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.

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.

Seroepidemiological study of Neospora caninum infection in dogs found in dairy farms and urban areas of Aguascalientes, Mexico.

The objective of this study was to describe the seroepidemiology of Neospora caninum infection in dogs in close contact with dairy cattle and dogs from urban areas of Aguascalientes, Mexico, as well as to estimate the possible association between age, gender and size of these groups and seroprevalence. Sera were obtained from samples of 152 dogs in dairy farms and 116 in the urban area, and were subjected to ELISA test. General seroprevalence reached 32%, while the seroprevalence in farm dogs (41%) was significantly higher than in dogs from the urban area (20%) (p<0.05). Regarding age groups, general seroprevalence was greater among dogs between 11 and 15 years of age (67%) remaining equal between males and females (32 and 31%, respectively). Nevertheless, females resident in farms had a higher seroprevalence (42%) than female resident in the urban area. Regarding size, large-sized animals had greater seroprevalence in farms (58%), while in the urban area medium-sized animals were those that had the highest seroprevalence (27%). It was found that dairy farm dogs had a higher risk of infection (OR=2.79; p=0.0004), and that in said group, the age range from <1 to 5 years of age was identified as a risk factor (OR=3.11, p=0.001).

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.

Relationships between stable fly infestation with some physical facility characteristics and sanitation practices in several dairy farms in the State of Aguascalientes, Mexico.

The possible relationships between stable fly infestation with dairy farm facilities and sanitation practices were studied using path analysis. Twelve dairies located in four counties of Aguascalientes dairy region were selected. The dairies were monitored from May to November 2003. In each occasion, fly infestation, individual physical facility characteristics, and sanitation practices were recorded. In all, 11 independent variables were involved in the study and related variables were grouped together and analyzed in two blocks by path analysis for each one of five population events (begin of fly season, first peak, fluctuation, second peak and decrease). There were significant regression coefficients only in the second peak for two variables, the distance to the silos and the distance to the dung heap (r(2)=0.96 for the full model). Among the 11 variables examined in the study, none had a statistical significant indirect contribution to fly infestation; direct contribution was observed for distance to the silos and for distance to the dung heap variables. However, only the distance to the silos variable was significantly related to stable fly Infestation.

Survey of Neospora caninum infection by nested PCR in aborted fetuses from dairy farms in Aguascalientes, Mexico.

The aim of this study was to determine by nested PCR probe the presence of Neospora caninum in fetal brain tissue from aborted dairy fetuses in Aguascalientes, a state in the central part of Mexico. Forty-four fetal brains from eight dairy farms were analyzed using single tube nested PCR probe with primers NF1, NS2, NR1 and SR1, with histopathology as the reference technique. From the 44 bovine fetuses studied, 35 (80%) were diagnosed as being infected by N. caninum using PCR probe and 20 (45%) were considered positive by histopathology, because they had lesions suggestive of bovine neosporosis. Fair agreement was observed (31%) between both probes. The age of aborting cows ranged from 1.6 to 2.9-year-old in 17 (49%) of the positive cases diagnosed by PCR. Gestational ages of aborted fetuses ranged from <3 to 9 months, with average of 5.6 months, and no statistical significant difference was observed when ages of infected and no infected fetuses were compared (P > 0.05). The frequency of N. caninum positive cases observed could be considered to be higher than other areas of Mexico and other countries.

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.

Analysis of BoLA class II microsatellites in cattle infested with Boophilus microplus ticks: class II is probably associated with susceptibility.

