in the State of Yucatan with time due to animal production intensification, an increasing wildlife population near rural communities because of natural habitat reduction and fragmentation. The diversity of ticks across host taxa documented here highlights the relevance of ecological information to understand tick-host dynamics. This knowledge is critical to inform public health and veterinary programs for the sustainable control of ticks and tick-borne diseases.

Persistence and Retention of Porcine Reproductive and Respiratory Syndrome Virus in Stable Flies (Diptera: Muscidae).

We investigated the acquisition of porcine reproductive and respiratory syndrome (PRRS) virus by the stable fly (Diptera: Muscidae; Stomoxys calcitrans (L.)) through a bloodmeal, and virus persistence in the digestive organs of the fly using virus isolation and quantitative reverse-transcription PCR (qRT-PCR). Stable flies were fed blood containing live virus, modified live vaccine virus, chemically inactivated virus, or no virus. Stable flies acquired PRRSV from the bloodmeal and the amount of virus in the flies declined with time, indicating virus did not replicate in fly digestive tissues. Virus RNA was recovered from the flies fed live virus up to 24 h postfeeding using virus isolation techniques and 96 h using qRT-PCR. We further examined the fate of PRRSV in the hemolymph of the flies following intrathoracic injection to bypass the midgut barrier. PRRSV was detected in intrathoracically inoculated adult stable flies for 10 d using qRT-PCR. In contrast to what we observed in the digestive tract, detectable virus quantities in the intrathoracically inoculated stable flies followed an exponential decay curve. The amount of virus decreased fourfold in the first 3 d and remained stable thereafter, up to 10 d.

Acaricide and ivermectin resistance in a field population of Rhipicephalus microplus (Acari: Ixodidae) collected from red deer (Cervus elaphus) in the Mexican tropics.

In the Neotropics the control of tick infestations in red deer (Cervus elaphus) is achieved primarily through the use of acaricides and macrocyclic lactones. In Mexico, resistance to one or multiple classes of acaricides has been reported in Rhipicephalus microplus infesting cattle, but information on acaricide susceptibility in R. microplus infesting red deer is lacking. In this study we report the level of resistance to different classes of acaricides and ivermectin in R. microplus collected from red deer in the Mexican tropics. Engorged R. microplus females were collected from a red deer farm in Yucatan, Mexico. The larval packet test was used to detect resistance to the organophosphates (OPs) chlorpyrifos and coumaphos, synthetic pyrethroids (SPs) cypermethrin and permethrin, and the phenylpyrazol, fipronil. Resistance to the formamidine amitraz (Am), and ivermectin was ascertained using the larval immersion test. Data were subjected to probit analysis to determine lethal concentrations and resistance ratios to kill 50% (RR50) and 99% (RR99) of the tick population under evaluation in relation to susceptible reference strains. Additionally, allele specific polymerase chain reaction was used to detect the sodium channel F1550I mutation associated with SP resistance in R. microplus. The R. microplus population from red deer in Yucatan showed very high resistance to the two SPs evaluated (RRs>72.2 for cypermethrin; RR for permethrin resistance was so high a dose-response curve was not possible). All individual larvae tested to detect the sodium channel F1550I mutation associated with SP resistance in R. microplus were homozygous. The same tick population showed different levels of resistance to OPs (chlorpyrifos: RR50=1.55, RR99=0.63; coumaphos: RR50=6.8, RR99=5.9), fipronil (RR50=1.8, RR99=0.9), and amitraz (RR50=2.3, RR99=4.4). Resistance to ivermectin was regarded as moderate (RR50=7.1, RR99=5.0). This is the first report of R. microplus ticks collected from red deer in Mexico with different levels of resistance to four acaricide groups and ivermectin.

In vitro and in vivo evaluation of cypermethrin, amitraz, and piperonyl butoxide mixtures for the control of resistant Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in the Mexican tropics.

A study was conducted to evaluate the efficacy of cypermethrin, amitraz, and piperonyl butoxide (PBO) mixtures, through in vitro laboratory bioassays and in vivo on-animal efficacy trials, for the control of resistant Rhipicephalus (Boophilus) microplus on cattle in the Mexican tropics. Also, to examine mechanisms of resistance to cypermethrin in this tick population, the frequency of a mutated sodium channel gene (F1550I) was determined using a PCR assay. Results of laboratory bioassays using modified larval packet tests revealed that cypermethrin toxicity was synergized by PBO (from 46.6-57.0% to 83.7-85.0% larval mortality; P<0.05). The cypermethrin and amitraz mixture showed an additive effect (from 46.6-57.0% to 56.0-74.3% larval mortality). Strong synergism was observed with the mixture of cypermethrin+amitraz+PBO and this mixture was the most effective killing resistant tick larvae in vitro (96.7-100% of larval mortality). Tick larvae surviving exposure to cypermethrin or mixtures either with amitraz and PBO in vitro showed 2.9-49.6 higher probability to present the mutated allele than those killed by acaricide treatment (P<0.05). In the in vivo trial, the mixtures containing cypermethrin+PBO (80.6-97.3%), and cypermethrin+amitraz (87.0-89.7%) were more efficacious than cypermethrin alone (76.3-80.5%). The highest level of efficacy was obtained with the mixture of cypermethrin+amitraz+PBO, which yielded >95% control that persisted for 28 days post-treatment against R. microplus infesting cattle when tested under field conditions in the Mexican tropics. Although this mixture is a potentially useful tool to combat pyrethroid resistance, a product based on an acaricide mixture like the one tested in this study has to be used rationally.

