Environmental determinants of Ixodes ricinus ticks and the incidence of Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis, in Scotland.

Lyme borreliosis (LB) is the most common arthropod-borne disease of humans in the Northern hemisphere. In Europe, the causative agent, Borrelia burgdorferi sensu lato complex, is principally vectored by Ixodes ricinus ticks. The aim of this study was to identify environmental factors influencing questing I. ricinus nymph abundance and B. burgdorferi s.l. infection in questing nymphs using a large-scale survey across Scotland. Ticks, host dung and vegetation were surveyed at 25 woodland sites, and climatic variables from a Geographical Information System (GIS) were extracted for each site. A total of 2397 10 m2 transect surveys were conducted and 13 250 I. ricinus nymphs counted. Questing nymphs were assayed for B. burgdorferi s.l. and the average infection prevalence was 5·6% (range 0·8-13·9%). More questing nymphs and higher incidence of B. burgdorferi s.l. infection were found in areas with higher deer abundance and in mixed/deciduous compared to coniferous forests, as well as weaker correlations with season, altitude, rainfall and ground vegetation. No correlation was found between nymph abundance and infection prevalence within the ranges encountered. An understanding of the environmental conditions associated with tick abundance and pathogen prevalence may be used to reduce risk of exposure and to predict future pathogen prevalence and distributions under environmental changes.

Defining the concept of 'tick repellency' in veterinary medicine.

Although widely used, the term repellency needs to be employed with care when applied to ticks and other periodic or permanent ectoparasites. Repellency has classically been used to describe the effects of a substance that causes a flying arthropod to make oriented movements away from its source. However, for crawling arthropods such as ticks, the term commonly subsumes a range of effects that include arthropod irritation and consequent avoiding or leaving the host, failing to attach, to bite, or to feed. The objective of the present article is to highlight the need for clarity, to propose consensus descriptions and methods for the evaluation of various effects on ticks caused by chemical substances.

Transcriptome analysis of the synganglion from the brown dog tick, Rhipicephalus sanguineus.

Tick control strategies rely heavily on chemicals (acaricides), most of which target the central nervous system. With increasing resistance, new acaricides are urgently needed but knowledge of tick neurobiology is surprisingly limited, notably the number of neural-specific gene sequences. One thousand and eight expressed sequence tags (ESTs) were obtained from a normalized cDNA library from Rhipicephalus sanguineus synganglia. Putative functional identities were assigned to 44% whereas 34% were unknown/novel sequences. Of particular interest were ESTs encoding a chitinase-like enzyme, an acetylcholinesterase and four transmembrane receptors including two glutamate-gated chloride channel receptors, a leucokinin-like receptor and a nicotinic acetylcholine receptor alpha-subunit. This study highlights the benefits of using both neural tissues and normalized libraries in an EST-approach for identifying potential acaricide targets expressed as rare transcripts.

RNA-interference methods for gene-knockdown in the sea louse, Lepeophtheirus salmonis: studies on a putative prostaglandin E synthase.

Harnessing the full utility of extensive gene sequences recently available for the economically important sea louse, Lepeophtheirus salmonis, requires the adaptation of modern molecular biology approaches to this non-model organism. Using a putative microsomal prostaglandin E synthase type-2 (PGES2) as a candidate gene, we investigated gene-knockdown by double-stranded RNA interference (dsRNAi) in the small free-living and the larger parasitic stages of L. salmonis. dsRNA was administered to nauplius and copepodid stages by immersion for 7 h. Pre-adult and adults received dsRNA by intra-haemocoelic injection. The extent, speed and persistence of the knockdown effects were determined by RT-PCR. LsPGES2 was abundantly expressed in all life stages, including the non-parasitic stages. Administration of dsRNA to nauplius and copepodids by immersion had no effect on mortality rates and moulting through to copepodids was observed. Dramatic knockdown of LsPGES2 was observed within 7 h and persisted for at least 48 h. Injection of dsRNA had no effect on mortality in pre-adults and adults, but knockdown of LsPGES2 was apparent within 24 h, reaching 95% over the 72 h and was persistent for at least 120 h. The methods developed resulted in rapid and persistent knockdown in L. salmonis suitable for studies in the different stadia.

The effect of host movement on viral transmission dynamics in a vector-borne disease system.

