Encapsulation of entomopathogenic fungal conidia: evaluation of stability and control potential of Rhipicephalus microplus

The use of chemical acaricides is the primary strategy to control tick infestations. Nonetheless, chemical resistance in ticks has been reported. Thus, complementary methods such as biological control using entomopathogenic fungi (EPF) have been investigated. EPF, although efficient, have their viability compromised when applied under natural conditions, which indicates that formulation development is essential. Some researchers have demonstrated the efficacy of ionic gelation in protecting EPF against deleterious abiotic factors. In the present study, we conducted the ionic gelation technique to encapsulate Metarhizium anisopliae (Metschn.) Sorokin (Hypocreales: Clavicipitaceae) conidia in 2% (EC 2%) and 3% (EC 3%) sodium alginate. Next, the quantity and viability of encapsulated conidia (EC) were determined. The morphology of particles was characterized by using Scanning Electron Microscopy (SEM). EC and non-encapsulated conidia (NEC) were stored at room temperature (26.8 °C) and in the freezer (-11.9 °C) to shelf-life testing. For UV-B irradiance tolerance and thermotolerance tests, EC and NEC were exposed to UV-B (6.0 or 8.0 kJ m ^− 2) and heat (42 ºC). In addition, biological parameters of Rhipicephalus microplus Canestrini (Acari: Ixodidae) engorged females exposed to EC were evaluated. The particles presented a spherical shape, more homogeneous (EC 2%) or heterogeneous (EC 3%). Encapsulation decreased (4.8×) the conidial concentration and did not affect their viability. On the other hand, encapsulation increased the shelf life of conidia at room temperature as well as their UV-B tolerance and thermotolerance (6 h). The fungal particles decreased the biological parameters of females more significantly than the NEC. As far as we know, we reported for the first time the use of the ionic gelation to encapsulate entomopathogenic fungi toward controlling R. microplus.     

Effects of carvacrol and thymol on the antioxidant and detoxifying enzymes of Rhipicephalus microplus (Acari: Ixodidae)

The present study was conducted to evaluate the effects of carvacrol and thymol on the antioxidant and detoxifying enzymes of larvae from two populations of R. microplus: Jaguar (tick population resistant to six classes of acaricides) and Porto Alegre (susceptible tick population). Carvacrol and thymol were tested at concentrations ranging from 0.14 to 5.0 mg mL^?1 in both populations to determine the LC₅₀. In addition, the LC₁, LC₂₅, and LC₇₅ were estimated using the LC₅₀ and HillSlope of each compound. Larvae of both populations of R. microplus were then treated with the LC₁, LC₂₅, LC₅₀, and LC₇₅ of each monoterpene, and those that survived were processed to evaluate the effects of the compounds on the antioxidant and detoxifying systems of larvae; these effects were assessed by determining the activity of the enzymes, glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX). Larvae from the Jaguar population treated with different lethal concentrations of carvacrol and thymol displayed a dose-dependent increase in CAT, GPX, SOD, and GST after treatment with LC₂₅. Further, larvae treated with the LC₇₅ had the highest levels of enzyme activity for carvacrol (1.76 mg mL^?1) and thymol (1.32 mg mL^?1). CAT, GPX, SOD, and GST activity in Porto Alegre population larvae treated with carvacrol and thymol also increased significantly up to the LC₅₀ of each monoterpene. However, at the LC₇₅ of carvacrol and thymol, a decrease in the activity of all enzymes was observed for this tick population. These findings indicate that carvacrol and thymol induced increased activity of all evaluated enzymes at different lethal concentrations in R. microplus larvae from two populations. Such findings unveil the possible mechanisms of action of these natural acaricides.     

长角血蜱和微小扇头蜱的形态与分子生物学鉴定

目的 建立鉴定长角血蜱和微小扇头蜱的形态学和分子生物学相结合的方法.方法 采集自湖北和河南省布尼亚新病毒疫区家养动物体表寄生的蜱及草丛、灌木中的蜱;先以形态学鉴定,后用PCR方法扩增得到蜱的12S rDNA,测序后进行同源性和系统进化分析.结果 形态学方法鉴定采集到两种蜱:长角血蜱(Haemaphysalis longicornis)和微小扇头蜱(Rhipicephalus microplus).蜱的12S rDNA经克隆、测序,用PAUP 4.0软件构建系统发生树,湖北、河南省两种蜱的12S rDNA序列分别与长角血蜱和微小扇头蜱聚类,与形态学鉴定结果一致.结论 在传统形态学分类的基础上结合分子生物学鉴定方法能更准确地鉴定蜱的种类.     

