Southern Cattle Tick Research Articles

Target-based discovery of antagonists of the tick (Rhipicephalus microplus) kinin receptor identifies small molecules that inhibit midgut contractions.

A GPCR (G protein-coupled receptor) target-based approach was applied to identify antagonists of the arthropod-specific tick kinin receptor. These small molecules were expected to reproduce the detrimental phenotypic effects that had been observed in Rhipicephalus microplus females when the kinin receptor was silenced by RNA interference. Rhipicephalus microplus, the southern cattle tick, cattle fever tick, or Asian blue tick, is the vector of pathogenic microorganisms causing the deadly bovine babesiosis and anaplasmosis. The widespread resistance to acaricides in tick populations worldwide emphasizes that exploring novel targets for effective tick control is imperative. Fifty-three structural analogs of previously identified tick kinin antagonists were screened in a 'dual-addition' calcium fluorescence assay using a CHO-K1 cell line expressing the tick kinin receptor. Seven molecules were validated as non-cytotoxic antagonists, four of which were partial (SACC-0428764, SACC-0428780, SACC-0428800, and SACC-0428803), and three were full antagonists (SACC-0428799, SACC-0428801, and SACC-0428815). Four of these antagonists (SACC-0428764, SACC-0428780, SACC-0428799, and SACC-0428815) also inhibited the tick midgut contractions induced by the myotropic kinin agonist analog 1728, verifying their antagonistic bioactivity. The small molecules were tested on recombinant human neurokinin (NK) receptors, the one most similar to the invertebrate kinin receptors. Most molecules were inhibitors of the NK1 receptor, except SACC-0412066, a previously identified tick kinin receptor antagonist, which inhibited the NK1 receptor only at the highest concentration tested (25 μm). None of the molecules inhibited the NK3 human receptor. Molecules identified through this approach could be useful probes for studying the tick kinin signaling system and midgut physiology. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Haemaphysalis longicornis (Acari: Ixodidae) does not transmit Babesia bovis, a causative agent of cattle fever

The Asian longhorned tick (Haemaphysalis longicornis) was first reported in the United States in 2017 and has since been detected in at least 17 states. This tick infests cattle and can produce large populations quickly due to its parthenogenetic nature, leading to significant livestock mortalities and economic losses. While H. longicornis has not been detected in Texas, species distribution models have identified southern Texas as a possible hospitable region for this tick. Southern Texas is currently home to the southern cattle tick (Rhipicephalus microplus), which can transmit the causative agent of cattle fever (Babesia bovis). With the potential for H. longicornis and B. bovis to overlap in southern Texas and their potential to negatively impact the national and global livestock industry, it is imperative to identify the role H. longicornis may play in the cattle fever disease system. A controlled acquisition and transmission experiment tested whether H. longicornis is a vector for B. bovis, with the R. microplus-B. bovis system used as a positive control. Transstadial (nymphs to adults) and transovarial (adults to larvae) transmission and subsequent transstadial maintenance (nymphs and adults) routes were tested in this study. Acquisition-fed, splenectomized animals were used to increase the probability of tick infection. Acquisition nymphs were macerated whole and acquisition adults were dissected to remove midguts and ovaries at five time points (4, 6, 8, 10, and 12 days post-repletion), with 40 ticks processed per time point and life stage. The greatest percentage of nymphs with detectable B. bovis DNA occurred six days post-repletion (20.0 %). For adults, the percentage of positive midguts and ovaries increased as days post-repletion progressed, with day 12 having the highest percentage of positive samples (67.5 % and 60.0 %, respectively). When egg batches were tested in triplicate, all H. longicornis egg batches were negative for B. bovis, while all R. microplus egg batches were positive for B. bovis. During the transmission phase, the subsequent life stages for transstadial (adults) and transovarial transmission/transstadial maintenance (larvae, nymphs, and adults) were fed on naïve, splenectomized calves. All life stages of H. longicornis ticks tested during transmission were negative for B. bovis. Furthermore, the transmission fed animals were also negative for B. bovis and did not show signs of bovine babesiosis during the 45-day post tick transmission period. Given the lack of successful transstadial or transovarial transmission, it is unlikely that H. longicornis is a vector for B. bovis.

