Guilherme M. Klafke

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.

First report of fluazuron resistance in Rhipicephalus microplus: A field tick population resistant to six classes of acaricides

The control of the cattle tick Rhipicephalus microplus is based mainly on the use of chemical acaricides, which has contributed to the emerging problem of selection of resistant tick populations. Currently, there are six main classes of acaricides commercially available in Brazil to control cattle ticks, with fluazuron, a tick growth regulator with acaricidal properties, being the only active ingredient with no previous reports of resistance. Ticks (designated the Jaguar strain) were collected in a beef cattle ranch located at Rio Grande do Sul state, Southern Brazil, after a complaint of fluazuron treatment failure. To characterise the resistance of this strain against acaricides, larval tests were performed and showed that the Jaguar strain was resistant to all of the drugs tested: cypermethrin (resistance ratio, RR=31.242), chlorpyriphos (RR=103.926), fipronil (RR=4.441), amitraz (RR=11.907) and ivermectin (3.081). A field trial was conducted to evaluate the efficacy of fluazuron treatment in heifers that had been experimentally infested with the Jaguar or a susceptible strain. Between 14 and 28 days after treatment, the average efficacy in cattle experimentally infested with the susceptible strain was 96%, while for the Jaguar strain the efficacy was zero. Additionally, the Jaguar strain response to fluazuron was evaluated in vitro using a modified adult immersion test (AIT) and the artificial feeding assay (AFA). With the AIT, 50 ppm of fluazuron inhibited 99% of larvae hatching in the susceptible strain (POA) and less than 50% in the Jaguar strain. Results of the AFA showed a larval hatching rate of 67% at 2.5 ppm of fluazuron with the Jaguar strain; conversely, only 3% of larvae of the susceptible strain hatched at the same fluazuron concentration. The results showed here demonstrated the first case of fluazuron resistance in R. microplus and the first tick population resistant to six classes of acaricides in Brazil.

ABC transporter efflux pumps: A defense mechanism against ivermectin in Rhipicephalus (Boophilus) microplus

ATP-binding cassette (ABC) transporters are efflux transporters found in all organisms. These proteins are responsible for pumping xenobiotic and endogenous metabolites through extra- and intracellular membranes, thereby reducing cellular concentrations of toxic compounds. ABC transporters have been associated with drug resistance in several nematodes and parasitic arthropods. Here, the ability of ABC transporter inhibitors to enhance ivermectin (IVM) sensitivity was tested in larvae and adult females of Rhipicephalus (Boophilus) microplus. Larvae of susceptible and IVM-resistant tick populations were pre-exposed to sub-lethal doses of the ABC transporter inhibitors Cyclosporin A (CsA) and MK571, and subsequently treated with IVM in a Larval Packet Test (LPT). ABC transporter inhibition by both drugs significantly reduced the concentration for 50% lethality (LC(50)) values of four IVM-resistant populations but IVM sensitivity of a susceptible population remained unchanged. IVM sensitivity in adults was assessed through an artificial feeding assay. The addition of CsA to a blood meal substantially affected IVM toxicity in adult female ticks from a resistant population by reducing oviposition and egg viability, although it did not alter IVM toxicity in susceptible females. Three partial nucleotide sequences with similarity to ABC transporters were retrieved from the DFCI Boophilus microplus Gene Index (http://compbio.dfci.harvard.edu/index.html). Their transcriptional levels in the midgut of resistant and susceptible females were determined by quantitative PCR, showing that one of these sequences was significantly up-regulated in IVM-resistant females and suggesting its participation in IVM detoxification. We believe this work reports the first known evidence for the participation of ABC transporters in IVM resistance in R. microplus.

Rhipicephalus(Boophilus) microplus resistant to acaricides and ivermectin in cattle farms of Mexico

Ticks and the diseases they transmit cause great economic losses to livestock in tropical countries. Non-chemical control alternatives include the use of resistant cattle breeds, biological control and vaccines. However, the most widely used method is the application of different chemical classes of acaricides and macrocyclic lactones. Populations of the cattle tick, Rhipicephalus (Boophilus) microplus, resistant to organophosphates (OP), synthetic pyrethroids (SP), amitraz and fipronil have been reported in Mexico. Macrocyclic lactones are the most sold antiparasitic drug in the Mexican veterinary market. Ivermectin-resistant populations of R. (B.) microplus have been reported in Brazil, Uruguay and especially in Mexico (Veracruz and Yucatan). Although ivermectin resistance levels in R. (B.) microplus from Mexico were generally low in most cases, some field populations of R. (B.) microplus exhibited high levels of ivermectin resistance. The CHPAT population showed a resistance ratio of 10.23 and 79.6 at lethal concentration of 50% and 99%, respectively. Many field populations of R. (B.) microplus are resistant to multiple classes of antiparasitic drugs, including organophosphates (chlorpyrifos, coumaphos and diazinon), pyrethroids (flumethrin, deltamethrin and cypermethrin), amitraz and ivermectin. This paper reports the current status of the resistance of R. (B.) microplus to acaricides, especially ivermectin, in Mexican cattle.