长角血蜱和微小扇头蜱的形态与分子生物学鉴定

目的 建立鉴定长角血蜱和微小扇头蜱的形态学和分子生物学相结合的方法.方法 采集自湖北和河南省布尼亚新病毒疫区家养动物体表寄生的蜱及草丛、灌木中的蜱;先以形态学鉴定,后用PCR方法扩增得到蜱的12S rDNA,测序后进行同源性和系统进化分析.结果 形态学方法鉴定采集到两种蜱:长角血蜱(Haemaphysalis longicornis)和微小扇头蜱(Rhipicephalus microplus).蜱的12S rDNA经克隆、测序,用PAUP 4.0软件构建系统发生树,湖北、河南省两种蜱的12S rDNA序列分别与长角血蜱和微小扇头蜱聚类,与形态学鉴定结果一致.结论 在传统形态学分类的基础上结合分子生物学鉴定方法能更准确地鉴定蜱的种类.

Abstract：

Objective To establish a method combined morphology and molecular marker for identifying Haemaphysalis longicomis and Rhipicephalus microplus. Methods Ticks were collected from domestic animals and wild environment in epidemic area of Hubei and Henan provinces where cases of fever with thrombocytopenia syndrome were prevalent. We classified the ticks by morphology characteristics before 12S rDNA of ticks were amplified by PCR and subsequently sequenced. Phylogenetic tree was constructed by PAUP4.0. Results The ticks belonged to Haemaphysalis longicomis and Rhipicephalus microplus through observation and analysed by the morphological characteristics of the ticks. 12S rDNA was cloned and sequenced while data confirmed the morphological identification of the results. Conclusion The method based on morphology that combined with molecular marker seemed a good method for the identificaton of ticks.     

Functional characterization of candidate antigens of Hyalomma anatolicum and evaluation of its cross-protective efficacy against Rhipicephalus microplus

Hyalomma anatolicum and Rhipicephalus microplus seriously affect dairy animals and immunization of host is considered as a sustainable option for the management of the tick species. Identification and validation of protective molecules are the major challenges in developing a cross-protective vaccine. The subolesin (SUB), calreticulin (CRT) and cathepsin L-like cysteine proteinase (CathL) genes of H. anatolicum were cloned, sequenced and analysed for sequence homology. Both Ha-SUB and Ha-CRT genes showed very high level of homogeneity within the species (97.6–99.4% and 98.2–99.7%) and among the tick species (77.3–99.3% and 85.1–99.7%) while for Ha-CathL the homogeneity was lower among ticks (57.5–89.5%). Besides tick species, both Ha-SUB and Ha- CRT genes showed high level of homogeneity with dipterans (47.2–53.4% and 72.0–74.4%) and nematodes (64.0% by CRT). The level of expression of the conserved genes in different stages of the tick species was studied. The differences in fold change of expression (FCE) of the targeted genes in life stages of tick were not statistically significant except Ha-SUB in eggs and in frustrated females, Ha-CRT in fed male and Ha-CathL in unfed and frustrated females where highest FCE was recorded. The functional properties of the genes were studied by RNAi technology and a significant level of gene suppression (p < 0.05) resulted in very low percentage of engorgement of treated ticks viz., 3.7%, 11.1% and 30.0% in Ha-SUB, Ha-CRT and Ha-CathL respectively, in comparison to control was recorded. The recombinant proteins rHa-SUB, rHa-CRT and rHa-CathL encoded by the genes were expressed in prokaryotic expression system. They were evaluated for cross-protective efficacy and found to be respectively, 65.4%, 41.3% and 30.2% protective against H. anatolicum and 54.0%, 37.6% and 22.2%, against R. microplus infestations.     

