METARHIZIUM ANISOPLIAE SENSU LATO NATIVE TO LIVESTOCK SOILS CAUSES HIGH MORTALITY ON RHIPICEPHALUS MICROPLUS LARVAE, ADULTS AND AFFECTS THEIR REPRODUCTION.

The acaricidal effect of 14 strains of Metarhizium anisopliae sensu lato isolated from soil of livestock farms in the Mexican tropics was evaluated against larvae and engorged females, and during the laying and hatching of eggs of Rhipicephalus microplus (Ixodida: Ixodidae). For each fungal strain, the larvae mortality percentage was evaluated through a larval immersion test, while the reproductive efficiency indices in engorged females were measured using adult immersion tests at a dose of 1 × 108 conidia/ml. All strains of M. anisopliae (s.l.) proved to be highly effective against R. microplus larvae (66-100%) and engorged females (100%). The strains also showed a good effect in inhibiting egg laying (16.45-56.38%) and a moderate effect in decreasing egg hatching (5.24-32.68%). Two strains demonstrated to be effective against all development phases of R. microplus in an integrated manner.

FIRST EVIDENCE OF THE TICKICIDE EFFECT OF ENTOMOPATHOGENIC FUNGI ISOLATED FROM MEXICAN CATTLE FARMS AGAINST AMBLYOMMA MIXTUM.

This is the first study to empirically evaluate the mortality of Amblyomma mixtum larvae caused by native entomopathogenic fungi (EPF) from cattle farm soils and the impact of the physicochemical conditions of those soils on the ability of EPF to kill ticks. The efficacy of 25 EPF strains isolated from cattle farms soils in Veracruz, Mexico, against A. mixtum ticks was evaluated using a larval immersion test. Physicochemical analyses of the soils where the EPF were isolated were carried out, and the results were correlated with the obtained mortality. The MaV25 strain showed a mortality of 36.55% (P < 0.05), followed by MaV57 with 27.30%, MaV08 with 26.21%, and MaV31 with 24.32% (P < 0.05). Nitrogen and potassium are nutritional components of soils that seem to be associated with mortality caused by the evaluated fungal strains. This is the first study in Mexico where the effect of EPF against A. mixtum is evaluated and also is the first study in the world that uses native strains from livestock soils against larvae of this tick. It is necessary to determine the virulence characteristics of EPF on A. mixtum to improve knowledge of fungus-tick interactions (Metarhizium anisopliae-A. mixtum).

Acaricidal activity of Metarhizium anisopliae isolated from paddocks in the Mexican tropics against two populations of the cattle tick Rhipicephalus microplus.

The acaricidal effects of 55 strains of Metarhizium anisopliae (Metschnikoff) Sorokin, 1883 (Hypocreales: Clavicipitaceae) isolated from paddocks of cattle farms were evaluated in two Rhipicephalus microplus (Canestrini 1887) (Ixodida: Ixodidae) populations, of which one was multi-resistant and one was susceptible to chemical acaricides. Percentage mortality and reproductive efficiency indices in R. microplus were evaluated by adult immersion tests at a dose of 1 × 108 conidia/mL for each fungal strain. Some strains were selected to calculate lethal concentrations to kill 50% (LC50 ) and 99% (LC99 ) of engorged ticks. Strains MaV22, MaV26 and MaV55 induced 100% mortality in R. microplus on day 14. Strains MaV05, MaV09 and MaV22 caused mortality of >90% from day 12 onward in both tick populations. The most effective acaricidal fungal strain, MaV55, inhibited egg laying by 54.86 and 55.86% in acaricide-resistant and -susceptible R. microplus populations, respectively. None of the fungal strains had statistically significant effects on larval hatching. In conclusion, nine strains of M. anisopliae demonstrated high acaricidal effects against R. microplus and reduced its egg laying. No differences in acaricidal effects were observed between the two populations of ticks tested.

First report of cattle farms with gastrointestinal nematodes resistant to levamisole in Mexico.