Many vector-borne pathogens whose primary vectors are generalists, such as Ixodid ticks, can infect a wide range of host species and are often zoonotic. Understanding their transmission dynamics is important for the development of disease management programmes. Models exist to describe the transmission dynamics of such diseases, but are necessarily simplistic and generally limited by knowledge of vector population dynamics. They are typically deterministic SIR-type models, which predict disease dynamics in a single, non-spatial, closed patch. Here we explore the limitations of such a model of louping-ill virus dynamics by challenging it with novel field data. The model was only partially successful in predicting Ixodes ricinus density and louping-ill virus prevalence at 6 Scottish sites. We extend the existing multi-host model by forming a two-patch model, incorporating the impact of roaming hosts. This demonstrates that host movement may account for some of the discrepancies between the original model and empirical data. We conclude that insights into the dynamics of multi-host vector-borne pathogens can be gained by using a simple two-patch model. Potential improvements to the model, incorporating aspects of spatial and temporal heterogeneity, are outlined.

Educating youth swine exhibitors on influenza A virus transmission at agricultural fairs.

Influenza A virus (IAV) is a major zoonotic pathogen that threatens global public health. Novel strains of influenza A viruses pose a significant risk to public health due to their pandemic potential, and transmission of influenza A viruses from animals to humans is an important mechanism in the generation and introduction of IAVs that threaten human health. The purpose of this descriptive correlational study was to develop real-life training scenarios to better inform swine exhibitors of the risks they may encounter when influenza A viruses are present in swine. Educational activities were implemented in five Ohio counties where exhibition swine had historically been shedding influenza A viruses during the county fair. A total of 146 youth swine exhibitors participated in the educational programme, and an increase in the knowledge base of these youth was documented. It is expected that educating youth exhibitors about exposure to influenza A virus infections in the swine they are exhibiting will result in altered behaviours and animal husbandry practices that will improve both human and animal health.

Prevalence of Influenza A Virus in Exhibition Swine during Arrival at Agricultural Fairs.

The exhibition swine at agricultural fairs provides a critical human-swine interface that allows for the bidirectional transmission of influenza A virus (IAV). Previous IAV surveillance at the end of fairs has resulted in frequent detection of IAV-infected swine; little is known, however, about the frequency with which swine arrive at fairs already infected with IAV. We investigated the IAV prevalence among exhibition swine entering fairs to better understand the epidemiology of IAV in this unique human-swine interface. In 2014, snout wipes were collected from 3547 swine during the first day of nine agricultural exhibitions in Indiana and Ohio. Samples were screened for IAV using rRT-PCR and positive samples were inoculated into cultured cells for virus isolation. The overall IAV prevalence detected among swine arriving at exhibitions was 5.3% (188/3547) via rRT-PCR and 1.5% (53/3547) via virus isolation, with IAV being detected and recovered from swine at 5 of the 9 exhibitions. Within the fairs with IAV-positive swine, the individual exhibition IAV prevalence ranged from 0.2% (1/523) to 34.4% (144/419) using rRT-PCR and 0.2% (1/523) to 10.3% (43/419) with virus isolation. Single IAV subtypes were detected at three of the fairs but subtype diversity was detected among the pigs at two fairs as both H1N1 and H3N2 were recovered from incoming swine. At two of the exhibitions, a temporal relationship was observed between the order of the individual swine in sampling and the associated IAV rRT-PCR results, indicating the fomite transmission of IAV through common contact surfaces may occur. With the knowledge that a small proportion of swine arrive at fairs shedding IAV, resources should be directed towards preventive strategies focused on limiting transmission during fairs to protect swine and humans during exhibitions.

The Inability to Screen Exhibition Swine for Influenza A Virus Using Body Temperature.