Comparison of the efficacy of different methods to apply acaricides for control of Rhipicephalus (Boophilus) microplus

The present study compared the efficacy of different methods to apply an acaricide formulation to control Rhipicephalus (Boophilus) microplus. To compare the methods, an acaricide blend containing three active ingredients (a pyrethroid and two organophosphates) was used. In experiment 1 (farm 1: Goiânia, GO, Brazil), three methods were tested: a backpack sprayer (BS), power sprayer (PS) and spray race (SR). In experiment 2 (farm 2: São José do Rio Pardo, SP, Brazil), two methods were tested: BS and PS. In both experiments, 10 cattle with similar tick burdens were used. On day 0 in both experiments, the animals were treated with the acaricide. On day +1 (only in experiment 1), +3, +7, +14, +21, +28 and +35 (only in experiment 2), tick counts were performed to determine the control efficacy. The time application, pressure (KPa), volume applied (L) and ergonomic aspects of each spraying system were also evaluated. The adult immersion test (AIT) using three different acaricide blends (combinations of pyrethroid + organophosphate) was performed to compare the susceptibility of strains of each farm. In experiment 1, all treatments significantly reduced (p < 0.05) the number of ticks on the animals, and PS resulted in the greatest acaricide efficacy since day +1. In experiment 2, both treatments (PS and BS) reduced (p < 0.05) the tick burden, and as observed in experiment 1, PS resulted in the best reduction. The application times were 4.5, 150 and 330 s, while pressures were 306.8, 4,826.3 and 220.6 KPa for SR, PS and BS, respectively. In the AIT, the efficacy values were between 99.8 and 100% for the tick strain form farm 1 (Goiânia), while for tick strain from farm 2 (São José do Rio Pardo), the efficacy was between 67.2 and 80.9%. We conclude that the sprayer methods chosen influences the efficacy of the acaricide. All sprayer methods were efficient for acaricide application; the best efficacy was obtained using the PS, while the SR resulted in good efficacy and lower application time. The strain from farm 2 was less susceptible to all acaricides tested.     

Rhipicephalus microplus: An overview of vaccine antigens against the cattle tick

Rhipicephalus microplus, popularly known as the cattle tick, is the most important tick of livestock as it is responsible for significant economic losses. The use of chemical acaricides is still the most widely used control method despite its known disadvantages. Vaccination would be a safe alternative for the control of R. microplus and holds advantages over the use of chemical acaricides as it is environmental-friendly and leaves no residues in meat or milk. Two vaccines based on the Bm86 protein were commercialized, TickGARD® and Gavac®, with varying reported efficacies in different countries. The use of other vaccines, such as Tick Vac®, Go-Tick®, and Bovimune Ixovac® have been restricted to some countries. Several other proteins have been analyzed as possible antigens for more effective vaccines against R. microplus, including peptidases, serine proteinase inhibitors, glutathione S-transferases, metalloproteases, and ribosomal proteins, with efficacies ranging from 14% to 96%. Nonetheless, more research is needed to develop safe and efficient tick vaccines, such as the evaluation of the efficacy of antigens against other tick species to verify cross-reactivity and inclusion of additional antigens to promote the blocking of the infection and spreading of tick-borne diseases. This review summarizes the discoveries of candidate antigens for R. microplus tick vaccines as well as the methods used to test their efficacy.     

Phenotypic evaluation of genetic resistance to the tick Rhipicephalus (Boophilus) microplus in Argentine Creole cattle

The objective of this work was to characterize the Argentine Creole cattle breed through the identification of individual phenotypic variations in the levels of infestation with Rhipicephalus (Boophilus) microplus. We evaluated 179 heifers exposed to successive artificial infestations from 2015 to 2018, achieving a total of 663 observations. Tick counts were assessed with the linear mixed model, considering year of evaluation, time of infestation, dam's age and nutritional status during the evaluated period as fixed effects. The average tick count value obtained allowed to classify the breed as highly resistant to the tick charge (99.3%). Although the previous nutritional condition of the animals did not affect the individual charge response, weight gain during the trial showed a significantly negative correlation. We conclude that the Argentine Creole breed is an attractive genetic alternative for cattle breeding in endemic regions, either as a pure breed or a cross-breed.     