The objectives of the present study were: (1) to report the percentage of cattle farms with gastrointestinal nematodes (GINs) resistant to levamisole in Veracruz, Mexico, (2) to identify the genera of GINs involved in resistance, and (3) to identify factors associated with these resistances. The faecal egg count reduction test (McMaster technique) was used to detect the presence of resistant GINs. A questionnaire was given to owners to understand the history of anthelmintic use. The percentage of cattle farms with GINs resistant to levamisole was 36.4% (4/11). The percentage of faecal egg count reduction on resistant farms was 91%, 82%, 42% and 88%. A similar number of cattle farms (4/11) were identified as potentially having levamisole resistance. Only three farms had GIN populations susceptible to levamisole. Cooperia spp. was the genus most commonly found to be resistant, followed by Haemonchus spp., Ostertagia spp. and Oesophagostomum spp. No factors were identified that influenced the presence of GIN resistance. However, there were identified inappropriate anthelmintic practices in cattle farms that should be improved. None of the farmers weighed their animals in order to dose them correctly with anthelmintics. Six cattle farms (54.5%) applied anthelmintics to new arriving animals. This is the first report of levamisole resistant GINs in Mexico. Improving the use of anthelmintics and measures of quarantine for infected cattle will help control the spread of resistance.

Amblyomma cajennense (Acari: Ixodidae) tick populations susceptible or resistant to acaricides in the Mexican Tropics.

The objectives of the present study were: (i) to identify the frequency of cattle farms with a cohabitation of Amblyomma cajennense and Rhipicephalus microplus, (ii) to determine the status of susceptibility or resistance to acaricides used in Veracruz, Mexico, on A. cajennense populations and (iii) to identify factors associated with A. cajennense resistant to acaricides. Fifty farms were visited to determine the presence of ticks (A. cajennense and R. microplus) and to collect engorged A. cajennense individuals. From these, 24 A. cajennense populations were evaluated in resistance bioassays using discriminating doses of acaricides. The acaricides tested were organophosphates (chlorpiriphos, coumaphos and diazinon), pyrethroids (flumethrin, deltamethrin and cypermethrin), amidines (amitraz) and fipronil (a broad spectrum N-phenylpyrazole insecticide). A. cajennense infesting bovines were identified in 86% (43/50) of the farms visited, and 100% of the farms sampled (43/43) had cohabitation between R. microplus and A. cajennense. Of the farm owners or managers surveyed, 87.5% could not distinguish the morphological difference between tick genera. Populations of A. cajennense were 100%, 91.7% and 12.5% resistant to diazinon, coumaphos and chlorpyriphos (organophosphates), respectively, and 12.5% to amitraz, as were those susceptible to flumethrin and fipronil. In conclusions, populations of A. cajennense showed a high frequency of resistance to the organophosphates tested and to amitraz. Factors associated with the resistance to acaricides in A. cajennense were not identified.

Ivermectin resistance status and factors associated in Rhipicephalus microplus (Acari: Ixodidae) populations from Veracruz, Mexico.

The objectives of the present study were to (1) determine the status of resistance or susceptibility to ivermectin (IVM) in Rhipicephalus microplus tick populations in Veracruz, Mexico, (2) determine the level of resistance (resistance ratios [RR] and lethal concentrations for 50% [LC(50)] and 99% [LC(99)]) mortality in each R. microplus population, and (3) identify factors associated with resistance. Populations of R. microplus were sampled from 53 cattle farms to evaluate their resistance using the larval immersion test. Mortality data were subjected to probit analysis to calculate LC(50) and LC(99). Resistance ratios were calculated in relation to a susceptible reference strain. A logistic regression model was used to evaluate the relation between resistance and possible associated factors. Thirteen tick populations were susceptible to ivermectin, eighteen had incipient resistance and twenty-two had significant resistance. RR(50) of the susceptible tick populations varied from 0.59 to 1.07. The populations that showed the highest level of resistance were: ANTE (RR(50)=8.21; RR(99)=46.0), PALO (RR(50)=6.25; RR(99)=35.47), P.VIE (RR(50)=5.89; RR(99)=180.3), AURO (RR(50)=5.36; RR(99)=13.82 and CEDR (RR(50)=4.11; RR(99)=26.47). Cattle farms that used macrocyclic lactones ≥ 4 times per year were more likely to develop R. microplus resistant to ivermectin (OR=13.0; p=0.0028). In conclusion, more than two-thirds of the farms sampled in Veracruz, Mexico, showed some level of ivermectin-resistant R. microplus populations and the number of ML applications per year is factor associated with the resistance of R. microplus to IVM.

