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.     

Status of ivermectin resistance in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) populations from north-western Himalayas,India

Status of ivermectin resistance was studied in 16 isolates of Rhipicephalus (Boophilus) microplus ticks collected from north-western Himalayan region of India using larval immersion test (LIT). Regression graphs of probit mortality of larval ticks plotted against log values of concentrations of ivermectin were utilized for the determination of slope of mortality, lethal concentrations (LC₅₀ and LC₉₅) and resistance ratios (RRs). Out of 16 isolates examined, 15 were found resistant against ivermectin. Level II resistance was detected in eleven isolates (RR₅₀ ranged from 5.72 to 7.62), whereas four isolates exhibited level I resistance (RR₅₀ ranged from 1.58 to 2.77). Only one isolate was found susceptible (RR₅₀: 1.23). Higher RR values were observed in tick isolates from low altitude areas where intensive animal husbandry activities are practiced as compared to tick isolates of high altitudes accredit the fact that managemental practices, availability of drug and its frequency of application play an important role in development of resistance.     

Rhipicephalus capensis (Acari: Ixodidae), A geographically restricted South African tick,returning with a human traveler to the United States

Accelerated frequency of recreational travel, globalization of business, and legal and illegal plant and animal trades have contributed to enduring introductions of exotic ticks into the United States. We herein report a new incursion of a female Rhipicephalus capensis on a human traveler returning to Connecticut from South Africa. Natural distribution of R. capensis is restricted to the Western Cape Province and southwestern portion of the Northern Cape Province of South Africa, an area called the Fynbos Biome, and adults of this species primarily parasitize large, wild ungulate hosts. Only one previous international introduction of this tick is documented on imported South African plant material into the United States in 1985. The specimen described here was identified initially by morphological means and subsequently, a partial DNA sequence for the mitochondrial ribosomal RNA gene was generated in a PCR assay, which showed 94.86% identity to an R. capensis sequence in GenBank. We also provide information on several other previously unreported or under-reported incursions by South African ticks into the United States in association with imported Fynbos floricultural materials and speckled Cape tortoises, Chersobius signatus. Documentation of these additional exotic tick species incursions highlights ongoing challenges of the international movement of humans, animals, and other goods carrying ticks of human and veterinary importance.     

Interaction between anti-tick vaccine and a macrocyclic lactone improves acaricidal efficacy against Rhipicephalus (Boophilus) microplus (Canestrini) (Acari: Ixodidae) in experimentally infested cattle

The southern cattle fever tick (SCFT) Rhipicephalus (Boophilus) microplus, is considered the most important ectoparasite of livestock in the world because of high financial losses associated with direct feeding and transmission of the hemoparasites Babesia bovis, B. bigemina, and Anaplasma marginale. Unfortunately, SCFT in many parts of the world have evolved resistance to all market-available pesticides thus driving development of new control technologies. Vaccination against ticks using the tick gut protein Bm86 has been shown to be effective against acaricide-resistant ticks. This technique has been successfully implemented in Puerto Rico for the control of acaricide-resistant R. microplus on dairy and beef cattle. Observations from Puerto Rico indicate a potentially positive interaction between anti-tick vaccination when used in conjunction with systemic acaricide treatment. In this project, controlled animal studies were completed directly comparing efficacy of anti-tick vaccination with and without systemic acaricide. Results show that the Bm86 anti-tick vaccine in combination with the macrocyclic lactone, Moxidectin, expressed a synergistic interaction, providing greater and longer efficacy than either treatment alone.     

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.     

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.     

Evaluation of cytotoxic effects of amitraz and fipronil on digestive,reproductive and neural processes of engorged Rhipicephalus microplus (Acari: Ixodidae) female

Fipronil and amitraz are potentially toxic compounds used for controlling ticks infesting pet and livestock. The use of fipronil on large animals was limited because of its high costs while amitraz is still persisting in the market since its introduction over four decades ago. Though resistance in ticks against these pesticides has been reported worldwide since 2000, the toxicity of these chemicals at cellular level in ticks is still poorly understood. The present study aimed to examine the gross and cellular impact of fipronil and amitraz on the gut, ovaries and synganglion of engorged Rhipicephalus microplus females. Fipronil and amitraz treated tick groups showed formation of a large number of vacuoles of different size throughout the cytoplasm of generative cells whereas sessile, residual and detached digestive cells were very low in numbers. The treatment of ticks resulted in the formation of vacuolations at periphery of all oocytes. Ultra-thin sections of the synganglion revealed severe rupture of neural lamella and perineurium with apoptosis of neural cells after fipronil treatment whereas in the amitraz treated ticks, severe destruction of neuropile region and extensive vacuolation of type I and II cells of cortical region as compared to the unexposed ticks were noted.     