Efficacy of entomopathogenic nematodes in insect cadaver formulation against engorged females of Rhipicephalus microplus (Acari: Ixodidae) in semi-field conditions

The present study evaluated, in the laboratory, the virulence of Heterorhabditis bacteriophora LPP30 against engorged females of Rhipicephalus microplus; in addition, we evaluated, in semi-field tests, the effects of four isolates of Heterorhabditis spp. (i.e., H. bacteriophora HP88, Heterorhabditis baujardi LPP7, Heterorhabditis indica LPP1 and H. bacteriophora LPP30) in insect cadaver formulation against the non-parasitic phase of R. microplus. In the first experiment (in vitro), engorged females were exposed, in Petri dish, to H. bacteriophora LPP30 at 75, 150, 300, 600, 1200 and 2400 nematodes/tick (10 ticks per concentration tested). In the second experiment (semi-field), five engorged females and four cadavers of Galleria mellonella infected with nematodes were placed in pots with soil and grown Brachiaria decumbens; the pots were hold outdoor, exposed to natural environment conditions during the tests. In the third experiment (semi-field), 65 days after the cadavers had been placed in the pots for the second experiment, new engorged females (five per pot) were placed in the pots of the groups treated with H. bacteriophora HP88 or H. baujardi LPP7, to assess their persistence in the soil. In the first test, the percent control was higher than 95% in all treatment groups. In the second experiment, at day 22, the mean mortality rate was 78% in the groups treated with H. bacteriophora LPP30 or H. indica LPP1, and reached 100% and 98% when treated with H. bacteriophora HP88 and H. baujardi LPP7, respectively. In this experiment, the egg-laying inhibition index was higher than 90% in the groups treated with H. bacteriophora HP88 (97.2%) or H. baujardi LPP7 (91.9%). In the third experiment with H. bacteriophora HP88 and H. baujardi LPP7, the egg-laying inhibition index was 59.1% and 43.1%, respectively. We concluded that the isolate LPP30 was highly virulent under laboratory conditions, whereas in semi-field tests, HP88 and LPP7 were the most effective isolates. Moreover, HP88 and LPP7 remained active against engorged females of R. microplus in the soil for 65 days after application of EPN-infected cadavers of G. mellonella.     

Efficacy of Rhipicephalus (Boophilus) microplus Bm86 against Hyalomma dromedarii and Amblyomma cajennense tick infestations in camels and cattle

The recombinant Bm86-based tick vaccines have shown their efficacy for the control of cattle ticks, Rhipicephalus (Boophilus) microplus and R. annulatus infestations. However, cattle ticks often co-exist with multi-host ticks such as Hyalomma and Amblyomma species, thus requiring the control of multiple tick infestations for cattle and other hosts. Vaccination trials using a R. microplus recombinant Bm86-based vaccine were conducted in cattle and camels against Hyalomma dromedarii and in cattle against Amblyomma cajennense immature and adult ticks. The results showed an 89% reduction in the number of H. dromedarii nymphs engorging on vaccinated cattle, and a further 32% reduction in the weight of the surviving adult ticks. In vaccinated camels, a reduction of 27% and 31% of tick engorgement and egg mass weight, respectively was shown, while egg hatching was reduced by 39%. However, cattle vaccination with Bm86 did not have an effect on A. cajennense tick infestations. These results showed that Bm86 vaccines are effective against R. microplus and other tick species but improved vaccines containing new antigens are required to control multiple tick infestations.     

Rotational and selective protocols using acaricides to control a multi-resistant strain of Rhipicephalus microplus under field conditions in Southern Brazil

The cattle tick, Rhipicephalus microplus, causes severe economic losses to beef and dairy production in several countries. The control of the cattle tick is based mostly on chemical acaricides. Currently, there are reports of tick populations resistant to all six classes of acaricides available on the market. The aim of this work was to evaluate two protocols for the control of the cattle tick, R. microplus, under field conditions. For that, an acaricide rotational-based approach and selective (partial)-based protocols were tested against a multi-resistant tick strain. Eighteen Brangus cattle were divided into three experimental groups: Group 1, a rotational-based approach aiming at six treatments per year (spring–spring period) using four different classes of acaricide (IPV6 protocol); Group 2, a selective (partial) and rotational-based protocol, in which only half of the animals with the highest tick counts were treated (IPV7S protocol); and Group 3 (non-treated). The protocols were able to maintain low tick infestation during the 57 weeks of study. The two experimental groups showed lower tick counts than the non-treated group, but not between them. Weekly efficacy of the experimental protocols reached 95% in Group 1 and 88% in Group 2. Herein, we proposed two protocols of cattle tick control (IPV6 and IPV7S) for use in multi-resistant tick populations in Southern Brazil.     

