Field efficacy of Metarhizium anisopliae oil formulations against Rhipicephalus microplus ticks using a cattle spray race.

Rhipicephalus microplus tick is the main ectoparasite of cattle in Brazil. The exhaustive use of chemical acaricides to control this tick has favored the selection of resistant tick populations. Entomopathogenic fungi, as Metarhizium anisopliae, has been described as a potential biocontroller of ticks. Therefore, the aim of this study was to evaluate the in vivo efficacy of two oil based formulations of M. anisopliae for the control of the cattle tick R. microplus under field conditions using a cattle spray race as a method of treatment. Initially, in vitro assays were carried out with an aqueous suspension of M. anisopliae, using mineral oil and/or silicon oil. A potential synergism between oils and fungus conidia for tick control was demonstrated. Additionally, the usefulness of silicon oil in order to reduce mineral oil concentration, while improving formulation efficacy was illustrated. Based on the in vitro results, two formulations were selected for use in the field trial: MaO1 (107 conidia/mL plus 5% mineral oil) and MaO2 (107 conidia/mL plus 2.5% mineral oil and 0.01% silicon oil). The adjuvants concentrations (mineral and silicon oils) were chosen since preliminary data indicate that higher concentrations caused significant mortality in adult ticks. For this, 30 naturally infested heifers were divided into three groups based on previous tick counts. The control group did not receive treatment. The selected formulations were applied on animals using a cattle spray race. Subsequently, tick load was evaluated weekly by counting. The MaO1 treatment significantly reduced the tick count only on day +21, reaching approximately 55% efficacy. On the other hand, MaO2 showed significantly lower tick counts on days +7, +14, and +21 after treatment, with weekly efficacy achieving 66%. The results showed a substantial reduction of tick infestation, up to day +28, using a novel formulation of M. anisopliae based in the mixture of two oils. Moreover, we have shown, for the first time, the feasibility of employing formulations of M. anisopliae for large-scale treatment methods, such as a cattle spray race, which in turn, may increase the use and adhesion to biological control tools among farmers.

Resistance profile and molecular characterization of pyrethroid resistance in a Rhipicephalus microplus strain from Colombia.

Intensive use of chemical acaricides for the control of cattle ticks (Rhipicephalus microplus) has led to the development of multiple acaricide resistance in Colombia. The present study aimed to characterize, using toxicological bioassays and molecular biology techniques, the resistance profile of a tick strain isolated from the Arauca state, Northeast Colombia. Commercial acaricides were used in adult immersion tests to determine its in vitro efficacies. Deltamethrin showed very low activity (4-7.3%), a mixture of cypermethrin and chlorpyrifos had intermediate efficacy (64-75.2%), and ethion presented the highest activity (88.5-100%). A colony (Arauquita strain) was established and larval immersion tests confirmed high resistance level to deltamethrin (241-fold) and susceptibility to ivermectin. A quantitative polymerase chain reaction-high resolution melt technique was used to identify single nucleotide polymorphisms (SNPs) in the para-sodium channel gene. All of the genotyped individuals were mutant, presenting one (n = 7), two (n = 7) or three (n = 9) SNPs previously associated with pyrethroid resistance. Sequencing revealed a novel mutation (F712L), that was found for the first time in R. microplus ticks from South America. This is the first description of mutations associated with pyrethroid resistance in R. microplus from Colombia. The acaricide resistance pattern found in the Arauquita strain is similar to other parts of Colombia.

Mutations in Rhipicephalus microplus GABA gated chloride channel gene associated with fipronil resistance.

The tropical cattle tick, Rhipicephalus microplus, is one of the most damaging parasites that affects cattle in tropical and subtropical regions in the world. Tick resistance to acaricides is dispersed worldwide and a number of associated mutations in target site genes have been described. Phenylpyrazole (e.g. fipronil) and cyclodiene (e.g. lindane, dieldrin) insecticides both have the same mode of action blocking the GABA-gated chloride channel encoded by the GABA-Cl gene. A conserved mutation, rdl (resistance to dieldrin) is found across a number of arthropods resistant to cyclodienes and phenylpyrazoles. In ticks, the mutation T290 L, was identified in the second transmembrane (TM2) domain of the GABA-gated chloride channel of Australian cattle tick populations that are resistant to dieldrin. Recently, cross-resistance between fipronil and lindane was reported in R. microplus populations obtained from Uruguay and Brazil. The objective of the present study was to identify mutations in the GABA-Cl gene associated with fipronil resistance. Genomic DNA was obtained from engorged females from fipronil-susceptible and resistant populations sampled from Uruguay and Brazil (n = 166). Initially, it was searched the T290 L mutation described in Australia; however, this mutation was not detected in individuals from resistant populations from either country. The sequencing of a fragment of the GABA-Cl gene revealed nucleotide polymorphisms in fipronil- and lindane-resistant ticks in two populations from Uruguay and five from Brazil. Five amino acid substitutions were present in the resistant strains. Two different substitutions were found in an alanine residue (A286S and A286 L) that is homolog to rdl mutations in fipronil-resistant individuals of other arthropod species. Four other amino acid substitutions (S281 T, V317I, T328 A and A329S) were present in some resistant strains, always with the mutation A286S. This is the first documentation of mutations in the GABA-Cl gene associated with fipronil-resistant in R. microplus.