Agricultural fairs create an unconventional animal-human interface that has been associated with swine-to-human transmission of influenza A virus (IAV) in recent years. Early detection of IAV-infected pigs at agricultural fairs would allow veterinarians to better protect swine and human health during these swine exhibitions. This study assessed the use of swine body temperature measurement, recorded by infrared and rectal thermometers, as a practical method to detect IAV-infected swine at agricultural fairs. In our first objective, infrared thermometers were used to record the body surface temperature of 1,092 pigs at the time of IAV nasal swab collection at the end of the exhibition period of 55 agricultural fairs. IAV was recovered from 212 (19.4%) pigs, and the difference in mean infrared body temperature measurement of IAV-positive and IAV-negative pigs was 0.83°C. In a second objective, snout wipes were collected from 1,948 pigs immediately prior to the unloading of the animals at a single large swine exhibition. Concurrent to the snout wipe collection, owners took the rectal temperatures of his/her pigs. In this case, 47 (2.4%) pigs tested positive for IAV before they entered the swine barn. The mean rectal temperatures differed by only 0.19°C between IAV-positive and IAV-negative pigs. The low prevalence of IAV among the pigs upon entry to the fair in the second objective provides evidence that limiting intraspecies spread of IAV during the fairs will likely have significant impacts on the zoonotic transmission. However, in both objectives, the high degree of similarity in the body temperature measurements between the IAV-positive and IAV-negative pigs made it impossible to set a diagnostically meaningful cut point to differentiate IAV status of the individual animals. Unfortunately, body temperature measurement cannot be used to accurately screen exhibition swine for IAV.

Biosynthesis of salivary prostaglandins in the lone star tick, Amblyomma americanum.

Dopamine-induced saliva from ticks fed [3H]arachidonic acid contained the radiolabelled prostaglandins E2, F2 alpha, D2, and B2, the latter probably derived from PGE2 owing to the alkalinity of tick saliva. Prostaglandin synthetase (PGS) activity in the salivary gland homogenate from the lone star tick, Amblyomma americanum, could not be detected by standard radiometric methodologies successfully employed for tissues from many animal species, including numerous arthropods. Modifications to the assay conditions had no effect. The presence of a PGS-inhibitor in the salivary glands was ruled out. It is postulated that the PGS in A. americanum salivary glands may be considerably different from that found in other animals, including vertebrate hosts.

Origin of arachidonic acid in the salivary glands of the lone star tick, Amblyomma, americanum.

The contribution of synthesis and dietary sequestration to the high arachidonate content of the lone star tick, Amblyomma americanum, salivary glands was investigated by assessing the salivary metabolites of various radiolabeled fatty acid substrates administered to partially fed females. A portion of each of the fatty acids studied was incorporated into the fatty acid moiety of the phospholipid fraction. [14C]acetate was metabolized only into myristic, palmitic, palmitoleic, steric, and oleic acids. [3H]oleic acid, [14C]linoleic acid, [14C]gamma-linolenic acid and [14C]eicosatrienoic acids were incorporated into salivary gland phospholipids but underwent little change including elongation and/or desaturation to arachidonate. Ingested [3H]arachidonic acid was readily taken up by the salivary gland and distributed among the lipid classes in a pattern markedly different from that of the other fatty acids tested. We conclude that ticks are unable to synthesize arachidonic acid for incorporation into the salivary glands, but rather sequester it from the host bloodmeal.

Distribution of arachidonic acid among phospholipid subclasses of lone star tick salivary glands.

The subclass composition of choline- and ethanolamine-containing phospholipids was determined by analysis of acyl-linked fatty acids released by base hydrolysis of diradylglycerobenzoates formed from lone star tick salivary gland diacyl, alkylacyl and alkenylacyl phospholipids. The diacyl subclass comprises 87% of all choline-containing phospholipids, while th alkylacyl subclass comprises c. 9% and the alkenylacyl subclass c. 4%. The diacyl subclass comprises 72-77% of ethanolamine-containing phospholipids and about 14 and 13% of this subclass of phospholipid are alkylacyl and alkenylacyl lipids, respectively. Arachidonic acid (20:4) is the most abundant fatty acid (28% of all fatty acids) esterified in the alkylacyl form of phosphatidylcholine (PC) and it comprises 17% of the fatty acids in alkenylacyl-PC. The alkylacyl form of phosphatidylethanolamine (PE) is also rich in 20:4 (24%) while the alkenylacyl-PE subclass contains only 9% 20:4. Despite the relatively high amounts of 20:4 within the ether-linked phospholipids, the majority of the salivary gland 20:4 (> 83%) is found in the diacyl phospholipid subclass because of the preponderence of this subclass in tick salivary glands. Isolated salivary glands incorporated [3H]-20:4 primarily (> 98%) into the sn-2 position of diacyl PC > PE, with some incorporation into triglycerides. Continued incubation in the absence of labeled 20:4 demonstrated remodeling of [3H]-20:4 from PC into PE, and from the diacyl subclass to the alkylacyl subclass in the choline containing phospholipids.