In vitro and in silico studies of the acaricidal and anticholinesterase activities of Randia aculeata seeds against the southern cattle tick Rhipicephalus (Boophilus) microplus.

Rhipicephalus (Boophilus) microplus is a leading cause of significant economic losses in the livestock industry, and tick populations have developed multiple forms of resistance to acaricides; therefore, the potential of novel natural bioactive compounds that are effective for targeting ticks must be addressed. The aim of this study was to evaluate the acaricidal and anticholinesterase activities of R. aculeata seeds and to identify naturally occurring compounds that potentially inhibit anticholinesterase through in silico docking. The acaricidal activity of the extract of R. aculeata seeds against larval and adult R. microplus ticks was assessed through immersion tests. Inhibition of anticholinesterase activity was measured spectrophotometrically. Extracts of R. aculeata seeds showed activity against larvae and engorged females of R. microplus, and a reduction in the reproductive index were also observed. Rutin, chlorogenic acid, quercetin, and epicatechin exhibited noteworthy interactions with the active site residues of RmAChE. These findings could significantly contribute to the exploration of novel natural products that can potentially inhibit RmAChE and could be used in the development of new acaricides for tick control.

Efficacy of hydroethanolic extract of Randia aculeata seed against the southern cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) on naturally infested cattle under field conditions.

The cattle tick Rhipicephalus (Boophilus) microplus is a major problem of concern for cattle industry in tropical and subtropical areas. Control of cattle tick is based mainly on the use of chemical acaricides, which has contributed to the emerging problem of selection of resistant tick lineages. Plants have been used as an alternative to conventional acaricidal drugs. On the other hand, the acaricidal activity of hydroethanolic extract of Randia aculeata seed (EHRA) has been demonstrated against R. microplus under laboratory conditions. However, the utility of EHRA seed as a potential acaricidal needs to be determined under field conditions. For this reason, the aim of this study was to evaluate the efficacy of the EHRA against R. microplus sprayed on naturally infested calves, determine the effect of theEHRA seedon acetylcholinesterase activity in R. microplus larval and identify the chemical composition of EHRA. Forty-five male calves were divided in three groups and treated with: G1 water; G2 EHRA 20% w/v and G3 coumaphos 0.2% v/v. Acetylcholinesterase (AChE) activity in R. microplus larvae was determined by a colorimetric assay. The chemical composition of EHRA was accessed through HPLC/MS. Significantly fewer ticks were observed after 24h on the treated group compared to control group. EHRA significantly inhibitedin vitroAChE activity inR.microplus at all tested concentrations. Chlorogenic acid, vanillinic acid, p-coumaric acid, caffeic acid. rutin, quercetin, (-)-epicatechin, 4-hydroxybenzoic acid, quercetin, vanillin, 2,4-dimethoxy-6-methylbenzoic acid, scopoletin and ferulic acid were identified in the extract. The results provided new data for the elucidation of the mechanisms of EHRA acaricide action and to further evaluate the use as a new alternative control agent againstR. microplus under in vivo conditions.

Identification of the G184C, C190A and T2134A mutations in the para-sodium channel gene of the southern cattle tick Rhipicephalus (Boophilus) microplus associated with resistance to cypermethrin in northern Veracruz, Mexico