Mutation in the Sodium Channel Gene Corresponds With Phenotypic Resistance of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) to Pyrethroids.

The brown dog tick, Rhipicephalus sanguineus sensu lato (Latreille), is a cosmopolitan ectoparasite and vector of pathogens that kill humans and animals. Pyrethroids represent a class of synthetic acaricides that have been used intensely to try to control the brown dog tick and mitigate the risk of tick-borne disease transmission. However, acaricide resistance is an emerging problem in the management of the brown dog tick. Understanding the mechanism of resistance to acaricides, including pyrethroids, is important to adapt brown dog tick control strategies. The main objective of this study was to determine if target-site mutations associated with pyrethroid resistance in other pests could be associated with phenotypic resistance detected in a brown dog tick population from Florida. We amplified segment 6 of the domain III of the voltage-sensitive sodium channel protein, using cDNAs synthesized from pyrethroid-susceptible and pyrethroid-resistant tick strains. A single nucleotide point mutation (SNP) identified in a highly conserved region of domain III S6 in the resistant ticks resulted in an amino acid change from phenylalanine to leucine. This mutation is characteristic of resistance phenotypes in other tick species, and is the first report of this mutation in R. sanguineus. Molecular assays based on this knowledge could be developed to diagnose the risk for pyrethroid resistance, and to inform decisions on integrated brown dog tick management practices.

Low temperature affects cattle tick reproduction but does not lead to transovarial transmission of Anaplasma marginale.

Anaplasma marginale is an obligate intracellular pathogen that infects the erythrocytes of calves, causing bovine anaplasmosis. This rickettsia is biologically transmitted by several species of ticks. In tropical and subtropical regions of the world, Rhipicephalus microplus is the main vector. Due to their mobility and longevity, the adult males play an important role in the transmission of A. marginale to calves. Some studies have demonstrated that A. marginale can be intrastadially and interstadially transmitted in R. microplus, but the transovarial transmission has not been demonstrated so far. In the present study, we investigated the effects of low temperature on both the A. marginale migration from infected females to their offspring and reproductive parameters of the tick R. microplus. The larvae of R. microplus fed on a calf infected with the strain Jaboticabal of A. marginale. At the end of the parasitic phase, fully engorged females were incubated at either 18°C or 28°C for oviposition. Although A. marginale was detected in the salivary glands of the females, demonstrating that the ticks were successfully infected, the presence of rickettsia was not detected in the offspring. However, the preoviposition period of the non-infected females maintained at 18°C was longer than that of those maintained at 28°C. In addition, the average weight of the mass of eggs as well as the egg production efficiency (ratio of the egg mass weight to the female weight) of the females maintained at 18°C were significantly lower than those of the females incubated at 28°C. There was no larval hatching from the eggs maintained exclusively at 18°C, even at 65 days after female detachment. Hatching occurred only when the eggs maintained at 18°C were transferred to 28°C at 20 days after female detachment (18°C/28°C). We also verified a significantly higher larvae conversion efficiency (ratio of the larvae mass weight to the egg mass weight) in the group of females maintained exclusively at 28°C compared to those from the 18°C/28°C group. Collectively, our results reinforce that low temperature exerts negative effects on female fertility and egg development in R. microplus, although it has no influence on A. marginale transmission to the progeny. In the field, the detrimental effects of temperatures on tick reproductive fitness lead to a reduction of tick population, which may cause a decrease in the incidence of bovine anaplasmosis.

Cross-resistance between fipronil and lindane in Rhipicephalus (Boophilus) microplus.

The southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini), is one of the most damaging parasites of cattle in tropical and subtropical regions. Several chemical groups have been used for its control, including cyclodienes (lindane and dieldrin). In Uruguay and Brazil these products were used at the beginning of the 1960s and during a few years. Fipronil and lindane act on the same target site. In both countries, southern cattle tick resistance to fipronil has sometimes developed quickly after only a few acaricide treatments (three to seven). The objective of the present study was to determine cross-resistance between fipronil and lindane in southern cattle ticks from Uruguay and Brazil. Initially, the FAO's (Food and Agricultural Organization) larval packet test with lindane was applied to a fipronil-resistant strain and to susceptible field populations. Mozo and POA strains were used as the susceptible controls. A larval immersion test was used to assess fipronil toxicity. Of fifteen fipronil-resistant field populations that were tested with lindane, eleven were lindane-resistant and three were susceptible. The last three populations had incipient resistance to fipronil. Finally, cross-resistance between fipronil and lindane in the southern cattle tick is reported in this study for the first time.

Applicability of in vitro bioassays for the diagnosis of ivermectin resistance in Rhipicephalus microplus (Acari: Ixodidae).

The applicability of laboratory bioassays to diagnose ivermectin (IVM) resistance in Rhipicephalus microplus was evaluated. Adult immersion tests (AITs), larval immersion tests (LITs) and larval packet tests (LPTs) were performed to characterise the effects of ivermectin toxicity on adults and larvae of a susceptible reference strain. The AIT was determined to be a reasonable assay but requires a large number of individuals to attain interpretable results. The LIT and LPT were validated with an IVM resistant strain, revealing resistance ratios (RRs) of 6.73 and 1.49, respectively. In a field survey, nine different populations of cattle tick from the states of São Paulo and Mato Grosso do Sul, Brazil, were analysed with the LIT. Populations without previous exposure to ivermectin exhibited RRs between 0.87 and 1.01. Populations previously exposed to IVM showed RRs between 1.83 and 4.62. The LIT was more effective at discriminating between resistant and susceptible populations than the LPT. The use of the LIT is recommended for the diagnosis of ivermectin resistance in R. microplus.

Resistance to cypermethrin, deltamethrin and chlorpyriphos in populations of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) from small farms of the State of São Paulo, Brazil.

A field survey of resistance was conducted based on the larval packet test technique with synthetic pyrethroids (cypermethrin and deltamethrin) and organophosphates (chlorpyriphos) in Rhipicephalus (Boophilus) microplus field populations from six different regions of the State of São Paulo (Brazil). 82.6% of the populations showed resistance to cypermethrin, 86.36% to deltamethrin and 65.25% to chlorpyriphos, with 50% presenting resistance to both SP and OP acaricide. According to the questionnaires completed by the producers, OP+SP mixtures followed by SP-only formulations were the products most commonly used for controlling the cattle tick in the surveyed areas. The present study showed high occurrence of resistance to SP and OP in the State of São Paulo, Brazil and revealed the type of strategy adopted by small dairy farms in this state. This information is fundamental in order to establish the monitoring of resistance on each farm individually, contributing to the rational use of acaricides for the control of R. (B.) microplus.

Diagnoses of fipronil resistance in Brazilian cattle ticks (Rhipicephalus (Boophilus) microplus) using in vitro larval bioassays.

Fipronil is a phenylpyrazolic insecticide that is widely used in agriculture and has been recently used to control the cattle tick, Rhipicephalus (Boophilus) microplus. Because of the serious problems associated with resistance to the available acaricides, this product has been used as an important alternative to control acaricide-resistant ticks. The objective of this work was to analyse the fipronil sensitivity of ticks that were collected from farms with a history of fipronil use by larval bioassays. A total of 11 Brazilian tick populations were studied: one population from Rio Grande do Sul, one population from Mato Grosso do Sul and nine populations from São Paulo. To validate the assays, susceptible reference strains, POA (Porto Alegre, Brazil) and Mozo (Dilave, Uruguay), and ticks from six different farms that never used fipronil were tested. The resistance of various tick populations to technical grade fipronil (95.3%) was primarily evaluated using the larvae immersion test (LIT) and the larval packet test (LPT), when a sufficient number of larvae was collected. Using the LIT, the resistance ratios (RR(50)) of the tick populations from Rio Grande do Sul and Mato Grosso do Sul were 14.9 and 2.6, respectively, and the populations derived from São Paulo had RR(50)s ranging from 2.5 to 6.9. Four populations were evaluated with the LPT, and two populations displayed lower RR(50), while other populations displayed higher RR(50) than those determined by the LIT. This article reports the first cases of fipronil resistance in Brazil and highlights the LIT as a more sensitive technique for the evaluation of fipronil resistance in R. (B) microplus ticks. We suggest the use of the LIT as an evaluation tool for monitoring fipronil resistance in the control programmes of R. (B) microplus.