Effects of pyriproxyfen and buprofezin on immature development and reproduction in the stable fly.

The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), is one of the most significant biting flies that affect cattle. The use of traditional insecticides for stable fly control has only a limited success owing to the insect's unique feeding behaviours and immature development sites. A laboratory study was conducted to evaluate the effects of two insect growth regulator (IGR) products, pyriproxyfen and buprofezin, on the development of the immature stages of the stable fly and the effects of pyriproxyfen on oviposition and egg hatch. Both pyriproxyfen and buprofezin had significant inhibitory effects on immature development. The LC(50) s of pyriproxyfen and buprofezin were 0.002 and 18.92 p.p.m., respectively. Topical treatment of adult females with different doses of pyriproxyfen had significant negative effects on both female oviposition and egg hatching when 1- and 3-day-old females were treated, and the effects were dose dependent. A significant reduction in the mean number of eggs laid was observed only at the highest pyriproxyfen dose (8 µg/fly) and egg hatch was unaffected by pyriproxyfen treatment when 5-day-old females were treated. Results from the present study indicate that pyriproxyfen has the potential to be used as part of an integrated stable fly management programme.

Facilitative ecological interactions between invasive species: Arundo donax stands as favorable habitat for cattle ticks (Acari: Ixodidae) along the U.S.-Mexico border.

The cattle tick, Rhipicephalus (Boophilus) spp. is a key vector of protozoa that cause bovine babesiosis. Largely eradicated from most of the United States, the cattle tick continues to infest south Texas, and recent outbreaks in this area may signal a resurgence of cattle tick populations despite current management efforts. An improved understanding of the dynamic ecology of cattle fever ticks along the U.S.-Mexico border is required to devise strategies for sustainable eradication efforts. Management areas of the cattle tick overlap considerably with dense, wide infestations of the non-native, invasive grass known as giant reed (Arundo donax L.). Here we show that stands of giant reed are associated with abiotic and biotic conditions that are favorable to tick survival, especially when compared with other nearby habitats (open pastures of buffelgrass (Pennisetum ciliare) and closed canopy native forests). Overhead canopies in giant reed stands and native riparian forests reduce daily high temperature, which was the best abiotic predictor of oviposition by engorged females. In sites where temperatures were extreme, specifically open grasslands, fewer females laid eggs and the resulting egg masses were smaller. Pitfall trap collections of ground dwelling arthropods suggest a low potential for natural suppression of tick populations in giant reed stands. The finding that A. donax infestations present environmental conditions that facilitate the survival and persistence of cattle ticks, as well or better than native riparian habitats and open grasslands, represents an alarming complication for cattle fever tick management in the United States.

Papular dermatitis induced in guinea pigs by the biting midge Culicoides sonorensis (Diptera: Ceratopogonidae).

Histological, ultrastructural, and virological examinations were performed on abdominal skin from guinea pigs after a blood meal by colony-bred biting midges, Culicoides sonorensis. Small, superficial, cutaneous, crateriform ulcers with necrosis of superficial dermis developed at feeding sites and healed within 24-48 hours. Animals developed nonpruritic erythematous papules 5 days after feeding that persisted until the study ended at 12 days after feeding. Papules corresponded histologically to foci of epidermal hyperplasia and superficial interstitial dermatitis with intraepidermal micropustules and scattered intraepidermal polykaryons. The principal ultrastructural changes were spongiosis in germinal epithelium and neutrophilic-histiocytic exocytosis. No viral agents or broken mouthparts were identified in lesions. The dermatitis may represent a host reaction to persisting insect salivary secretion and should be considered as an additional consequence of blood feeding in future studies involving biting midges.

Oral infection of Culicoides sonorensis (Diptera: Ceratopogonidae) by vesicular stomatitis virus.

Vesicular stomatitis virus serotype New Jersey (VSNJV) was mixed with bovine blood or fetal bovine serum (FBS) and fed across silicone membranes to laboratory populations of Culicoides sonorensis Wirth & Jones. In an initial study, virus was detected after 13 d in 21% of the midges that received an FBS/VSNJV mixture. In subsequent time-course experiments, engorged females were collected and maintained at 20.0 degrees C and assayed for VSNJV immediately after feeding and at 1, 3, 7, 10 and 13 d after feeding. Virus was detected after 13 d in 3% of the midges that received a bovine blood/VSNJV mixture and in 9% of the midges that received an FBS/VSNJV mixture. The results indicate that C. sonorensis should be considered as a potential biological vector of VSNJV.

Anticoagulant activity in salivary glands of the insect vector Culicoides variipennis sonorensis by an inhibitor of factor Xa.