The southern cattle tick (Rhipicephalus microplus) represents one of the ectoparasites with the greatest distribution worldwide. Infestations by this arthropod can cause a decrease in the production of meat and milk, as well as anaemia and the transmission of bacterial and parasitic agents. For this reason, several active molecules have been developed to control these arthropods. A widely used group of ixodicides are pyrethroids, especially cypermethrin, which have knockdown effects on ticks. Resistance to cypermethrin has been reported in ticks since the 2000s; it was registered for the first time in Mexico in 2009. Even though multiple studies have evaluated resistance with conventional tests, there are few studies in Mexico that have identified the presence of single nucleotide polymorphisms (SNPs) associated with resistance. Hence, the aim of this work was to monitor three mutations associated with resistance in the sodium/chlorine channel in eight populations of ticks from northern Veracruz. Engorged adult females were collected from which genomic DNA was extracted. Subsequently, three mutations in domains II and III of para‑sodium channel gene were detected by conventional PCR and sequencing. Global alignments were done with the reference sequences deposited in GenBank. A total of 116 engorged females were analysed, of which 10 tested positive for G184C and C190A of domain II of the para‑sodium channel gene. T2134A was present in domain III in a single production unit. This is the first work where molecular monitoring of cypermethrin resistance has been carried out in the northern zone of the state of Veracruz.

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens. The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells. The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a "dual-addition" approach can detect functional agonist and antagonist "hit" molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z' factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 µM final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

Pyrokinin receptor silencing in females of the southern cattle tick Rhipicephalus (Boophilus) microplus is associated with a reproductive fitness cost

BackgroundRhipicephalusmicroplus is the vector of deadly cattle pathogens, especially Babesia spp., for which a recombinant vaccine is not available. Therefore, disease control depends on tick vector control. However, R.microplus populations worldwide have developed resistance to available acaricides, prompting the search for novel acaricide targets. G protein-coupled receptors (GPCRs) are involved in the regulation of many physiological processes and have been suggested as druggable targets for the control of arthropod vectors. Arthropod-specific signaling systems of small neuropeptides are being investigated for this purpose. The pyrokinin receptor (PKR) is a GPCR previously characterized in ticks. Myotropic activity of pyrokinins in feeding-related tissues of Rhipicephalussanguineus and Ixodesscapularis was recently reported.MethodsThe R.microplus pyrokinin receptor (Rhimi-PKR) was silenced through RNA interference (RNAi) in female ticks. To optimize RNAi, a dual-luciferase assay was applied to determine the silencing efficiency of two Rhimi-PKR double-stranded RNAs (dsRNA) prior to injecting dsRNA in ticks to be placed on cattle. Phenotypic variables of female ticks obtained at the endpoint of the RNAi experiment were compared to those of control female ticks (non-injected and beta-lactamase dsRNA-injected). Rhimi-PKR silencing was verified by quantitative reverse-transcriptase PCR in whole females and dissected tissues.ResultsThe Rhimi-PKR transcript was expressed in all developmental stages. Rhimi-PKR silencing was confirmed in whole ticks 4 days after injection, and in the tick carcass, ovary and synganglion 6 days after injection. Rhimi-PKR silencing was associated with an increased mortality and decreased weight of both surviving females and egg masses (P < 0.05). Delays in repletion, pre-oviposition and incubation periods were observed (P < 0.05).ConclusionsRhimi-PKR silencing negatively affected female reproductive fitness. The PKR appears to be directly or indirectly associated with the regulation of female feeding and/or reproductive output in R.microplus. Antagonists of the pyrokinin signaling system could be explored for tick control.Graphical abstract

Climate Change and Alternative Hosts Complicate the Eradication of Cattle Fever Ticks (Acari: Ixodidae) in the Southern United States, a Review