The aim of this study was to determine the role of certain bovine lymphocyte antigens (BoLA) regions in the resistance or susceptibility to Boophilus microplus tick infestation in two different breeds of cattle. The breeds were maintained, one in natural conditions and the second one in an experimental setting at the research station in Martinez de la Torre, Veracruz, Mexico. The study took place from June to August 2001 (natural infestation) using 33 crossbreed steers (crossbreed is here defined as 3/4 European = 1/2 Simmenthal x 1/4 Holstein x 1/4 Zebu, a cross resulting from F1 x Simmenthal), ranging from 15 to 20 months old. Fifty-nine F1 cows (1/2 Holstein x 1/2 Zebu) were included in the experimental setting, infested and followed during 25 days in November 2001 and 2002. Experiment A included thirty-one 2-7-year-old F1 cows, and experiment B included twenty-eight 18-24-month-old F1 heifers. Both groups were analysed separately and were not comparable because of the different infestation methods and genetic background. All ticks > or =4mm long were counted on the total body of F1 animals and on one side of the 3/4 European steers. In this case, susceptible animals were defined when having ticks = X + 1S.D. (29 +/- 16). In the experimental setting susceptibility was defined when the number of ticks was over the 75 percentile (> or =79). DNA was extracted from peripheral blood samples of all animals. The BoLA DRB3, DRBP1, RM185 and BM1815 microsatellite loci were amplified using a PCR method. Genescan software was used for analysis in an ABI sequencer. The SPSS statistical program was used and the comparisons were assessed using the Fisher's exact test. In the naturally infested animals, DRB3-184 was found positively associated with tick infestation (P = 0.018; Pc = NS; OR = 5; EF = 28%). DRBP1-128 was also found to be increased (P = 0.03; Pc = NS; OR = 6; EF = 42%). In the experimentally infested animals, two more loci were found to be associated, BM1815-152 (P = 0.01; Pc = NS; OR = 15; EF = 74%) and DRBP1-130 (P = 0.05; Pc = NS; OR = 4; EF = 77%). None of them remained significant after correction, indicating that a larger sample size is needed to confirm the results. This is the first study showing MHC genes associated with tick infestation based on class II microsatellite polymorphisms. Further studies are needed to confirm the susceptibility traits and to determine haplotype segregation in families.

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.

Serological survey of Neospora caninum infection in dairy cattle herds in Aguascalientes, Mexico.

A serological survey for antibody activity to Neospora caninum was carried out in Aguascalientes, a state in the central part of Mexico. One-hundred and eighty-seven serum samples from 13 dairy herds were tested by the ELISA test. The herd prevalence was 100% and the overall prevalence was 59% (n=110). Seventy-six of 97 seropositive cows had previous records of abortion. There was a statistically significant difference between the groups (P<0.05). However, the odds ratio was 1.4, suggesting an association between abortion and seropositivity. Neosporosis in dairy cattle appears to be widespread in Mexico, warranting more epidemiological studies to determine the distribution of the causative protozoan.

Anti-tick effects of Stylosanthes humilis and Stylosanthes hamata on plots experimentally infested with Boophilus microplus larvae in Morelos, Mexico.

The anti-tick effects of the tropical pasture legumes Stylosanthes humilis and Stylosanthes hamata were evaluated and compared with two common grasses, Cenchurus ciliaris and Andropogon gayanus, in the state of Morelos, Mexico, on plots experimentally infested with larvae of the tick Boophilus microplus. The effect was evaluated by recovery of larvae from the experimental plots by flagging during a 4 week period. The anti-tick effect due to Stylosanthes was significantly higher for S. humilis and S. hamata (p < 0.05) and slightly better for S. humilis (3% survival) than for S. hamata (12% survival). Further studies are required to determine the potential role of Stylosanthes plants for tick control in Mexico.

Seasonal distribution of Rhipicephalus sanguineus ticks (Acari: Ixodidae) on dogs in an urban area of Morelos, Mexico.

Between 1993 and 1995, we examined 1742 dogs at two veterinary clinics in Cuernavaca City in Mexico for the presence of Rhipicephalus sanguineus ticks. The overall tick infestation prevalence was 20% and there were no significant differences (p > 0.05) between different years. The prevalence was somewhat higher in spring, summer and autumn (20% or more) than in winter (13.7%). A positive correlation (p < 0.01) was found between prevalence of ticks and rainfall in spring, summer and autumn, whereas in winter there was only a correlation with temperature. Different stages of the tick were found and it was calculated that 2.5 generations could be completed each year. It is concluded that the environmental conditions of Cuernavaca City favour development and maintenance of R. sanguineus ticks throughout the year and that its role as a vector of diseases poses a threat to dogs and may have potential zoonotic risks.