Uptake, incorporation and redistribution of arachidonic acid in isolated salivary glands of the lone star tick.

The ability of isolated salivary glands from the lone star tick, Amblyomma americanum, to take up, incorporate and redistribute [3H]arachidonic acid was examined. Uptake of arachidonic acid was concentration dependent--a single salivary gland incorporated up to approximately 2.8 micrograms arachidonic acid in 60 min. Over 90% of the [3H]arachidonate entering the glands was esterified and found only in the phospholipid (approximately 80%) and triglyceride (approximately 10%). Essentially no radioactivity was associated with the diglyceride fraction and none with phosphatidic acid indicating de novo phospholipid synthesis was negligible. Phospholipid synthesis via acylation of lysophospholipids (the Lands pathway) was indicated by the rapidity of the synthesis (< 2 min) and the sensitivity to sulfhydryl-blocking agents. Within the phospholipids, [3H]arachidonate was incorporated only into phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Initially [3H]arachidonate was incorporated primarily into PC, but as the incubation proceeded PE contained an increasing proportion of the label. The proportion of [3H]arachidonate incorporated into triglyceride increased at higher media concentrations of arachidonic acid. The roles of lysophosphatide acyltransferase, transacylase and diglycerol acyltransferase in the distribution of arachidonate in tick salivary glands are discussed.

A specific prostaglandin E2 receptor and its role in modulating salivary secretion in the female tick, Amblyomma americanum (L.).

Prostaglandins of the 2-series (e.g. PGE2) are typically synthesized from arachidonic acid (AA) after AA is released from cellular phospholipids after activation of an intracellular phospholipase A2 (PLA2). Treatment of isolated salivary glands with PLA2 inhibitor oleyloxyethyl phosphorylcholine (OPC) or prostaglandin synthetase inhibitors reduced dopamine-induced fluid secretion and cyclic AMP (cAMP) levels in isolated salivary glands. PGE2 and its analog, 17-phenyl trinor PGE2, partly reversed the inhibition of secretion and cAMP level by OPC, suggesting that prostaglandins may have an autocrine effect in modulating tick salivary gland function. A specific PGE2 receptor was identified in the plasma membrane fraction of the salivary glands. The receptor exhibits a single, high affinity PGE2 binding site with a KD approximately 29 nM, is saturable, reversible, and specific for PGE2 and coupled to a cholera toxin-sensitive guanine nucleotide regulatory protein. Assay of adenylate cyclase activity in salivary gland membranes showed that PGE2 neither stimulated nor inhibited adenylate cyclase activity, indicating that the PGE2 effects on cAMP levels and possibly secretion are indirect, and that the PGE2 receptor stimulates an alternate "second messenger" pathway.

Prostaglandin E(2)-stimulated secretion of protein in the salivary glands of the lone star tick via a phosphoinositide signaling pathway.

Previous studies identified a prostaglandin E(2) (PGE(2)) receptor in the salivary glands of partially fed female lone star ticks, Amblyomma americanum (L.). In the present studies, protein secretion from dispersed salivary gland acini was shown to be specific for PGE(2), as compared with PGF(2alpha) or the thromboxane analog U-46619, in accordance with their respective binding affinities for the PGE(2) receptor. Furthermore, the selective PGE(2) EP1 receptor agonist, 17-phenyl trinor PGE(2), was as effective as PGE(2) in stimulating secretion of anticoagulant protein. Calcium ionophore A-23187 (1 to 100 microM) stimulated secretion of anticoagulant protein in a dose-dependent manner but the voltage-gated Ca(2+)-channel blocker verapamil (1 to 1000 microM) and the receptor-mediated Ca(2+)-entry antagonist, SK&F 96365 (1 and 10 microM), and 5mM ethylene glycol bis(beta-aminoethyl ether)-N,NN', N'-tetraacetic acid (EGTA) had no appreciable effect on inhibiting PGE(2)-stimulated secretion of anticoagulant protein. PGE(2) (0.1 microM) and the non-hydrolyzable analog of guanosine triphosphate (GTP), GTPgammaS (10 microM), directly activated phospholipase C (PLC) in a membrane-enriched fraction of the salivary glands after PLC was first incubated with the PGE(2) EP1 receptor antagonist AH-6809, which presumably antagonized endogenous PGE(2) (0.3 microM) in the broken-cell-membrane-enriched fraction. TMB-8, an antagonist of intracellular inositol trisphosphate (IP(3)) receptors, inhibited PGE(2)-stimulated secretion. The results support the hypothesis that PGE(2) stimulates secretion of tick salivary gland protein via a phosphoinositide signaling pathway and mobilization of intracellular Ca(2+).