AbstractPotential reinvasion of the United States by cattle fever ticks, Rhipicephalus (Boophilus) annulatus (Say) and R. (B.) microplus (Canestrini), which are endemic in Mexico, threatens the domestic livestock industry because these ticks vector the causal agents (Babesia bovis (Babes) (Piroplasmida: Babesidae) and B. bigemina Smith &amp; Kilborne) of bovine babesiosis. The Cattle Fever Tick Eradication Program safeguards the health of the national cattle herd preventing reemergence of bovine babesiosis by keeping the United States cattle fever tick-free. Free-living southern cattle tick, R. (B.) microplus, larvae have been collected from vegetation in the wildlife corridor of Cameron Co.- Willacy Co., Texas. Finding R. microplus larvae on vegetation complements reported infestations in wildlife hosts inhabiting the south Texas coastal plains. Substantial population expansion of native white-tailed deer Odocoileus virginianus (Zimmermann) (Artiodactyla: Cervidae), and exotic nilgai antelope Boselaphus tragocamelus (Pallas) (Artiodactyla: Bovidae), both of which are definitive hosts for the cattle fever tick, support local tick populations independent of cattle. Increasing prevalence of native and exotic wild ungulates, widespread tick acaracide resistance, and climate change, undermine efforts to control bovine babesiosis. Thus, ecological conditions have changed since cattle fever ticks were eradicated from the United States in 1943 using cattle-centric control strategies. These changes complicate efforts by the Cattle Fever Tick Eradication Program to keep cattle in the United States free of these cattle fever disease vectors. Technologies that could be applied to integrated eradication efforts are discussed.

Tick CAPA propeptide cDNAs and receptor activity of endogenous tick pyrokinins and analogs: Towards discovering pyrokinin function in ticks

Pyrokinins (PKs) are pleiotropic neuropeptides with significant roles in invertebrate physiology. Although functions of PKs are known in insects, there is a lack of knowledge of PK-encoding genes and PKs functions in ticks. Herein the first tick cDNAs of the capability (capa) gene were cloned from the southern cattle tick, Rhipicephalus microplus (Acari: Ixodidae), and the blacklegged tick, Ixodes scapularis. Each cDNA encoded one periviscerokinin and five different pyrokinins. Two PKs were identical in sequence in the two species. The three PKs unique to R. microplus (Rhimi-CAPA-PK1, -PK2, and -PK5) were tested on the recombinant R. microplus pyrokinin receptor using a calcium bioluminescence assay. The Rhimi-CAPA-PKs acted as agonists with EC50s ranging from 101–188 nM. Twenty PK analogs designed for enhanced bioavailability and biostability were tested on the receptor. Five of these were designed based on the sequences of the three unique Rhimi-CAPA-PKs. Eight PK analogs were also agonists; four of them were full agonists that exhibited comparable efficacy to the native Rhimi-CAPA-PKs, with EC50 ranging from 401 nM-1.9 μM. The structure-activity relationships (SAR) of all analogs were analyzed. Our results suggested that a positively charged, basic lysine at the variable position X of the PK active core (FXPRLamide) conferred enhanced affinity to the analogs in their interaction with the tick receptor. These analogs are promising tools to elucidate the pyrokinin function in ticks in vivo as these analogs are expected to have prolonged hemolymph residence time in comparison to the native peptides.

Acaricidal activity of the hexanic and hydroethanolic extracts of three medicinal plants against southern cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

The southern cattle tick Rhipicephalus microplus is a major problem for the cattle industry in tropical and subtropical areas worldwide. Chemical products are commonly applied to control it; however, their indiscriminate use has resulted in the appearance of resistant lineages. In the last decades, plants have been used as an alternative to conventional acaricidal drugs, as several plant compounds repel activity, decrease the reproductive potential and reduce the survival rate of ticks. For this reason, the in vitro efficacy of hexanic and hydroalcoholic extracts of Randia aculeata, Moringa oleifera and Carica papaya were evaluated against the larvae and engorged females of R. microplus. Larval packet tests and adult immersion tests were performed with seven concentrations of each of the extracts. The extracts obtained with hydroethanolic solution (polar solvent) exhibited a higher acaricidal activity than extracts prepared with n-hexane (non-polar solvent). Hydroethanolic extracts of R. aculeata seed and shell showed the highest larvicidal activity against R. microplus (100 and 91% mortality, respectively) at a concentration of 100mg/mL. Randia aculeata (seed and shell), M. oleifera and C. papaya treatments at the same concentration (100mg/mL) also resulted in adult mortality of 85, 75, 66 and 55%, respectively. The adult immersion test showed that hydroethanolic extracts derived from R. aculeata seed significantly reduced the index of egg laying and increased the percentage inhibition of oviposition of female ticks at a concentration of 100mg/mL. These results indicate that the tested extracts exhibit acaricidal activity and could be considered as potential agents for the development of alternative natural acaricides against R. microplus.