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.

Multiple resistance to acaricides in field populations of Rhipicephalus microplus from Rio Grande do Sul state, Southern Brazil

Acaricide resistance is a major obstacle to the control of Rhipicephalus microplus. Historically, the indiscriminate use of chemical compounds has contributed to the selection of populations resistant to different classes of acaricides. Therefore, multiple acaricide resistance is an important threat to the chemical control of the cattle tick. To investigate the occurrence and extent of multiple resistance to acaricides in Southern Brazil we performed larval tests with cypermethrin, chlorpyriphos, amitraz, fipronil and ivermectin on 104 cattle tick field samples from different ranches in Rio Grande do Sul, between the years 2013 and 2015. Adult immersion tests with a commercial formulation mixture of chlorpyriphos and cypermethrin were performed on 75 samples. Four levels of resistance were established according to the mortality of larvae: Level I: mortality between 82% and 95%; Level II: mortality between 57% and 82%; Level III: mortality between 25% and 57%; and Level IV: mortality lower than 25%. Resistance to cypermethrin was detected in 98.08% of the samples evaluated, mostly at resistance level IV. The frequency of samples resistant to amitraz, chlorpyriphos, ivermectin and fipronil was 76.92%, 60.58%, 60.58% and 53.85% respectively. Multiple resistance to three or more compounds was found in 78.85% of the samples. The results obtained in this study are alarming and reveal a new scenario for the challenge of tick control using chemicals. This is an issue of high importance to cattle production systems where this tick is responsible for a high economic impact.

Spotted Fever Group Rickettsia in the Pampa Biome, Brazil, 2015–2016

To the Editor: Several cases of tickborne rickettsiosis have been reported in South America in recent years (1,2). In Brazil, 2 spotted fever group (SFG) Rickettsia species, R. rickettsii and Rickettsia sp. strain Atlantic Rainforest, have been identified as causes of human disease. Rio Grande do Sul is the southernmost state of Brazil and the only part of the country located in the Pampa biome. Despite confirmed cases of human spotted fever in that state since 2005, little information is available regarding Rickettsia species. We report an eco-epidemiologic investigation of R. parkeri in Amblyomma tigrinum ticks on dogs from a household (and neighborhood) where a case of human spotted fever was diagnosed.

Does Rhipicephalus microplus tick infestation increase the risk for myiasis caused by Cochliomyia hominivorax in cattle

The larval phase of Cochliomyia hominivorax (screwworm) is an obligate parasite of vertebrate animals, particularly mammals, and widespread in South America, where it remains one of the most important parasitic diseases of domestic animals. The skin of cattle highly infested by ticks, with cutaneous lesions, exudation of tissue fluid and blood scent seems to produce the ideal environment for fly attraction. However, an association between these parasites was never investigated. The aim of this work was to verify if there is an association between Rhipicephalus microplus tick load and the occurrence of C. hominivorax myiasis in cattle, and to quantify the risk. Sixty bovine (Bos taurus taurus, Angus breed) under field conditions were observed for 24 weeks, during which weekly tick counts and examination for the presence of myiasis were performed. There was a significant association between a high tick burden (24-week mean above 50 ticks per animal) and myiasis occurrence (P=0.0102). The calculated relative risk (RR) for C. hominivorax myiasis occurrence in cattle with high tick burden was 3.85 (CI95%=1.23-12.13); indicating that cattle highly parasitized by R. microplus have about four times more risk of myiasis than those with a low parasite load. As far as we aware, this is the first statistically based evidence of the relationship between R. microplus parasitic load and occurrence of myiasis by C. hominivorax. This result could be useful for the design of integrated control strategies for these parasites and to provide more information for the understanding of cattle tick parasitism in cattle production.

Selection of an ivermectin-resistant strain of Rhipicephalus microplus (Acari: Ixodidae) in Brazil

Resistance to ivermectin (IVM) in field populations of Rhipicephalus microplus of Brazil has been observed since 2001. In this work, four selection methods (infestations with: (1) IVM-treated larvae; (2) larvae from IVM-treated adult female ticks; (3) larvae from IVM-treated adult female ticks on an IVM-treated host; and (4) larvae obtained from IVM-treated females that produced eggs with a high eclosion rate) were used on a field population with an initial ivermectin (IVM) resistance ratio at LC50 (RR50) of 1.37 with the objective to obtain experimentally a highly-resistant strain. After ten generations, using these methods combined, the final RR50 was 8.06. This work shows for the first time that it was possible to increase IVM resistance in R. microplus in laboratory conditions. The establishment of a drug resistant R. microplus strain is a fundamental first step for further research into the mechanisms of ivermectin-resistance in R. microplus and potentially methods to control this resistance.