First report of a Rhipicephalus microplus tick population multi-resistant to acaricides and ivermectin in the Mexican tropics.

We document the presence of a Rhipicephalus microplus tick population resistant to acaricides (organophosphates (OP), synthetic pyrethroids (SP), amitraz) and macrocyclic lactones (ML) (ivermectin). Engorged females of R. microplus were collected from a cattle farm in Veracruz, Mexico, to evaluate acaricide and ivermectin resistance. The modified larval packet test (LPT) was used to detect OP (chlorpiriphos and diazinon) and SP (flumethrin, deltamethrin and cypermethrin) resistance and the larval immersion test (LIT) to detect resistance to amitraz and ivermectin. Both, LPT and LIT were performed twice at different times with different collected samples. Mortality data with ivermectin were subjected to probit analysis to obtain lethal concentrations and resistance ratios (RR) using an ivermectin-susceptible strain (Deutch) as a reference. The R. microplus population showed resistance to all acaricides tested, with different mortalities at the discriminate dose: chlorpiriphos (1%), diazinon (24.2%), flumethrin (92.8%), deltamethrin (94.2%), cypermethrin (98.0%) and amitraz (1.5%). The studied tick population also showed resistance to ivermectin with a resistance ratio at 99% of 9.58 and 6.52 in the first and second evaluation, respectively. We report for the first time a R. microplus population in Mexico with different levels of resistance to OP, SP, amidines (Am) and ivermectin. The uncontrolled use of these products in the study area may promote the complete failure of tick control within a short period of time.

In vitro acaricidal effect of tannin-rich plants against the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

The objectives of this study were to evaluate the in vitro acaricidal effects of lyophilized extracts of four tannin rich plants (Acacia pennatula, Piscidia piscipula, Leucaena leucocephala and Lysiloma latisiliquum) against diverse stages of Rhipicephalus (Boophilus) microplus, and to asses whether tannins were involved in the acaricidal effect using polyethylene glycol (PEG) to block tannins. Larval immersion (LIT) and adult immersion (AIT) tests were used to evaluate the acaricidal effect of each of the lyophilized extracts against larval and adult stages of R. microplus respectively. Larvae and adult ticks were exposed to increasing concentrations of each plant extract (0, 1200, 2400, 4800, 9600 and 19,200 µg ml(-1)) for 10 min. Larval mortality was recorded at 48 h post-incubation. Adult mortality was recorded daily over 14 days, at which point their reproductive efficiency was evaluated. PEG was added to the extracts to verify whether tannins were involved in the acaricidal effect. The effect on egg laying inhibition and larval mortality was analyzed using the GLM procedure in SAS. A Kruskal-Wallis test was used to assess the effect of PEG on LIT results. Calculation of the lethal concentration 50 (LC50) was performed using a probit analysis. All extracts reduced the viability of R. microplus larval stages (P<0.001), and viability was restored with the addition of PEG suggesting an important role of tannins in the acaricidal effect (P<0.001). The LC50 values of L. latisiliquum and P. piscipula plant extracts were 6.402 and 2.466 µg ml(-1). None of the tannin-rich plant extracts affected adult mortality (P>0.05). Lysiloma latisiliquum extract inhibited egg hatching of R. microplus (P<0.01). Tannin-rich plant extracts from A. pennatula, P. piscipula, L. leucocephala and L. latisiliquum showed potential acaricidal activity. Further in vivo studies are needed to confirm this finding.