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.     

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.     

Contextual influences on contraceptive use in the Eastern Cape, South Africa

This paper uses linked individual and health facility data from the 1998 South Africa Demographic and Health Survey and the 1998 Eastern Cape Facility Survey to explore community and health facility influences on modern contraceptive use. Several pathways of influence between the community and individual contraceptive adoption are identified, centering primarily on the community climate of female autonomy. Few significant effects of the health facility environment on contraceptive adoption are identified. The residual variation in contraceptive use highlights the deficits that exist in current datasets for capturing community influences on contraceptive behavior.     

Direct and indirect inferences on parasite mating and gene transmission patterns. Pangamy in the cattle tick Rhipicephalus (Boophilus) microplus.

Mating system plays a determinant role in the maintenance and distribution of genetic variation. Difficulties in applying standard methods of indirect inferences onto parasitic life-cycles partly explain the current lack of knowledge on parasite mating systems. The present study develops a combination of direct and indirect inference methods circumventing such difficulties, and illustrates in particular how such a combination modifies our understanding of the biology of the southern cattle tick, Rhipicephalus microplus. To directly assess how females and males pair in natural populations, we sampled 225 mating pairs, genotyped them at six microsatellite loci, and analysed the correlation between mating status and genetic relatedness. This analysis revealed pangamy, i.e. a random association of male and female genotypes. In addition, indirect methods inferred that sib-groups of ticks exploit distinct individual-cows, and hence that ticks probably move among cows during their parasitic lifetime. Altogether, these results negate the expectation of a high frequency of sib-mating, and show the coexistence of genetically diverging stocks within tick cohorts and populations. These results have several consequences for our understanding of R. microplus. For instance, while existing models of pesticide resistance management look perfectly applicable to this species, the epidemiological models of the micropathogens it vectors needs revision. More largely, the methods developed here would help clarify the evolutionary patterns of any dioceous parasite.     

Interpretation of the larval immersion test with ivermectin in populations of the cattle tick Rhipicephalus (Boophilus) microplus from Colombian farms

Interpreting in vitro bioassays used to determine resistance against acaricides in Rhipicephalus microplus can be challenging without parallel in vivo studies that assess for lost efficacy. The larval immersion test (LIT) is currently the most widely used bioassay to detect ivermectin resistance. The objective of this study was to compare results of the LIT and a field trial using ivermectin in naturally infested cattle. Criteria to consider ticks as resistant with the LIT were based on discriminating doses (DD) and the ratio of lethal concentration (LC) in test populations over the LC of the susceptible Deutch strain, known as the resistance ratio (RR). Ticks were collected from 4 farms, two where ivermectin provided good control of tick infestations and two that claimed lack of efficacy. In two farms where administration of a long-acting ivermectin formulation reduced body tick counts to 45 and 25% of the initial counts at 10-days post-treatment, the RR₅₀ and RR₉₉ were approximately 6 and 20, respectively. The LC50 value approximated the DD for the two farms with claimed resistance, suggesting that about half of the population in each farm was resistant. These LIT values are equal to those reported for the most resistant ticks, which supports the use of the LIT to predict control failure in field situations. The two farms where ivermectin provided good control of tick infestations had LC₅₀s similar to the susceptible strain, although for one farm the LC₉₉ and RR₉₉ suggested incipient resistance.     

