J.A. Rosado-Aguilar

Acaricidal activity of extracts from Petiveria alliacea (Phytolaccaceae) against the cattle tick, Rhipicephalus (Boophilus) microplus (Acari: ixodidae)

The acaricidal activity of crude extracts and fractions from stems and leaves of Petiveria alliacea (Phytolaccaceae) was carried out on larvae and adults of the cattle tick Rhipicephalus (Boophilus) microplus using the larval immersion test (LIT) and adult immersion test (AIT), respectively. Methanolic extracts of stems and leaves of P. alliacea showed 100% mortality on the LIT bioassay. On the other hand, methanolic extracts of leaves and stem on the AIT test showed 26% and 86% of mortality, respectively, egg laying inhibition of 40% and 91%, respectively and hatchability inhibition of 26% and 17%, respectively. Purification of the active stem methanolic extract showed that the activity was present in the n-hexane non-polar fraction. Bioassay-guided purification of the n-hexane fraction produced 10 semi-purified fractions; fraction B had the highest activity against tick larvae (100% mortality). Gas chromatography-mass spectrometry demonstrated that the chemical composition of the active fraction B samples were mainly composed of benzyltrisulfide (BTS) and benzyldisulfide (BDS). These metabolites might be responsible for the acaricidal activity of stem extract of P. alliacea. However, further experiments to evaluate the acaricidal activity of BTS and BDS on larvae and adults of R. (B.) microplus are needed. Our results showed that P. alliacea is a promising biocontrol candidate as acaricide against R. (B.) microplus resistant strains.

Factors affecting the prevalence of mange-mite infestations in stray dogs of Yucatán, Mexico.

The aim of the present study was to determine the factors affecting the prevalence of mange-mite infestations in stray dogs of Yucatán, Mexico. The study was carried out in 200 stray dogs of Mérida capital city of Yucatán, Mexico. Four samples (head, thoracic-abdominal area, extremities and ear) were taken from each animal by skin scraping and examined microscopically in 10% KOH solution to detect the presence of mites. Mites were also collected from the external ear canal of dogs using cotton-tipped swabs. The prevalence of different mite species was calculated. A primary screening was performed using 2xK contingency tables of exposure variables. All variables with P< or =0.20 were analyzed by a logistic-binomial regression. The overall prevalence was 34%. Demodex canis (23.0%) was the most frequent mite, followed by Sarcoptes scabei var. canis (7.0%) and Otodectes cynotis (3.5%). The following factors were found: body condition (bad, OR: 5.35, CI 95%: 1.66-17.3; regular, OR: 3.72, CI 95%: 1.39-9.99) and the presence of macroscopic lesions of dermatosis (OR: 42.80, CI 95%: 13.65-134.24).

Survey of Rhipicephalus microplus resistance to ivermectin at cattle farms with history of macrocyclic lactones use in Yucatan, Mexico

Engorged females of Rhipicephalus microplus were collected from 30 cattle farms in Yucatan, Mexico to evaluate ivermectin resistance. The larval progeny of each tick sample were produced in laboratory and evaluated using the larval immersion test to obtain the larval mortality. Concentration-mortality data were subjected to probit analysis to generate lethal concentrations (LC). Resistance ratio (RR) of each tick sample was calculated by dividing its LC with that of an ivermectin-susceptible strain (Deutch). Field populations of R. microplus demonstrated various levels of resistance to ivermectin. The top three resistant populations were CHPAT (RR(50)=10.23, RR(99)=179.6), SDGO (RR(50)=7.37, RR(99)=115.3) and FND (RR(50)=7.09, RR(99)=50.22). The level of ivermectin resistance in most tick samples from cattle farms in Yucatan, Mexico were relatively low. However, the intensive use of macrocyclic lactones to control both endo- and ectoparasites in the region will likely lead to a more serious resistance problem that may cause control failure in the future.

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.

Evolution of acaricide resistance: phenotypic and genotypic changes in field populations of Rhipicephalus (Boophilus) microplus in response to pyrethroid selection pressure.