Identification of Rickettsia spp., Anaplasma spp., and an Ehrlichia canis-like agent in Rhipicephalus microplus from Southwest and South-Central China

Rhipicephalus microplus is considered to be the most important tick infesting cattle, buffalo, horse, goats as well as other animals. They transmit diseases between domestic animals and act as vectors of a variety of zoonotic pathogens. Although pathogens harbored by R. microplus have been extensively studied, the Rickettsiales pathogens vectored by R. microplus in some areas of China remained largely unexplored. From August to October 2020, a total of 291 R. microplus ticks were collected from goats and cattle in three Southern China provinces, Guangxi (n = 138), Sichuan (n = 120) and Hubei (n = 33) provinces. Phylogenetic analysis based on COI gene sequences shows that these ticks are divided into three distinct clades, indicating the remarkable genetic diversity of R. microplus ticks in China. These samples were subsequently screened for the presence of Rickettsia, Anaplasma and Ehrlichia using conventional PCR and sequencing. Subsequently, five bacterial species were identified. Out of the 120 tick DNA samples from Sichuan province, 35.83% (43/120) were positive for Rickettsia sp. belonging to spotted fever group (SFG), 12.50% (15/120) were positive for Anaplasma marginale and 0.83% (1/120) was identified as A. platys. From the 138 DNA samples from Guangxi province, an Ehrlichia canis-like and Rickettsia sp. were detected, with a positive rate of 11.59% (16/138) and 2.17% (3/138), respectively. A. capra DNA was detected in 4 out of 33 (12.12%) samples from Hubei province. Notably, the 16S, gltA and groEL sequences of the E. canis-like are closely related to the E. canis strain previously identified from China, and form a distinct cluster in the phylogenetic trees. Collectively, our results expand the knowledge on tick-borne Rickettsiales pathogens in China. Because the state of engorgement of ticks was not recorded, it is not clear at this stage whether these pathogens are infecting the ticks or are simply present in the blood meal. Given the public health significance of SFG Rickettsia, A. capra, A. platys and E. canis, a thorough investigation of the diversity and presence of pathogens in R. microplus in areas with tick-associated diseases are needed.     

Successive treatments with ivermectin (3.15%) to control the tick Rhipicephalus (Boophilus) microplus in cattle: Pharmacokinetic and efficacy assessment

This study aimed to evaluate the pharmacokinetics, the potential accumulation in the body of treated animals and the efficacy of ivermectin long-acting formulation (3.15%) against the cattle tick Rhipicephalus (Boophilus) microplus in a scheme of three successive treatments. Fifteen 12-month-old heifers, naturally infested with R. microplus, were divided into two groups (G). Cattle from GI (n = 10) were subjected to three treatments with ivermectin 3.15% (IVOMEC GOLD®, Merial Argentina S.A.) at a rate of 1 mL/50 kg on days 0, 35, and 70. Cattle from GII (n = 5) were not treated. From day 1 to 202 post-treatment blood samples were taken to measure ivermectin concentrations by HPLC and female ticks (4.5–8 mm) were counted to evaluate the efficacy of the treatment. The level of tick resistance to ivermectin was evaluated before and after finishing the scheme of successive treatments by larval immersion test (LIT) bioassay from engorged females collected from GI. The area under the concentration vs. time curves (AUC_0–35d) obtained post-second treatment was 1.51 ± 0.39-fold higher than those observed post-first treatment (P<0.05). The mean plasma concentrations of ivermectin 3.15% at 20 days after the first, second and third treatment were 17.0, 27.5 and 37.8 ng/mL, respectively (P<0.01). The elimination half-life of ivermectin post-third treatment was significantly longer than that was previously reported after a single dose (P<0.01). Values of therapeutic efficacy percentage reached 75.6% post-first treatment and between 95.9 and 100% after the second treatment. Ticks evaluated by LIT showed a significant increase in lethal concentrations after treatments. Although the efficacy level was high, the successive treatments with long-acting ivermectin formulation generate a significant accumulation of drug in plasma and could increase the levels of resistance to this drug in the tick population.     

