RESUMO
Rhipicephalus microplus, the "common cattle tick", is the most important ectoparasite in livestock worldwide due to the economic and health losses it produces. This tick is a vector for pathogens of several tick-borne diseases. In Latin American countries, damages reach approximately USD 500 million annually due to tick infections, as well as tick-borne diseases. Currently, resistant populations for every chemical group of acaricides have been reported, posing a serious problem for tick control. This study aims to find new alternatives for controlling resistant ticks with compounds derived from small synthetic organic molecules and natural origins. Using BME26 embryonic cells, we performed phenotypic screening of 44 natural extracts from 10 Mexican plants used in traditional medicine, and 33 compounds selected from our chemical collection. We found 10 extracts and 13 compounds that inhibited cell growth by 50% at 50 µg/mL and 100 µM, respectively; the dose-response profile of two of them was characterized, and these compounds were assayed in vitro against different life stages of Rhipicephalus microplus. We also performed a target-directed screening of the activity of triosephosphate isomerase, using 86 compounds selected from our chemical collection. In this collection, we found the most potent and selective inhibitor of tick triosephosphate isomerase reported until now. Two other compounds had a potent acaricidal effect in vitro using adults and larvae when compared with other acaricides such as ivermectin and Amitraz. Those compounds were also selective to the ticks compared with the cytotoxicity in mammalian cells like macrophages or bovine spermatozoids. They also had a good toxicological profile, resulting in promising acaricidal compounds for tick control in cattle raising.
Assuntos
Acaricidas , Doenças dos Bovinos , Rhipicephalus , Doenças Transmitidas por Carrapatos , Animais , Bovinos , Acaricidas/farmacologia , Triose-Fosfato Isomerase , Extratos Vegetais/química , Doenças dos Bovinos/parasitologia , Larva , MamíferosRESUMO
Ticks are important ectoparasites with a worldwide distribution. The most commonly used method for tick control involves the use of acaricides. The main problem is that its indiscriminate use has led to the selection of resistant tick populations. Glutathione transferases (GSTs) are enzymes that play an important role in the detoxification of several types of compounds used in commercial tick control products. This work aims to find new bioactive molecules through in vitro assays with a panel of 160 molecules with putative inhibitory activity on the Rhipicephalus microplus GST enzyme (RmGST). Also, selected molecules were tested against GSTs from other tick species; Rhipicephalus decoloratus, Amblyomma variegatum, Rhipicephalus appendiculatus, and Haemaphysalis longicornis. The first screening on RmGST identified 30 compounds with the ability to modify the enzymatic activity of this enzyme. These compounds included different chemical families, like chalcones, diarylideneketones, flavone, thiazoles, thiourea, steroids, thiadiazines, indazoles, and hydrazine. The most potent compounds against RmGST belong to the diarylideneketones family with an inhibition concentration of 50% of activity (IC50) between 7-50 µM. Interestingly, one of the most potent compounds was also an inhibitor of the GST from other tick species. Experiments with R. microplus adults and larvae showed toxicity at 150 µM, suggesting a potential acaricidal effect of these molecules.
Assuntos
Acaricidas , Rhipicephalus , Infestações por Carrapato , Acaricidas/farmacologia , Animais , Glutationa Transferase , Larva , Infestações por Carrapato/parasitologiaRESUMO
The resistance of Rhipicephalus microplus to acaricides is a serious control problem, so its early diagnosis by a molecular technique is important. This study aims to develop a multiplex allele-specific polymerase chain reaction (PCR) for single-nucleotide polymorphisms (SNPs) in the para-sodium channel gene and in the GABA-Cl gene, associated with pyrethroids (cypermethrin and flumethrin) and fipronil resistance, respectively. We used 22 tick field isolates from farms with tick control problems (sampling convenience). These farms are located in departments of northern Uruguay. Three mutations in the sodium channel gene (Domain II S4-5: C190A and G215T; domain III S6: T2134A) and one in the GABA-Cl gene (A286S/L: CG856CC/TG) were studied. Mutations G215T and T213A were not detected. In all field isolates, the resistant allele (R) for C190A mutation (knockdown resistance, kdr) was detected, mainly in heterozygous individuals (SR) (11.1% to 86.7%). The highest incidence of the kdr mutant allele occurred in the Tacuarembó tick field isolates, where on 7 out of 10 farms >30% of individuals were SR and on one farm > 30% of individuals were RR. The next highest was Artigas (half of farms had>30% SR individuals and a quarter had >30% RR individuals). The resistance to dieldrin locus (rdl) mutation (CG856CC/TG) was absent in five field isolates. The highest incidenceof the mutant allele was observed in ticks from farms in Rivera (all farms had SR in >30% of individuals and two farms had RR in >12.5 and >16.7% of individuals) followed by farms in Tacuarembó (3 of 10 farms had >30% SR and 2 with >30% RR). Less than half of the farms had rdl in homozygous individuals. No significant association was observed between phenotypic bioassays and the rdl resistance allele. Several field isolates were phenotypically susceptible to the presence of the rdl allele. Several causes are possible (bioassay sensitivity, discriminating concentration). Individuals with simultaneous kdr and rdl mutations were present in 17 field isolates, and their frequency varied between 0.06% and 60%. Genotypic analysis shows that tick resistance to both acaricides, especially pyrethroids, is a serious problem. It is important to monitor the resistance using molecular techniques to plan efficient control measures. This is the first report describing kdr and rdl detection in R. microplus in Uruguay.
Assuntos
Resistência a Inseticidas/genética , Rhipicephalus/genética , Canais de Sódio/genética , Ácido gama-Aminobutírico/genética , Acaricidas/farmacologia , Animais , Mutação , Patologia Molecular/métodos , Polimorfismo de Nucleotídeo Único , Pirazóis/farmacologia , Piretrinas/farmacologia , Rhipicephalus/efeitos dos fármacos , Infestações por Carrapato/epidemiologia , Uruguai/epidemiologiaRESUMO
Attempts to eliminate Rhipicephalus microplus from Uruguay have been unsuccessful, and, currently, the country is divided into two areas: a tick-free area and a tick-infested area. In the tick-infested area, different farms face different situations. Some farms are in regions where, due to environmental conditions or a lack of infrastructure, it is difficult to eliminate R. microplus, and the only option is to control it. In contrast, other farms can attempt complete removal. Before deciding whether a farmer should attempt to eliminate R. microplus, the probability of reintroduction must be evaluated. The objective of this study was to develop a probabilistic model based on a Bayesian Belief Network (BBN) to assess the likelihood of a farm becoming infested with R. microplus via the introduction of tick-infested cattle. Only the tick-infested area was considered in the development of this model. Nine variables related to environmental conditions and biosecurity measures, with a focus on cattle movement, were considered. Three different sources of data were used to populate the BBN model: data from the literature; a representative national survey from 2016; and a survey developed to identify biosecurity practices on farms. Model sensitivity and specificity were assessed, and an overall accuracy of 92% was obtained. The model was applied to 33 farms located in the tick-infested area. For one farm, the probability of introduction of R. microplus was 1%; for three farms, the probability was between 21% and 34%; for seven farms, it was between 66% and 76%; and for 22 farms, the probability was greater than 83%. This model was useful for estimating the probability of the introduction of R. microplus into farms, making it possible to assess the impact that the evaluated biosecurity measures have on the probability of introduction and, thus, guiding more objective decision making about the control or elimination of R. microplus from farms.