Efficacy of a water-based botanical acaricide formulation applied in portable spray box against the southern cattle tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae), infesting cattle

Ectoparasitism result in annual losses to livestock producers estimated to reach billions of dollars in tropical and subtropical parts of the world where the southern cattle tick, Rhipicephalus (Boophilus) microplus, is established because this invasive pest is also a vector of pathogens causing bovine babesiosis and anaplasmosis. Reintroduction of R. microplus could be economically devastating for the cattle industry in the United States. Novel technologies are necessary to manage populations of R. microplus that are resistant to multiple classes of synthetic chemicals widely used as acaricides to control ticks infesting livestock, and to mitigate the environmental impact of these synthetic acaricides. Bioassays with a water-based formulation of a commercially available botanical acaricide (Essentria® IC3) at 6.25 % against a laboratory strain of R. microplus was 100 % lethal against unfed larvae, and 94 % mortality was recorded against engorged female ticks. These results prompted documentation of efficacy on infested cattle using an acaricide delivery system mimicking field conditions, which is required to consider the use of a product by integrated tick management programs. Twelve tick naïve cattle were artificially infested with unfed larvae on days -19, -12 and -5. On day -2, ticks from the left-side of the body of each animal were manually counted. Depending on pre-treatment tick counts the animals were assigned into three separate treatment groups: i) water (untreated control); ii) coumaphos 0.3 % in water (positive control); and iii) botanical acaricide 6.25 % in water. Five days after the last infestation, cattle were doused in a spray box and moved to individual stalls to collect tick data for calculation of treatment efficacy. The percentage control, based on the reproduction of surviving engorged female ticks after treatment with the 6.25 % Essentria in water-based spray was 70 % against R. microplus. Coumaphos treatment had a mean percent control of 100 %. Whereas this application of Essentria® IC3 cannot be recommended as a stand-alone method to eradicate R. microplus, the 6.25 % botanical acaricide water-based spray could be part of integrated tick management to control populations of R. microplus resistant to organophosphates like coumaphos and other classes of commercially available synthetic acaricides.

A random small molecule library screen identifies novel antagonists of the kinin receptor from the cattle fever tick, Rhipicephalus microplus (Acari: Ixodidae).

The southern cattle tick, Rhipicephalus microplus, is a primary vector of the deadly bovine disease babesiosis. Worldwide populations of ticks have developed resistance to acaricides, underscoring the need for novel target discovery for tick control. The arthropod-specific R. microplus kinin receptor is such a target, previously validated by silencing, which resulted in female reproductive fitness costs, including a reduced percentage of eggs hatching. In order to identify potent small molecules that bind and activate or inhibit the kinin receptor, a high-throughput screening (HTS) assay was developed using a CHO-K1 cell line expressing the recombinant tick kinin receptor (BMLK3 ). A total of ~20 000 molecules from a random in-house small molecule library were screened in a 'dual-addition' calcium fluorescence assay. This was followed by dose-response validation of the hit molecules identified both from HTS and an in silico screen of ~390 000 molecules. We validated 29 antagonists, 11 of them were full antagonists with IC50 values between 0.67 and 8 μmol L-1 . To explore the structure-activity relationships (SAR) of the small molecules, we tested the activities of seven analogs of the most potent identified antagonist, additionally discovering three full antagonists and four partial antagonists. These three potent antagonists (IC50 < 3.2 μmol L-1 ) were validated in vitro using the recombinant mosquito kinin receptor and showed similar antagonistic activities. In vivo, these three compounds also inhibited the mosquito hindgut contraction rate induced by a myotropic kinin agonist analog 1728. Antagonists identified in this study could become pesticide leads and are reagents for probing the kinin signaling system. © 2021 Society of Chemical Industry.

Status of Rhipicephalus microplus resistance to ivermectin, fipronil and fluazuron in Mato Grosso do Sul, Brazil.

