Potential effects on Rhipicephalus microplus tick larvae fed on calves immunized with a Subolesin peptide predicted by epitope analysis.

The cattle tick Rhipicephalus microplus economically impacts cattle production in tropical and subtropical regions of the world. The recombinant R. microplus Subolesin antigen has been shown to protect cattle against tick infestations. In this study, we searched in silico protective epitopes in the subolesin gene and a recombinant peptide containing the predicted epitopes was expressed, and evaluated against a tick challenge. Two different epitope types, linear B-cells and conformational discontinuous epitopes, were predicted using bioinformatics strategies, in order to synthesize the recombinant peptide. Two Eightmonth-old calves European crossbred were immunized with two subcutaneous doses of the subolesin recombinant peptide, emulsified with Montanide ISA 50 V as an adjuvant, at 30-day intervals. The tick challenge was conducted with 5 000 R. microplus larvae/animal. ELISA test was used to evaluate the IgG immune response elicited against the peptide. After tick challenge, reduction in the number of engorged females (79%), and reduction in egg hatching (30%) was observed in tick population fed on immunized calves with regards to an untreated control group. The results showed a potential higher effect on tick reproduction for the recombinant peptide compared to other studies reported with Subolesin protein, demonstrating that the use of bioinformatics strategies to predict protective epitopes may lead to improve the immune response elicited against tick recombinant peptides and therefore to prevent cattle tick infestations.

Acaricidal effect of essential oils from Lippia graveolens (Lamiales: Verbenaceae), Rosmarinus officinalis (Lamiales: Lamiaceae), and Allium sativum (Liliales: Liliaceae) against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

Acaricidal effects of three essential oils extracted from Mexican oregano leaves (Lippia graveolens Kunth), rosemary leaves (Rosmarinus officinalis L.), and garlic bulbs (Allium sativum L.) on 10-d-old Rhipicephalus (Boophilus) microplus (Canestrini) tick larvae were evaluated by using the larval packet test bioassay. Serial dilutions of the three essential oils were tested from a starting concentration of 20 to 1.25%. Results showed that both Mexican oregano and garlic essential oils had very similar activity, producing high mortality (90-100%) in all tested concentrations on 10-d-old R. microplus tick larvae. Rosemary essential oil produced >85% larval mortality at the higher concentrations (10 and 20%), but the effect decreased noticeably to 40% at an oil concentration of 5%, and mortality was absent at 2.5 and 1.25% of the essential oil concentration. Chemical composition of the essential oils was elucidated by gas chromatography-mass spectrometry analyses. Mexican oregano essential oil included thymol (24.59%), carvacrol (24.54%), p-cymene (13.6%), and y-terpinene (7.43%) as its main compounds, whereas rosemary essential oil was rich in a-pinene (31.07%), verbenone (15.26%), and 1,8-cineol (14.2%), and garlic essential oil was rich in diallyl trisulfide (33.57%), diallyl disulfide (30.93%), and methyl allyl trisulfide (11.28%). These results suggest that Mexican oregano and garlic essential oils merit further investigation as components of alternative approaches for R. microplus tick control.

Phenotype changes inherited by crossing pyrethroid susceptible and resistant genotypes from the cattle tick Riphicephalus (Boophilus) microplus.

Dialelic crosses and backcrosses of pyrethroid resistant (RR) and susceptible (SS) Rhipicephalus (Boophilus) microplus tick strains were carried out and the substitution (Phe-Ile) within the sodium channel gene was monitored in order to analyze the effects of the genotype on the pyrethroid resistance phenotype as measured by the larval packet test (LPT). Parental strains: susceptible (SS) and resistant (RR); dialelic crosses: RS (♂RR × â™€SS), and SR (♂SS × â™€RR); and backcrosses: RS × SS, RS × RR, SR × SS and SR × RR were infested on 280 kg calves. Resistance type (monogenic or polygenic) and effective dominance were determined based on the discriminant concentration (DC) for cipermethrine (0.5%), deltamethrine (0.09%) and flumethrine (0.01%). Allele specific PCR (AS-PCR) was used for genotyping, looking at a sodium channel mutation (Phe-Ile substitution). The mortality rates and allele frequency of susceptible and pyrethroid resistant reference strains were 0% mortality and 90% RR alleles for resistant strain, and 100% mortality and 0% RR alleles as measured by the larval packet test (LPT) and allele specific PCR (AS-PCR) respectively. Backcrossed strain SR × RR showed an effective dominance (D(ML)) of 0.605 for cypermethrin, 0.639 for deltamethrin and 0.498 for flumethrin, while survival of backcrosses RS × SS, RS × RR and SR × SS showed a significant tendency to recesivity. Backcrossed strain SR × RR (69.4%) also showed a higher RR genotype frequency with regards to RS × SS (25.5%), RS × RR (36.7%) and SR × SS (32.0%), however, susceptible allele was inherited in general as an incomplete dominant trait. Monogenic inheritance hypothesis was tested and the results showed monogenic inheritance for cypermethrin and flumethrin (P < 0.05) but not for deltamethrin (P > 0.05). However, significant correlation was found between RR genotype and the survival rate for all three pyrethroids used (P < 0.05), suggesting that a single substitution on the sodium channel gene can be responsible for resistance to pyrethroids as a class, due to the high frequency for RR genotypes. Combination with different mutations or metabolic resistance mechanisms cannot be excluded.