There have been few, if any, studies of arthropod field populations quantifying the kinetics of evolution of phenotypic and genotypic resistance to chemicals in response to the presence or absence of selection pressure. A prospective intervention study was undertaken over 2 years in Mexico to measure changes in resistance phenotype and genotype in the presence or absence of pyrethroid selection pressure on field populations of Rhipicephalus (Boophilus) microplus ticks on 11 farms with varying degrees of pyrethroid resistance. The resistance phenotype was evaluated by bioassay in a larval packet test expressed as the resistance factor (RF) derived from probit analysis of dose mortality regressions, and resistance genotype by an allele-specific PCR (AS-PCR) to determine the frequency of a sodium channel mutation (F1550I) associated with pyrethroid resistance. To validate the AS-PCR, a Pyrosequencing™ method was developed to detect the F1550I mutation. There was good concordance with the genotypes identified by both Pyrosequencing™ and AS-PCR (Kappa: 0.85). On five farms cypermethrin (CY) was exclusively used at intervals and on six farms amitraz was used. On two of the five CY-treated farms, the experiment was prematurely terminated due to unacceptably high levels of tick resistance. For all five farms, after 8-24 months of continued selection pressure with CY, the RF had increased 2-125-fold. The frequency of the resistance allele increased on all five farms from a starting range of 5-46% to a range of 66-95% after 8-24 months. On six farms treated with amitraz neither the RF nor the frequency of the resistance allele changed. A clear correlation between the phenotype and genotype was found in three of four treated farms confirming that the F1550I mutation is a major cause of synthetic pyrethroid resistance in Mexico. These results show that the pyrethroid resistance trait is stable (> 2 years) and that resistance is acquired much faster than it is lost. Hence, alternation of pyrethroid acaricide with other chemicals is likely to lead to the stepwise acquisition of synthetic pyrethroid resistance but not additional prolongation of its efficacious lifespan.

Uso de lactonas macrocíclicas para el control de la garrapata Rhipicephalus (Boophilus) microplus en el ganado bovino

En esta revision se describe en detalle la composicion quimica, farmacocinetica, presentacion y dosis, seguridad y tiempo de retiro y la seguridad ambiental de los grupos que componen a las LM: avemectinas (ivermectina, doramectina, eprinomectina, abamectina) y milbemicinas (moxidectina). Ademas se presenta una discusion sobre su eficacia y el problema de resistencia de Rhipicephalus (Boophilus) microplus a las LM. Se concluye que las LM por su alta liposolubilidad se distribuyen ampliamente en los tejidos, siendo eficaces para el control de R. (B.) microplus en el ganado bovino. La eficacia de la ivermectina, doramectina, eprinomectina, abamectina y moxidectina de corta accion es similar (> 90% de eficacia a las cuatro semanas postratamiento, PT) para el control de fases adultas de R. (B.) microplus; sin embargo, existen en el mercado LM de larga accion que presentan eficacia > 95% con persistencia hasta 70 dias PT.

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.

The prevalence of pyrethroid resistance phenotype and genotype in Rhipicephalus (Boophilus) microplus in Yucatan, Mexico.

A field survey was conducted to evaluate susceptibility of Rhipicephalus (Boophilus) microplus to cypermethrin on 49 farms in three areas of Yucatan, Mexico. The modified larval packet test was used to evaluate larval mortality at different cypermethrin concentrations. Dose-mortality regressions, lethal concentrations (LC(50)-LC(99)), confidence intervals and slope were estimated by probit analysis. Phenotype was defined as susceptible, tolerant or resistant when the resistance factor (RF) derived from both LC(50) and LC(99) determinations were <3, 3-5 or >5, respectively. An allele specific PCR (AS-PCR) was used to determine the frequency of a sodium channel mutation (F1550I, Phe→Ile) associated with pyrethroid resistance. Overall, 26.5%, 40.8% and 32.6% of tick populations were susceptible, tolerant and resistant to cypermethrin, respectively. A substantial inter-population variation in the level of cypermethrin response was evident (resistance factors ranged from 0.3 to 2599 and from 0.7 to >5000 when were indicated by the LC(50) and LC(99), respectively). The F1550I mutation (R allele) in R. microplus was present in all studied areas. The increasing presence of the R allele correlated well with increased levels of response indicated by both the LC(50) (r(2)=0.659, p=0.001) and LC(99) (r(2)=0.688, p=0.001) to cypermethrin. These results indicated that the F1550I mutation is a major common mechanism responsible for pyrethroid resistance in field populations of R. microplus ticks in the Mexican tropics. Both bioassay and AS-PCR showed that the prevalence of cypermethrin-resistant/tolerant R. microplus is high in Yucatan, Mexico and the relationship between the RF and the frequency of the R allele supports the role of F1550I as one of the most important mechanisms conferring pyrethroid resistance in these R. microplus populations.