Southern cattle tick resistance to pour-on and injectable acaricides has yet to be evaluated on a broader scope, and the paucity of information on the subject may hinder efforts to control this parasite. The objective of this study was to evaluate the resistance profile of ten populations of Rhipicephalus microplus to the acaricides fluazuron, fipronil and ivermectin in cattle herds in Mato Grosso do Sul, Brazil. The larval immersion test (LIT) was used to evaluate susceptibility to ivermectin and fipronil and the adult immersion test (AIT) was performed to evaluate fluazuron. Samples were randomly obtained in ten farms, and in general, we found resistance in five samples to fluazuron and in four samples to ivermectin and fipronil. Six samples showed incipient resistance to ivermectin and fipronil. Five of the ten evaluated samples showed resistance and/or incipient resistance to all the active ingredients, and the other five to two active ingredients. Among the samples classified as resistant, the average resistance ratio for ivermectin was 2.75 and 3.26 for fipronil. These results demonstrate the advanced status of resistance to the most modern chemical groups for the control of R. microplus in the state of Mato Grosso do Sul.

A point mutation in the β-adrenergic-like octopamine receptor: possible association with amitraz resistance.

Amitraz is a unique formamidine-class acaricide/insecticide that effectively controls ticks, mites, and insect pests. However, the recent emergence of amitraz-resistant cattle ticks is a serious problem that requires an urgent solution. A nonsynonymous single nucleotide polymorphism (A181T) leading to an amino acid substitution (I61F) in the β-adrenergic-like (β-AL) octopamine receptor (OAR) of amitraz-resistant southern cattle ticks (Rhipicephalus microplus) (RmβAOR) was proposed to be a cause of the amitraz resistance. However, it remains unclear whether this substitution exerts any functional effect on the action of amitraz. To make this clear, the functional role of this mutation was examined using an orthologous OAR (BmOAR2) from the silkworm (Bombyx mori). Both amitraz and its metabolite N2 -(2,4-dimethylphenyl)-N1 -methyformamidine (DPMF) elevated intracellular cyclic AMP levels as orthosteric OAR agonists in HEK-293 cells stably expressing BmOAR2. The I45F mutant of BmOAR2 (equivalent to I61F in RmβAOR) was generated and tested for its sensitivity to amitraz and DPMF. The assay result showed that the I45F mutation reduces the potency of DPMF to a level similar to that of the endogenous agonist (R)-OA in wild-type BmOAR2. The amino acid substitution found in the first transmembrane segment of RmβAOR most likely causes target-site insensitivity to DPMF, which might contribute to the resistance of R. microplus to amitraz. This needs to be further confirmed using RmβAOR. © 2020 Society of Chemical Industry.

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.

Rhipicephalus (Boophilus) microplus (Ixodida: Ixodidae) Larvae Collected From Vegetation in the Coastal Wildlife Corridor of Southern Texas and Research Solutions for Integrated Eradication.

The potential for reinvasion of the United States by cattle fever ticks, Rhipicephalus (Boophilus) annulatus and Rhipicephalus microplus (Canestrini), which remain established in Mexico, threatens the viability of the domestic livestock industry because these ticks vector the causal agents (Babesia bovis and Babesia bigemina) of bovine babesiosis. The Cattle Fever Tick Eradication Program safeguards the health of the national cattle herd preventing the reemergence of bovine babesiosis by keeping the United States free of cattle fever ticks. Here, the collection of free-living southern cattle tick, R. microplus, larvae by sweeping flannel flags over vegetation in the wildlife corridor of Cameron and Willacy Counties, TX, is reported. Finding R. microplus larvae on vegetation complements reports of infestations in wildlife hosts inhabiting the southern Texas coastal plains. Land uses and environmental conditions have changed since cattle fever ticks were eradicated from the United States by 1943. These changes complicate efforts by the Cattle Fever Tick Eradication Program to keep cattle in the United States free of the cattle fever tick disease vectors. Current scientific research on technologies that could be used for area-wide management of fever tick larvae in south Texas and how this could be applied to integrated eradication efforts are discussed.

