Phenotypic and Target-Directed Screening Yields New Acaricidal Alternatives for the Control of Ticks.

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.

Novel tick glutathione transferase inhibitors as promising acaricidal compounds.

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.

Molecular survey of pyrethroid and fipronil resistance in isolates of Rhipicephalus microplus in the north of Uruguay.

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.

Pyrazol(in)e derivatives of curcumin analogs as a new class of anti-Trypanosoma cruzi agents.

Aim: We report the synthesis and biological evaluation of a small library of 15 functionalized 3-styryl-2-pyrazolines and pyrazoles, derived from curcuminoids, as trypanosomicidal agents. Methods & results: The compounds were prepared via a cyclization reaction between the corresponding curcuminoids and the appropriate hydrazines. All of the derivatives synthesized were investigated for their trypanosomicidal activities. Compounds 4a and 4e showed significant activity against epimastigotes of Trypanosoma cruzi, with IC50 values of 5.0 and 4.2 µM, respectively, accompanied by no toxicity to noncancerous mammalian cells. Compound 6b was found to effectively inhibit T. cruzi triosephosphate isomerase. Conclusion: The up to 16-fold higher potency of these derivatives compared with their curcuminoid precursors makes them a promising new family of T. cruzi inhibitors.

Probability of Rhipicephalus microplus introduction into farms by cattle movement using a Bayesian Belief Network.

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.

Influence of selective treatment of bulls on the infestation of Haematobia irritans on untreated cows.

The distribution of Hematobia irritans is not homogeneous in cattle populations. Generally, between 15% and 30% of bovines carry more than 50% of the flies, and higher infestations occur in bulls. In this study, the effect of treating the bulls with ear tags containing 40% diazinon on the infestation of flies on the herd during the breeding season was evaluated. Four groups of cattle were made, each containing one bull and 35 Braford cows. Groups 1 and 2 were on pastures in highlands. Groups 3 and 4 were on pastures in lowlands. At the beginning of the trial, each bull from Groups 1 and 3 was treated with an ear tag. Bulls from Groups 2 and 4 remained untreated. Weekly, for 10 weeks, horn flies were counted from each bull and 15 cows from each group. A repeated measures linear mixed model was used with cows as random effects. The number of horn flies in the treated groups was lower than that in the untreated group, suggesting that selective treatment of bulls could be an effective strategy to control horn flies on the herd. The number of flies was higher in the lowland than that in the highland groups, suggesting an effect of the environment on horn fly infestations. In addition, we estimated the cow level predictions from the model to estimate the cow effect on fly counts, and we compared those values with the observed weekly quartiles for each group. Fly-susceptible cows were defined as those for which the infestation of flies appeared in the upper quartile more than 50% of the weeks and in the lower quartile less than 20% of the weeks. In contrast, a fly-resistant cow was defined as one for which the fly counts appeared in the lower quartile more than 50% of the weeks and in the upper quartile less than 20% of the weeks. In all, 8.3% of the cows were susceptible and 15% were resistant. These results may be used to selectively treat bulls or to treat the bulls and the most susceptible cows during the breeding season.