Prostaglandin E2 in the salivary glands of the female tick, Amblyomma americanum (L.): calcium mobilization and exocytosis.

A cholera toxin-sensitive, prostaglandin E2 (PGE2) specific receptor has been identified in the plasma membrane fraction of tick salivary glands. In the present study, we report that stimulation of dispersed salivary glands of the lone star tick Amblyomma americanum (L.) with 1 nM to 10 microM PGE2 increased the intracellular concentration of inositol trisphosphate (IP3) in a dose-dependent manner. Incubation of dispersed tissue with 1 nM to 10 microM PGE2 also stimulated release of 45Ca2+ from preloaded tissue. PGE2 (10 microM) did not stimulate an influx of 45Ca2+. Therefore, the PGE2 receptor in the salivary glands appears to activate a phosphoinositide phospholipase C signalling pathway to increase formation of intracellular IP3 and, thus, mobilize Ca2+ from intracellular stores. Incubation of dispersed salivary glands with 1 nM to 1 microM PGE2 stimulated secretion of anticoagulant protein, but not at < 1 nM or > 1 microM PGE2. In addition, the mammalian PGE2 EP1 receptor antagonist AH-6809 affected secretion of anticoagulant by dispersed salivary gland tissue at a low concentration supporting the hypothesis that the PGE2 receptor in tick salivary glands is EP1-like. We propose that a major function for PGE2 in tick salivary glands is to mobilize Ca2+ and stimulate secretion (exocytosis) of bioactive proteins into the tick's saliva during feeding.

Identity and synthesis of prostaglandins in the lone star tick, Amblyomma americanum (L.), as assessed by radio-immunoassay and gas chromatography/mass spectrometry.

High concentrations of PGE(2) and PGF(2alpha) were identified by radio-immunoassay (RIA) and/or gas chromatography/mass spectrometry (GC/MS) in the hemolymph, salivary glands and saliva of the lone star tick Amblyomma americanum (L.). Binding studies indicated that PGE(2) was free and not bound to any proteins in the hemolymph. A small amount of 6-keto-PGF(1alpha) (breakdown product of PGI(2); prostacyclin) was also found in the salivary glands but not in the hemolymph or saliva. Neither PGD(2) nor PGA(2)/B(2) was detected in any tick material investigated. Although PGE(2) was found in the gut contents, only small amounts of label crossed the gut into the hemolymph during artificial feeding with labeled PGE(2), indicating that the high amounts of PGE(2) in hemolymph and salivary glands are not sequestered from the host blood meal. Isolated salivary glands and salivary gland homogenates demonstrated robust synthesis of PGE(2) at high concentrations of exogenous arachidonic acid. Synthesis by the salivary glands was monitored by measuring increasing PGE(2) with increasing arachidonic acid by RIA, GC/MS and labeled PGE(2) in the presence of labeled arachidonic acid. Synthesis was inhibited in a dose-dependent manner by indomethacin indicating that the cyclooxygenase synthesizing prostaglandins in ticks shares similarities to the enzyme found in mammals.

Identification of SNARE and cell trafficking regulatory proteins in the salivary glands of the lone star tick, Amblyomma americanum (L.).