Biocidal potential of Eugenia pyriformis essential oil in the control of Rhipicephalus (Boophilus) microplus in the free-living cycle

ABSTRACT: Brazil has one of the largest commercial cattle herds in the world, which naturally coexist with an enormous number of parasitic species. Southern cattle tick, Rhipicephalus (Boophilus) microplus, is among these species, interfering with animal productivity and causing losses to the beef and dairy cattle sector. The use of chemical acaricides in the control of this mite has resulted in the emergence of resistant populations. In this sense, alternative control measures using plants as sources of botanical acaricides have shown to be effective. Eugenia pyriformis Cambess is a Brazilian plant with antioxidant and antimicrobial activity; however, there are no reports on its acaricidal activity in the literature. The present study aimed to evaluate the acaricidal and larvicidal potential of E. pyriformis leaf essential oil (EO) on southern cattle tick at different stages of the reproductive cycle. E. pyriformis leaves were collected and dried, and had their EO extracted by hydrodistillation (3h) using a modified Clevenger apparatus. Chemical analysis was performed by gas chromatography coupled with mass spectrometry (GC/MS), and 32 compounds belonging to the sesquiterpene class were identified: hydrocarbons (17.98%) and oxygenated forms (81.96%), with spathulenol (43.65%) and caryophyllene oxide (12.17%) as the most common. The EO was evaluated by the Adult Immersion Test at the concentrations (500.00 to 3.12mg/mL) in which the following parameters were measured: mortality of females (%), hatchability of eggs (%), and product efficiency (%). Larvae were assessed by the Larval Packet Test at concentrations ranging from 25.00 to 0.00004mg/mL. Lethal concentrations (LC) required for killing 50 and 99.9% of adult females and larvae were determined using Probit analysis. LC50 and LC99.9 of EO were 0.06 and 24.60mg/mL and 1,208.80 and 2,538mg/mL for larvae and adult females, respectively. Action of the EO in the free-living cycle of R. (B.) microplus larvae was another parameter assessed. To this end, the larvae were deposited in pots containing Brachiaria decumbens and, after migration to the leaf apex, a solution containing LC99.9 (24.60mg/mL) of the EO was sprayed. After 24h, 72.25% of the larvae had died, indicating stability of the EO when subjected to uncontrolled temperature and humidity conditions. The mechanism of action through which the EO killed the larvae and adult females was investigated by the Bioautographic Method, which showed inhibition of 3.15mg/mL of the EO on the acetylcholinesterase (AChE) enzyme. The results found in the present experiment indicate that E. pyriformis essential oil is an alternative in the control of southern cattle tick in the larval (parasitic) and free-living cycle (non-parasitic) stages under field conditions.

Acaricidal activity and effects of acetylcarvacrol on Rhipicephalus (Boophilus) microplus (Canestrini, 1888) engorged female ticks (Acari: Ixodidae).

ABSTRACTThis study aimed to verify the acaricidal potential of acetylcarvacrol (5-isopropyl-2-methylphenol acetate) in Rhipicephalus (Boophilus) microplus engorged female ticks, by determining the lethal concentration for 50, 90 and 99% of the ticks (LC50, LC90 and LC99, respectively). The acetylated derivative of carvacrol was obtained from the reaction with acetic anhydride. The final product was identified from its melting point and by infrared spectroscopy. In order to evaluate the acaricidal potential and the lethal concentrations, engorged females were exposed to the adult immersion test at the following concentrations of acetylcarvacrol diluted in 3% DMSO: 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 μL/mL. This experiment was performed in triplicate. The LC50, LC90 and LC99 (with 95% confidence intervals) for R. microplus females were 4.62 μL/mL (4.45–4.78), 7.97 μL/mL (7.68–8.26) and 8.73 μL/mL (8.39–9.07), respectively. Tegumentary alterations and dorsal circular deformation were observed in some ticks. Moreover, many individuals were considered in a knockdown state, with no ability to move, although still alive. The present study demonstrated that the acetylation of carvacrol enhances its activity on the southern cattle-tick, causing higher mortality rates even at low concentrations, besides numerous physiological and morphological alterations, opening new paths for tick control.