Blood feeding by the insect vector Culicoides variipennis sonorensis involves laceration of superficial host tissues, an injury that would be expected to trigger the coagulation cascade. Accordingly, the salivary glands of C.v. sonorensis were examined for the presence of an antihemostatic that prevents blood coagulation. Assays using salivary gland extracts showed a delay in the recalcification time of plasma devoid of platelets, indicating the presence of anticoagulant activity. Retardation in the formation of a fibrin clot was also observed after the addition of tissue factor to plasma that was preincubated with salivary gland extracts. Similarly, an inhibitory effect by salivary gland extracts was detected in assays that included factors of the intrinsic pathway. Inhibition of the catalytic activity of purified factor Xa toward its chromogenic substrate suggested that it was the target of the salivary anticoagulant of C.v. sonorensis. This was corroborated by the coincidence of anticoagulant and anti-FXa activities obtained by reverse-phase HPLC. The depletion of anti-FXa activity from salivary glands during blood feeding suggests that the FXa inhibitor functions as anticoagulant. Molecular sieving HPLC yielded an apparent molecular mass of 28 kDa for the salivary FXa inhibitor of C.v. sonorensis. Preventing the formation of thrombin through the inhibition of FXa likely facilitates blood feeding by maintaining the pool of blood fluid at the feeding site. The salivary FXa inhibitor of C.v. sonorensis could impair the network of host-defense mechanisms in the skin microenvironment by avoiding blood coagulation at the site of feeding.

Identification of a salivary vasodilator in the primary North American vector of bluetongue viruses, Culicoides variipennis.

Several species of Culicoides biting midges are important pests and vectors of pathogens affecting humans and other animals. Bluetongue is the most economically important arthropod-borne animal disease in the United States. Culicoides variipennis is the primary North American vector of the bluetongue viruses. A reddish halo surrounding a petechial hemorrhage was noticed at the site of C. variipennis blood feeding in previously unexposed sheep and rabbits. Salivary gland extracts of nonblood-fed C. variipennis injected intradermally into sheep and rabbits induced cutaneous vasodilation in the form of erythema. A local, dose-dependent erythema, without edema or pruritus, was noted 30 min after injection. Erythema was inapparent with salivary gland extracts obtained after blood feeding. This observation suggested that the vasodilatory activity was inoculated into the host skin at the feeding site. The vasodilatory activity was insoluble in ethanol and destroyed by trypsin or chymotrypsin, which indicated that vasodilation was due to a protein. The association of cutaneous vasodilation with a salivary protein was corroborated by reversed-phase, high-performance liquid chromatography (HPLC). Fractionation of salivary gland extracts by molecular sieving HPLC resulted in maximal vasodilatory activity that coeluted with a protein having a relative molecular weight (MWr) of 22.45 kD. The C. variipennis vasodilator appears to be biologically active at the nanogram level. This vasodilator likely assists C. variipennis during feeding by increasing blood flow from host superficial blood vessels surrounding the bite site. The identification of a salivary vasodilator in C. variipennis may have implications for the transmission of Culicoides-borne pathogens and in the development of dermatitis resulting from the sensitization of humans and animals to Culicoides salivary antigens.

Apyrase activity and adenosine diphosphate induced platelet aggregation inhibition by the salivary gland proteins of Culicoides variipennis, the North American vector of bluetongue viruses.

Salivary gland homogenates of Culicoides variipennis, the primary vector of bluetongue (BLU) viruses in North America, were analyzed for apyrase activity. Apyrase (ATP diphosphohydrolase, EC 3.6.1.5) is an anti-hemostatic and anti-inflammatory salivary enzyme of most hematophagous arthropods. The enzyme activity was measured by the release of orthophosphate using ATP, ADP, and AMP as substrates with Ca2+ as the divalent cation. ATPase (11.5 +/- 1 mU/pair of glands), ADPase (7.3 +/- 0.7 mU/pair of glands), and insignificant (P < 0.05) AMPase (0.07 mU/pair of glands) activities were detected in female salivary glands. Male salivary glands contained lower amounts of ATPase and ADPase activity (P < 0.05). The ATPase and ADPase activities were greatest at pH 8.5, and were similarly activated by Mg2+. Molecular sieving HPLC of salivary gland homogenates generated a single peak which coincided with ATPase and ADPase, but no AMPase, activity; the protein has an estimated molecular mass of 35,000 Da. ATPase and ADPase activity, and total protein concentration, were reduced (P < 0.05) in the salivary glands of females after taking a blood meal from a sheep. Salivary gland homogenates also inhibited ADP-induced platelet aggregation in vitro. It is concluded that the salivary ATPase and ADPase activities of C. variipennis reside in one enzyme, and that this enzyme is likely an apyrase. The apyrase activity is thought to be responsible for the inhibition of ADP-induced platelet aggregation, as indicated by the apparent discharge of apyrase from salivary glands into the host during blood feeding. This suggests that apyrase is one of the salivary proteins present in C. variipennis acting as antigens in the development of Culicoides hypersensitivity in ruminants and horses. Apyrase may inhibit an inflammatory response at the feeding site through the subsequent degradation of its end-product, AMP, to adenosine, a potent anti-inflammatory substance, by the ecto-5' nucleotidase activity of neutrophils.