Virulence of Mexican isolates of entomopathogenic fungi (Hypocreales: Clavicipitaceae) upon Rhipicephalus=Boophilus microplus (Acari: Ixodidae) larvae and the efficacy of conidia formulations to reduce larval tick density under field conditions.

The first objective was laboratory evaluation of the virulence of 53 Mexican isolates of fungi against larvae of Rhipicephalus (Boophilus) microplus. Thirty-three isolates of Metarhizium anisopliae var. anisopliae (Metschnickoff) Sorokin (Hypocreales: Clavicipitaceae) and 20 isolates of Isaria (Paecilomyces) fumosorosea (fumosoroseus) (Wize) (Eurotiales: Trichomaceae) were tested on 7-day-old larvae under laboratory conditions. Larvae were immersed in a suspension containing 10(8)conidia/mL and the CL(50) values were estimated. Then, field tests were conducted to determine the efficacy of formulations of the isolate with the highest virulence. M. anisopliae (Ma 14 isolate) was formulated with four carriers: Tween, Celite, wheat bran, and Citroline (mineral oil) and applied on pasture beds of Cynodon plectostachyus (L.), at a dose of 2 x 10(9)CFU/m(2). In the first trial, M. anisopliae was applied on plots naturally infested with larvae; in the second trial, tick populations in the experimental plots were eliminated and then re-infested with 20,000 7-day-old larvae. In the laboratory, all M. anisopliae isolates infected larvae with a mortality range between 2 and 100%; also, 13 of 20 I. fumosorosea isolates caused mortality rates between 7 and 94%. In the first field trial, 14 days post-application, conidial formulations in Celite and wheat bran caused 67.8 and 94.2% population reduction, respectively. In the second trial, the Tween formulation caused the highest larval reduction, reaching up to 61% (28 days post-application). Wheat bran formulation caused 58.3% larval reduction (21 days post-application) and was one of the most effective. The carriers and emulsifiers have a large impact on the effectiveness of conidial formulations.

Development of amitraz resistance in field populations of Boophilus microplus (Acari: Ixodidae) undergoing typical amitraz exposure in the Mexican tropics.

The purpose of this study was to determine the effect of amitraz selection pressure on the development of resistance in field populations of Boophilus microplus in the Mexican tropics. Three farms (FA(1), FA(2) and FA(3)) in Yucatan, Mexico, were selected in this study. Amitraz was applied as a whole body sprays to all cattle on each farm for tick control once monthly for 15 months. From each farm, 20-30 B. microplus engorged females were collected every 3 months. The modified larval immersion test was used to test susceptibility of B. microplus to amitraz. Larvae were exposed to serial dilutions of amitraz. Probit analysis was used to determine lethal dose at 50% mortality and associated 95% confidence limits. The resistance factors found in the three farms during the 15 months of amitraz selection pressure were FA(1) (1, 2, 4, 4 and 13), FA(2) (1, 6, 23, 21 and 22) and FA(3) (2, 13, 2, 6 and 6). It is concluded that amitraz selection pressure on field populations of B. microplus increased the resistance level in all populations studied in the Mexican tropics.

Evaluation of Metarhizium anisopliae (Hyphomycetes) for the control of Boophilus microplus (Acari: Ixodidae) on naturally infested cattle in the Mexican tropics.

The efficacy of Metarhizium anisopliae on the control of Boophilus microplus in cattle infested naturally in the Mexican tropics was evaluated. The study was carried out on a ranch in Veracruz, Mexico. Twenty steers were randomly allocated into two groups of 10 cattle. Animals were naturally infested with B. microplus. Animals in the treated group were sprayed with M. anisopliae (strain Ma34) at a concentration of 1x10(8)conidia/ml every 15 days (four treatments). The other group remained as untreated control. Standard engorged female ticks were recorded on days 0, 1, 3, 5, 7 and 14 post-treatment. From the second application treatment (day 7) to the end of the experiment, animals in the treated group had lower tick infestation (P<0.05) with an efficacy of 40.0-91.2%. The results demonstrate the efficacy of repeated treatment with M. anisopiae (Ma34 strain) to control natural infestation of engorged female B. microplus on cattle in the Mexican tropics.

Prevalence and potential risk factors for amitraz resistance in Boophilus microplus ticks in cattle farms in the State of Yucatan, Mexico.

Southern cattle tick, Boophilus microplus, control in the Mexican tropics is based on the application by spray, dip or pour-on of acaricides. In Mexico the first report of ticks resistant to amitraz was reported in 2002. Amitraz resistance affecting the cattle industry of Yucatan State, Mexico, has not been investigated. A cross-sectional study was carried out in order to investigate the possible association between management risk factors and the prevalence of B. microplus ticks resistant to amitraz in Yucatan, Mexico. Ninety-eight cattle farms were randomly selected and a sample of 20-30 engorged B. microplus females was collected from at least 10 cattle from each farm. Collected ticks were taken to the laboratory where the larval progeny were obtained. The modified larval immersion test was used to detect ticks resistant to amitraz. A discriminating dose of 0.0002% amitraz was used. A questionnaire was applied on each collaborating farm. For the statistical analysis a 2x2 contingency table was used to identify potential risk factors. The prevalence of farms with B. microplus resistant to amitraz was 19.40%. However, even though tick resistance to amitraz is a present problem there was no evidence that it was associated with the risk factors analyzed in this survey.