Acaricide and ivermectin resistance in a field population of Rhipicephalus microplus (Acari: Ixodidae) collected from red deer (Cervus elaphus) in the Mexican tropics

In the Neotropics the control of tick infestations in red deer (Cervus elaphus) is achieved primarily through the use of acaricides and macrocyclic lactones. In Mexico, resistance to one or multiple classes of acaricides has been reported in Rhipicephalus microplus infesting cattle, but information on acaricide susceptibility in R. microplus infesting red deer is lacking. In this study we report the level of resistance to different classes of acaricides and ivermectin in R. microplus collected from red deer in the Mexican tropics. Engorged R. microplus females were collected from a red deer farm in Yucatan, Mexico. The larval packet test was used to detect resistance to the organophosphates (OPs) chlorpyrifos and coumaphos, synthetic pyrethroids (SPs) cypermethrin and permethrin, and the phenylpyrazol, fipronil. Resistance to the formamidine amitraz (Am), and ivermectin was ascertained using the larval immersion test. Data were subjected to probit analysis to determine lethal concentrations and resistance ratios to kill 50% (RR50) and 99% (RR99) of the tick population under evaluation in relation to susceptible reference strains. Additionally, allele specific polymerase chain reaction was used to detect the sodium channel F1550I mutation associated with SP resistance in R. microplus. The R. microplus population from red deer in Yucatan showed very high resistance to the two SPs evaluated (RRs>72.2 for cypermethrin; RR for permethrin resistance was so high a dose-response curve was not possible). All individual larvae tested to detect the sodium channel F1550I mutation associated with SP resistance in R. microplus were homozygous. The same tick population showed different levels of resistance to OPs (chlorpyrifos: RR50=1.55, RR99=0.63; coumaphos: RR50=6.8, RR99=5.9), fipronil (RR50=1.8, RR99=0.9), and amitraz (RR50=2.3, RR99=4.4). Resistance to ivermectin was regarded as moderate (RR50=7.1, RR99=5.0). This is the first report of R. microplus ticks collected from red deer in Mexico with different levels of resistance to four acaricide groups and ivermectin.

In vitro and in vivo evaluation of cypermethrin, amitraz, and piperonyl butoxide mixtures for the control of resistant Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in the Mexican tropics

A study was conducted to evaluate the efficacy of cypermethrin, amitraz, and piperonyl butoxide (PBO) mixtures, through in vitro laboratory bioassays and in vivo on-animal efficacy trials, for the control of resistant Rhipicephalus (Boophilus) microplus on cattle in the Mexican tropics. Also, to examine mechanisms of resistance to cypermethrin in this tick population, the frequency of a mutated sodium channel gene (F1550I) was determined using a PCR assay. Results of laboratory bioassays using modified larval packet tests revealed that cypermethrin toxicity was synergized by PBO (from 46.6-57.0% to 83.7-85.0% larval mortality; P<0.05). The cypermethrin and amitraz mixture showed an additive effect (from 46.6-57.0% to 56.0-74.3% larval mortality). Strong synergism was observed with the mixture of cypermethrin+amitraz+PBO and this mixture was the most effective killing resistant tick larvae in vitro (96.7-100% of larval mortality). Tick larvae surviving exposure to cypermethrin or mixtures either with amitraz and PBO in vitro showed 2.9-49.6 higher probability to present the mutated allele than those killed by acaricide treatment (P<0.05). In the in vivo trial, the mixtures containing cypermethrin+PBO (80.6-97.3%), and cypermethrin+amitraz (87.0-89.7%) were more efficacious than cypermethrin alone (76.3-80.5%). The highest level of efficacy was obtained with the mixture of cypermethrin+amitraz+PBO, which yielded >95% control that persisted for 28 days post-treatment against R. microplus infesting cattle when tested under field conditions in the Mexican tropics. Although this mixture is a potentially useful tool to combat pyrethroid resistance, a product based on an acaricide mixture like the one tested in this study has to be used rationally.