Prostaglandin E(2) (PGE(2)) stimulates secretion of tick salivary gland proteins via a phosphoinositide signaling pathway and mobilization of intracellular Ca(2+) (). Highly conserved intracellular SNARE (soluble NSF attachment protein receptors) complex proteins are associated with the mechanism of protein secretion in vertebrate and invertebrate neuronal and non-neuronal cells. Proteins in the salivary glands of partially fed female lone star ticks cross-react individually with antibodies to synaptobrevin-2 (vesicle (v)-SNARE), syntaxin-1A, syntaxin-2 and SNAP-25 (target (t)-SNAREs), cytosolic alpha/beta SNAP and NSF (N-ethylmaleimide-sensitive fusion protein), Ca(2+) sensitive synaptotagmin, vesicle associated synaptophysin, and regulatory cell trafficking GTPases Rab3A and nSec1. V-SNARE and t-SNARE proteins form an SDS-resistant, boiling sensitive core complex in the salivary glands. Antibodies to SNARE complex proteins inhibit PGE(2)-stimulated secretion of anticoagulant protein in permeabilized tick salivary glands. We conclude that SNARE and cell trafficking regulatory proteins are present and functioning in the process of PGE(2)-stimulated Ca(2+) regulated protein secretion in tick salivary glands.

Identification of hemolytic activity in saliva of the lone star tick (Acari:Ixodidae).

Hemolytic activity was identified in the saliva of Amblyomma americanum (L.) when red blood cells from sheep were incubated with tick saliva in the presence of phosphatidylcholine and sodium deoxycholate. The hemolytic activity was destroyed by boiling or treating with trypsin. The hemolytic activity in tick saliva was calcium-dependent, and inhibited by a phospholipase A2 inhibitor oleyloxyethyl phosphorylcholine. Phosphatidylserine could replace phosphatidylcholine in the hemolytic assays but phosphatidylethanolamine and phosphatidylinositol were ineffective. Size exclusion chromatography of tick saliva revealed one peak of hemolytic activity, which correlated with the activity of tick salivary phospholipase A2, both having a molecular weight approximately 55,000 daltons. These results suggest that the hemolytic activity in tick saliva results from salivary phospholipase A2. The hemolytic activity in tick saliva may play a role in lysing host red blood cells, thus facilitating the tick digestive process.

Phospholipase A2 activity in salivary glands and saliva of the lone star tick (Acari: Ixodidae) during tick feeding.

Phospholipase A2 (PLA2) activity levels in tick [Amblyomma americanum (L.)] salivary glands and saliva were examined during tick feeding by using 14C-phosphatidylcholine as the substrate. Saliva produced by stimulating female ticks to salivate with dopamine contains PLA2 (ts-PLA2) activity. The ts-PLA2 activity level in saliva did not change significantly during tick feeding except for a decrease in the last rapid feeding phase (> 200 mg) and in replete ticks. Phospholipase A2 activity was higher in salivary glands of fed than unfed ticks, both in males and females; activity increased during tick feeding correlating with salivary secretory rates during tick feeding suggesting that much of the PLA2 activity measured in whole salivary glands is synthesized for subsequent secretion. During the time course of in vitro salivation, the first 10 microliters of saliva contained higher ts-PLA2 activity than saliva secreted thereafter. Phospolipase A2 was identified in the saliva of artificially fed ticks indicating that ts-PLA2 is a physiological component of tick saliva.

Identification and characterization of anticoagulant activities in the saliva of the lone star tick, Amblyomma americanum (L.).

Anticoagulant activities against both the extrinsic and intrinsic coagulation pathways were identified in the saliva of partially fed female lone star ticks, Amblyomma americanum (L.). The activities of factor Xa and thrombin in the common pathway of the coagulation cascade were inhibited by tick saliva. The greatest anticoagulant activities were found in the saliva of ticks weighing more than 200 mg. The anticoagulant activities in tick saliva could be detected without preincubation of tick saliva with sheep plasma, but preincubation significantly increased the activities. Tick saliva anticoagulant activities were abolished by boiling for 15 min or being treated with trypsin for 1 hr. Phosphatidylcholine (3 mM) and phospholipase A2 inhibitor oleyloxyethyl phosphorylcholine (0.2 mM) did not affect the anticoagulant activities significantly, suggesting that the phospholipase A2 activity found in tick saliva does not contribute to the anticoagulant activities. Size exclusion high performance liquid chromatography revealed that the molecular weights of the anticoagulant activities were approximately 16,000 D. The anticoagulant activities in tick saliva are believed to play an important role in facilitating tick feeding by helping overcome the host hemostatic system.