Inheritance of pyrethroid resistance and a sodium channel gene mutation in the cattle tick Boophilus microplus.

A substitution (Phe-->Ile) within the sodium channel gene sequence has been associated with pyrethroid resistance in Boophilus microplus. The aim of the present study was to analyze the inheritance of pyrethroid resistance and the mutant allele, on reciprocal crosses of a susceptible (SS) and a resistant (RR) strain. Bioassays and genotypes were determined to evaluate pyrethroid resistance. The resistance allele frequency of both parental strains were 100% and 2.27% for RR and SS, respectively. The reciprocal crosses show a predominance of the heterozygote genotype, in agreement with the significant decrease of the acaricide resistance to cypermethrin, deltamethrin, and flumethrin. However, the RS progeny showed a complete recessive survival (D (ML) = 0) for deltamethrin and flumethrin, suggesting a complete dominance of the susceptible allele and incomplete dominance for cypermethrin (D (ML) = 0.169). On the other hand, SR progeny showed a partially recessive survival for cypermethrin (D (ML) = 0.380), deltamethrin (D (ML) = 0.319), and flumethrin (D (ML) = 0.258), indicative of a partially dominant inheritance of the resistance. A possible maternal strain effect should be considered for practical purposes and prediction of acaricide resistance and further work needs to be done to elucidate the underlying inheritance of pyrethroid resistance and the sodium channel mutation in B. microplus.

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.

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.

Neospora caninum seropositivity and association with abortions in dairy cows in Mexico.

The seroprevalence of Neospora caninum infection was estimated from a sample of 813 cattle from 20 dairy herds in five regions in Mexico. The true prevalence of infection was estimated to be 42% (95% CI: 39, 46). Seropositivity was associated with abortion (odds ratio (OR)=2.0) and was higher among cows raised on-farm (41%), than among replacement cattle purchased outside the farm (28%). The ORs relating abortion to seropositivity in individual herds ranged from 1.3 to 10. Overall, 26% of the abortions were attributed to N. caninum.

Roles played by esterase activity and by a sodium channel mutation involved in pyrethroid resistance in populations of Boophilus microplus (Acari: Ixodidae) collected from Yucatan, Mexico.

Pyrethroid resistance in Boophilus microplus (Canestrini) (Acari: Ixodidae) was studied by correlating discriminating-dose (DD) bioassay results and esterase activity or the frequency of a sodium channel mutation known to be involved in pyrethroid resistance in nine field strains of B. microplus from Yucatan, Mexico. Two tick strains (P67 and B74) were identified as susceptible to cypermethrin, deltamethrin, and flumethrin by DD, one strain (P65) was susceptible to cypermethrin and resistant to deltamethrin and flumethrin, and six strains were resistant to cypermethrin, delta-methrin, and flumethrin (T11, M10, C54, R49, B71, and T66). By using polymerase chain reaction, only 6.0 and 6.2% of resistance allele (R) was found in the susceptible strains (P67 and B74, respectively). In the T66 strain, with 100% of larval survival to the DD of pyrethroids as measured by the larval packet test (LPT), 98.0% of the gene pool contained the R allele. Positive correlations between the larval survival and the percentage of the R allele were found (deltamethrin r2 = 0.8875, P < 0.01; cypermethrin r2 = 0.8563, P < 0.01; and flumethrin r2 = 0.8491, P < 0.01). There were no significant correlations between the level of larval survival and esterase-based hydrolytic activity. It was concluded that within the B. microplus populations studied, resistance to flumethrin, deltamethrin, or cypermethrin was because of the novel sodium channel mutation (Phe-->Ile amino acid substitution in the S6 transmembrane segment of domain III), and there was a correlation between tick mortality by pyrethroid exposure (larval survival) and the presence of R allele. It was not determined whether enhanced esterase-based hydrolytic activity was involved in pyrethroid resistance in the populations tested.