Efficacy of cypermethrin for the control of mosquito larvae and pupae, and impact on non-target organisms, including fish

In laboratory tests, cypermethrin was highly toxic to mosquito larvae and pupae. It was more toxic at low temperatures after a 24 hr exposure. Larvae of Aedes stimulans were less susceptible than Culex restuans. Technical cypermethrin was more toxic than an emulsifiable concentrate formulation. In outdoor simulated pools cypermethrin 40% EC was consistently effective against larvae and pupae of Ae. stimulans at 10 g AI/ha and Culex spp. at 50 g AI/ha. When stickleback fish were tested, no mortality occurred at the lowest effective dosage in each trial. The residual toxicity of cypermethrin increased with dosage and was much higher in a test at 8 degrees C than at 20 degrees C. In natural snowmelt pools, cypermethrin at 20 g AI/ha provided 92-100% control of Aedes spp. larvae and pupae by 7 days after treatment. Non-target amphipod, anostracan, cladoceran and insect populations were usually reduced 80-100% while copepods, ostracods and hydracarinid mites were generally less affected. No significant mortality of caged stickleback fish occurred in these pools.     

Vaccine efficacy of recombinant BmVDAC on Rhipicephalus microplus fed on Babesia bigemina-infected and uninfected cattle

Rhipicephalus microplus is the most widely distributed tick worldwide and causes significant economic losses in the livestock industry. It directly affects hosts (especially in large infestations) by feeding on blood and piercing the skin and indirectly affects hosts as a vector of pathogens that cause infectious diseases, such as bovine babesiosis. Current research on the control of ticks is focused on integrated tick control programmes, including vaccination treatment with acaricides and completely blocking pathogen transmission. Our previous studies showed that R. microplus VDAC (BmVDAC) expression is modulated by Babesia bigemina infection. VDAC is a mitochondrial protein with multiple functions in addition to its primary role as a central component of the apoptotic machinery. In this paper, we evaluated BmVDAC as an anti-tick vaccine and its capacity to block the infection of Babesia bigemina in ticks. Our results demonstrate that rBmVDAC is immunogenic and that antibodies specifically recognize the native protein from midguts of R. microplus. Immunization with rBmVDAC afforded an 82% efficacy against R. microplus infestation in the group of vaccinated cattle compared with the control group. In contrast, rBmVDAC showed a lower efficacy of 34% against tick infestation in cattle vaccinated with rBmVDAC, infested with R. microplus and infected with B. bigemina. The main effect on ticks fed in vaccinated and infected cattle was a 34% reduction in egg fertility (DF) compared to ticks fed on the control group. There was no reduction in the B. bigemina parasite levels of ticks fed on rBmVDAC-vaccinated cattle. These results suggest that the rBmVDAC protein could be tested as a vaccine for the control of tick infestation.     

Diacylglycerol: efficacy and mechanism of action of an anti-obesity agent

Obesity is at the forefront of global health issues and directly contributes to many chronic illnesses. Several dietary components show promise in the treatment of obesity, one of which is oil rich in diacylglycerols (DAGs). Present objectives are to examine scientific knowledge concerning DAG to assess evidence supporting the effects on substrate oxidation rates, body weight and fat mass, and blood lipids, and to assess safety, as well as elucidate potential mechanisms of action. DAG can be synthesized by an enzymatic process to produce mainly 1,3-isoform DAG. This 1,3-DAG oil is believed to have the ability to increase beta-oxidation, to enhance body weight loss, to suppress body fat accumulation, and to lower serum triacylglycerol levels postprandially. While certain animal and human studies indicate that consumption of 1,3-DAG has positive physiological effects, others report no effect. The mechanisms of action of DAG are suggested to decrease the resynthesis of chylomicrons as well as shunting them directly to the liver through the portal vein, where they are oxidized. This increased fat oxidation may influence control of food intake by increasing satiety. Further study into the precise mechanism is required to understand its effects. Safety studies show no risks in consuming a diet rich in DAG oil. Overall, consumption of oils with higher amounts of DAG, specifically 1,3-DAG, may be useful in the battle against obesity.     

Effect of cyclosporin A on the toxicity of ivermectin,eprinomectin and moxidectin in populations of Rhipicephalus microplus

Rhipicephalus microplus is mainly controlled by acaricides. However, reports of resistance to acaricides including macrocyclic lactones (MLs) have become frequent worldwide. Involvement of ABC transporters (ABCts) in populations resistant to ivermectin has been demonstrated. Thus, the aim of this study was to evaluate the efficacy of ivermectin, eprinomectin and moxidectin with and without use of synergistic cyclosporin A (CsA) in resistant populations of R. microplus using larval immersion tests (LITs). Engorged females were collected from four farms in the semiarid region of northeastern Brazil that had histories of continuous use of ivermectin. Questionnaires were applied to collect information about management aimed at controlling ticks on these farms. Resistance to MLs was observed on all of the farms. There was statistically significant synergism (p < 0.05) between CsA and ivermectin in all populations; between CsA and eprinomectin in only one population; and between CsA and moxidectin in two populations. It was concluded that, despite the involvement of ABCts in the mechanisms of resistance to ivermectin, metabolic detoxification does not seem to be the mechanism predominantly involved in resistance to eprinomectin and moxidectin in the populations of R. microplus evaluated.     