Hard ticks (Acari: Ixodidae) and tick-borne diseases of sheep and goats in Africa: A review

Ticks are leading vectors of economically important pathogens that affect small ruminants due to favourable climatic conditions across different regions of the African continent. They are responsible for both direct and indirect economic losses in the livestock industry. This review focuses on the species diversity of hard ticks, their biology, tick-borne diseases of sheep and goats including non-infectious disease, and risk factors to tick infestation in Africa. Furthermore, our review provides recent updates on distribution of ticks and tick-borne pathogens of small ruminants in Africa. It was observed that several species and subspecies of hard ticks belonging to the genera Hyalomma (Hy), Rhipicephalus (Rh), Ixodes (I) and Amblyomma (Am) were found infesting small ruminants across the different regions of the continent. Of these genera, Rhipicephalus ticks accounts for the majority of the registered species, with exactly 27 different species infesting small ruminant stocks comprising of different developmental instars and adults of the tick. Rhipicephalus decolaratus, Rh. e. evertsi and Rh. appendiculatus were the three most common Rhipicephalus species reported. Both protozoal (Babesia and Theileria) and bacterial (Anaplasma, Rickettsia, Ehrlichia, Coxiella and Mycoplasma) pathogens have being reported to be amplified in several hard tick species and/or small ruminant hosts. Furthermore, tick paralysis and lameness were non-infectious conditions attributed to tick infestations. Amblyomma hebraeum and Rh. glabroscutatum may cause lameness in goats, while Hy. rufipes is responsible for the same condition in Merino sheep. Host paralysis due to a neurotoxin released by female Rh. e. evertsi and I. rubicundus has been documented within the continent. We therefore advocate for the need of integrated control measures against tick-borne pathogens (TBPs) including their arthropod vectors, to be performed simultaneously to ease the burden of vector-borne diseases in small ruminant production.     

Permethrin-induced ultrastructural changes in oocytes of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) semi-engorged females

Currently, the most effective method for tick control is by the use of chemical acaricides such as permethrin (active ingredient of the Advantage^® Max3 acaricide, Bayer), a pyrethroid with neurotoxic action. However, data about its effect on other tick systems, such as glandular and reproductive, are still scarce. This study provides ultrastructural details of the toxic effects induced in oocytes of semi-engorged Rhipicephalus sanguineus females exposed to 206, 1031, and 2062 ppm of permethrin. The results showed that permethrin is an effective acaricide by inhibiting and/or neutralizing the tick reproductive process since oocytes of treated ticks presented major structural changes such as cytoplasm vacuolation, disruption of both membranes and cell organelles, leading to cell death. Data also showed that even in low concentrations permethrin induced changes in germ cells, making them non-viable. The defense mechanisms used by oocytes against the action of permethrin in the attempt of recovering the cellular integrity and of ensuring the viability of these cells were also described in this study.     

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.     

Action of permethrin on Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) semi-engorged females: Morpho-physiological evaluation of salivary glands

Currently the most effective method of tick control is the use of acaricides, among which stands out permethrin (active ingredient of acaricide Advantage^® Max3, Bayer), a neurotoxic pyrethroid. However, assessments of their effects on other tick systems such as glandular are still scarce. Thus, this study provides information, through histochemical techniques, about the toxic effect of this pyrethroid on the morphophysiology of salivary glands of semi-engorged Rhipicephalus sanguineus females exposed to different concentrations of permethrin (206, 1031, and 2062 ppm). The results showed that permethrin caused significant changes in the salivary gland metabolism accelerating the process of glandular degeneration, an event which would occur naturally and with great intensity only in the final engorgement stage. Furthermore, this study pointed out that permethrin reduces the salivary gland secretion ability through a drastic reduction of proteins, lipids, and polysaccharides in acinar cells. These changes impair the females to finalize the feeding process, what indirectly affects the reproductive process.     

Morphological records of oocyte maturation in the parthenogenetic tick Amblyomma rotundatum Koch, 1844 (Acari: Ixodidae)

Oocyte maturation in the thelytokous parthenogenetic tick Amblyomma rotundatum was examined for the first time using light and scanning electron microscopy. The panoistic ovary lacks nurse and follicular cells and is a single continuous tubular structure forming a lumen delimited by the ovarian wall. Oocytes of tick species are usually classified according to cytoplasm appearance, the presence of germinal vesicle, the presence of yolk granules, and the chorion. However, for this species, we also use oocyte size as an auxiliary tool since most oocytes were in stages I–III and were histologically very similar. Oocytes were classified into five development stages, and specific characteristics were observed: mature oocytes with thin chorion, pedicel cells arranged forming an epithelium with two or more oocytes attached by the same structure, and a large number of oocytes in the process of reabsorption.