Prevalence and potential risk factors for organophosphate and pyrethroid resistance in Boophilus microplus ticks on cattle ranches from the State of Yucatan, Mexico.

A cross-sectional study was carried out in order to investigate the association between management risk factors and the prevalence of organophosphate (OP) and synthetic pyrethroid (SP) resistance in Boophilus microplus ticks in Yucatan State, Mexico. Ninety-eight cattle ranches were randomly selected and a sample of 20-30 engorged female B. microplus ticks was collected at each. The modified larvae packet test was used to detect OP and SP tick resistance. A questionnaire was administered on each ranch. For risk factors, four reference types of response were used: Tuxpan (resistant to coumaphos, chlorfenvinphos and diazinon), Parkhurst (resistant to flumethrin, deltamethrin and cypermethrin), Lamington (resistant to flumethrin in absence of deltamethrin and cypermethrin) and Marmor (resistant to flumethrin and cypermethrin in absence of flumethrin). A primary screening was performed using 2 x 2 contingency tables of exposure variables. All variables with p < or = 0.20 were analyzed by a logistic regression. The prevalence of B. microplus resistance to OP and SP groups were 83.70% and 66.32%, respectively. The prevalence for a single chemical compound was: diazinon (80.60%), coumaphos (45.90%), chlorfenvinphos (35.70%), flumethrin (63.28%), deltamethrin (61.22%), and cypermethrin (59.18%). The prevalence type of response was: Tuxpan (34.16%), Parkhurst (58.16%), Lamington (4.08%), and Marmor (4.08%). The use of a different tick control program was the risk management factor associated with the Parkhurst type of response. It is concluded that the prevalence of cattle ranches with B. microplus resistant to OPs and SPs in Yucatan, Mexico, is a current problem, and a treatment interval of > or =6 times/year and use of a tick control program (macrocyclic lactones and pasture burn) different from standard recommended programs are risk factors associated with the Parkhurst type of response.

Factors that influence the prevalence of acaricide resistance and tick-borne diseases.

This manuscript provides a summary of the results presented at a symposium organized to accumulate information on factors that influence the prevalence of acaricide resistance and tick-borne diseases. This symposium was part of the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP), held in New Orleans, LA, USA, during August 10-14, 2003. Populations of southern cattle ticks, Boophilus microplus, from Mexico have developed resistance to many classes of acaricide including chlorinated hydrocarbons (DDT), pyrethroids, organophosphates, and formamidines (amitraz). Target site mutations are the most common resistance mechanism observed, but there are examples of metabolic mechanisms. In many pyrethroid resistant strains, a single target site mutation on the Na(+) channel confers very high resistance (resistance ratios: >1000x) to both DDT and all pyrethroid acaricides. Acetylcholine esterase affinity for OPs is changed in resistant tick populations. A second mechanism of OP resistance is linked to cytochrome P450 monooxygenase activity. A PCR-based assay to detect a specific sodium channel gene mutation that is associated with resistance to permethrin has been developed. This assay can be performed on individual ticks at any life stage with results available in a few hours. A number of Mexican strains of B. microplus with varying profiles of pesticide resistance have been genotyped using this test. Additionally, a specific metabolic esterase with permethrin-hydrolyzing activity, CzEst9, has been purified and its gene coding region cloned. This esterase has been associated with high resistance to permethrin in one Mexican tick population. Work is continuing to clone specific acetylcholinesterase (AChE) and carboxylesterase genes that appear to be involved in resistance to organophosphates. Our ultimate goal is the design of a battery of DNA- or ELISA-based assays capable of rapidly genotyping individual ticks to obtain a comprehensive profile of their susceptibility to various pesticides. More outbreaks of clinical bovine babesisois and anaplasmosis have been associated with the presence of synthetic pyrethroid (SP) resistance when compared to OP and amidine resistance. This may be the result of differences in the temporal and geographic patterns of resistance development to the different acaricides. If acaricide resistance develops slowly, herd immunity may not be affected. The use of pesticides for the control of pests of cattle other than ticks can affect the incidence of tick resistance and tick-borne diseases. Simple analytical models of tick- and tsetse-borne diseases suggest that reducing the abundance of ticks, by treating cattle with pyrethroids for example, can have a variety of effects on tick-borne diseases. In the worst-case scenario, the models suggest that treating cattle might not only have no impact on trypanosomosis but could increase the incidence of tick-borne disease. In the best-case, treatment could reduce the incidence of both trypanosomosis and tick-borne diseases Surveys of beef and dairy properties in Queensland for which tick resistance to amitraz was known were intended to provide a clear understanding of the economic and management consequences resistance had on their properties. Farmers continued to use amitraz as the major acaricide for tick control after the diagnosis of resistance, although it was supplemented with moxidectin (dairy farms) or fluazuron, macrocyclic lactones or cypermethrin/chlorfenvinphos.