Development and validation of software that quantifies the larval mortality of Rhipicephalus (Boophilus) microplus cattle tick

Rhipicephalus (Boophilus) microplus is controlled almost exclusively using synthetic acaricides, and reports of resistant populations have been described worldwide. Several time-consuming and laborious toxicological in vitro tests have been used to diagnose acaricidal resistance, especially those that require differential counting of live and dead larvae. Larval mortality is currently done manually and subjectively, which can limit the performance of a large number of tests and comparing results between different laboratories. The present study aimed to develop and validate a new automatic counting method to evaluate tick larval mortality. A software for differentiation of live and dead larvae was developed using different steps: obtaining videos; image segmentation using the firefly algorithm; detection of larvae with the fast radial symmetry transform technique (FRST); and tracking of the larvae at a given time. Larval immersion tests with ivermectin, cypermethrin, and fipronil were performed to validate the developed software. The larval mortality evaluation was performed by (1) recording for 60 s for each package and (2) manual counts of the same sample using three different analysts, each responsible for counting one replicate of each test. All videos obtained were copied and cut at 60, 40, and 20 s for later analysis in the counting software. The median lethal doses (LD₅₀) of the different compounds in each test were calculated for each method (automatic and manual) for different video times. There was no statistical difference in LD₅₀ between manual and automatic count techniques for ivermectin and fipronil. The LD₅₀ of cypermethrin calculated with manual evaluation was up to 2.2 times lower than that of automatic evaluation. The acquisition time of the videos was 2.9–4.4 times faster than the manual evaluation. The average processing time for each video was 5.73 min, regardless of their duration. Thus, the method developed for automatic counting of tick larvae was validated, and although it still has points to be optimized, it can be considered a viable alternative for determining the percentage of tick larvae mortality and could be applied to toxicological in vitro tests with acaricides, assisting in the diagnosis of resistant tick populations and studies of novel acaricide development.     

Towards an appropriate malaria control strategy: issues of concern and alternatives for action

A resurgence of malaria in India prompted formation of a Malaria Expert Group, which met in 1996 to formulate an appropriate malaria control strategy. An estimated 20-30 million episodes of malaria occur in India each year. Since malaria is an exclusively focal phenomenon, strategies should be responsive to the epidemiologic characteristics of the five ecotypes: tribal, rural, urban, industrial, and border. Also needed are flexible, individualized strategies for development project areas and Triple Insecticide Resistance Areas. Most malaria deaths are attributable to delayed diagnosis and treatment. Recommended are streamlined logistics for laboratory supplies, monitoring of anti-malarial drugs for possible resistance, training of nongovernmental organizations to open peripheral laboratories, rigorous training for laboratory technicians in malaria diagnosis, training of supervisors for laboratory work and cross-checking of findings, and establishment of graded laboratory facilities for different levels of the health care delivery system. Integration of malaria control into India's primary health care system will require community participation, appropriate technology, intersectoral coordination, and social equity.     

Control of Rhipicephalus (Boophilus) microplus infestations by the combination of subolesin vaccination and tick autocidal control after subolesin gene knockdown in ticks fed on cattle

Tick subolesin was shown in immunization trials using the recombinant protein to protect hosts against tick infestations. In this study, we demonstrated that subolesin vaccination and release of ticks after subolesin knockdown by RNA interference (RNAi) could be used for the control of Rhipicephalus (Boophilus) microplus tick infestations in cattle and suggested that the combination of these methods could increase the efficacy of cattle tick control under some circumstances. The greatest tick control was obtained when both release of ticks after subolesin knockdown and vaccination were used concurrently. However, modeling results suggested that vaccine efficacy could be increased if at least 80% of the ticks infesting cattle correspond to subolesin-knockdown ticks. The results of this proof-of-concept trial demonstrated the efficacy of the sterile acarine technique (SAT) through production of subolesin-knockdown larvae by dsRNA injection into replete females for the control of R. microplus tick infestations, alone or in combination with subolesin vaccination.