126 Detection of fecal chemistry changes in cattle infested with the Southern Cattle Tick, Rhipicephalus (Boophilus) microplus

Abstract Bovine babesiosis, a highly fatal tick-borne disease of cattle, was eliminated from the United States in the last century through tick eradication programs against 2 species of cattle fever ticks. The threat to the U.S. cattle industry continues through tick introductions from Mexico. The standard method of detecting Cattle Fever Ticks [Rhipicephalus (B.) annulatus and R. (B.) microplus] in the state-federal tick eradication program is physical examination of restrained cattle to find attached ticks. New methods of detecting tick-infested cattle could improve reliability and reduce animal stress. The objective of this study was to determine whether changes in fecal chemistry induced by tick infestation was detectable using near-infrared reflectance spectroscopy (NIRS). Fecal samples were collected daily from 6 stanchioned Bos taurus yearling heifers (initial mean weight 163.3 kg +/- 4.7 kg) at the USDA Cattle Fever Tick Research Laboratory, Edinburg, TX, before, during, and after an infestation of 5000 R. (B.) microplus larvae. Cluster analyses were conducted using GRAMS IQ for NIR spectra in the 576–1126 nm range to test for fecal chemistry changes different from pre-infestation condition, and coincident with the biological phases of the tick infestation. The first three factors of spectral variation accounted for 87.87% of spectral variation among all samples. Factors 1, 2, and 3 had F-Ratios for the Reduced Eigenvalues of 941.59, 387.44, and 221.79, respectively. Three-dimensional analysis for these 3 factors shows shifts in sample clusters away from pre-infestation and coincident with progressive tick blood-feeding and post-infestation recovery. We conclude that fecal NIRS may provide a tool for detection of tick-infested cattle; however, further testing is needed to determine the sensitivity of detection on cattle with varying levels of tick burden, and a protocol developed and evaluated for fecal sampling under field conditions

Baculoviral Expression of Presumptive OP-Resistance Mutations in BmAChE1 of Rhipicephalus (Boophilus) microplus (Ixodida: Ixodidae) and Biochemical Resistance to OP Inhibition.

The southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini), transmits bovine babesiosis and anaplasmosis, and is endemic to Mexico, Latin and South America. Rhipicephalus (B.) microplus infestations within the United States are a continuing threat to U.S. cattle producers. An importation barrier between Texas and Mexico keeps the ticks from re-entering the United States. All cattle imported into the United States are dipped in an organophosphate (OP) acaricide and hand inspected for presence of ticks. Tick resistance has developed to most available acaricides, including coumaphos, the OP used in the cattle dip vats. OP-resistance can result from one or more mutations in the gene encoding the enzyme, acetylcholinesterase (AChE), resulting in production of an altered AChE resistant to OP inhibition. Previous research reported a large number of BmAChE1 mutations associated with OP resistance. We report baculovirus expression of recombinant tick BmAChE1 (rBmAChE) enzymes containing a single resistance-associated mutation, to assess their contribution to OP inhibition resistance. Surprisingly, of the naturally occurring BmAChE1 resistance-associated mutations, only D188G resulted in markedly reduced sensitivity to OP-inhibition suggesting that OP-insensitivity in BmAChE1 may result from the D188G mutation, or may possibly result from multiple mutations, each contributing a small decrease in OP sensitivity. Furthermore, an OP-insensitivity mutation (G119S) found in mosquitoes was expressed in rBmAChE1, resulting in 500-2000-fold decreased sensitivity to OP inhibition. Recombinant BmAChE1 with the G119S mutation demonstrated the lack of any structural prohibition to broad and high-level OP-insensitivity, suggesting potential increases in tick OP-resistance that would threaten the U.S. importation barrier to ticks.