First report of acaricidal efficacy from plumbagin on larvae of Rhipicephalus microplus and Rhipicephalus sanguineus resistant to conventional acaricides.

The problem of resistance to acaricides in ticks such as Rhipicephalus microplus and R. sanguineus has motivated the search for control alternatives, such as the use of extracts and secondary metabolites from plants. Plumbagin is a natural product present in plants such as Plumbago zeylanica L., Diospyros kaki, and D. anisandra, of which acaricidal activity has been reported. Therefore, the objective of this study was to evaluate in vitro the acaricidal efficacy of plumbagin on larvae of R. microplus and R. sanguineus resistant to conventional acaricides. Larvae from engorged female ticks, collected from naturally infested dairy cattle and domiciled dogs, in Yucatan, Mexico, were used. The larval packet test and the larval immersion test were performed to detect acaricide susceptibility. Both tick populations were detected as resistant to cypermethrin and amitraz. Then, the modified larval immersion test was used and plumbagin was evaluated at concentrations of 1%, 0.5%, 0.25%, and 0.125% (%w/v), obtaining a mortality of 100% in the four concentrations for both tick species. Subsequently, lower doses of plumbagin were evaluated at concentrations of 0.0625%, 0.03125%, 0.015625% and 0.0078125%, obtaining mortalities of 100 to 36.26% for R. microplus and 100%-5.33% for R. sanguineus. Using Probit analysis, lethal concentrations at 50% (LC50), 99% (LC99) and confidence intervals at 95% (CI95%) were calculated. R. microplus showed a LC50 of 0.011% (CI95%: 0.010-0.011) and LC99 of 0.019% (CI95%: 0.018-0.022). R. sanguineus presented a LC50 of 0.017% (CI95%: 0.015-0.018) and CL99 of 0.031% (CI95%: 0.027-0.036). It was concluded that plumbagin has high acaricidal efficacy against larvae of R. microplus and R. sanguineus resistant to amitraz and cypermethrin. R. microplus larvae were significantly more susceptible to LC50 and LC99 compared to R. sanguineus. This is the first report on the acaricidal efficacy of plumbagin on larvae of R. microplus and R. sanguineus resistant to conventional acaricides.

First report of the lethal activity and synergism between deltamethrin, amitraz and piperonyl butoxide against susceptible and pyrethroid-resistant nymphs of Triatoma infestans.

In South America, Triatoma infestans (Hemiptera: Reduviidae) is the main vector of the parasite Trypanosoma cruzi, etiological agent of Chagas disease. The main strategy for vector control is to spray domestic structures with pyrethroids. Reports of populations of T. infestans with varying degrees of resistance to pyrethroids have made the search for alternative molecules for vector control necessary. In the first stage of this work we investigated the lethal activity of amitraz and deltamethrin against susceptible and pyrethroid-resistant nymphs of Triatoma infestans. Lethal dose at 50% (LD50) of susceptible nymphs were compared with those recorded in pyrethroid-resistant nymphs and the resistance ratio (RR50) was obtained. The RR50 of deltamethrin was approximately 300. In the case of amitraz, we observed similar triatomicidal activity in the two nymph populations (RR50: 0.7). In a second stage of the work, we determined the synergistic effect of amitraz and piperonyl butoxide (PBO) on the lethal activity of deltamethrin. The strong synergistic effect of PBO on the lethal activity of deltamethrin in resistant nymphs produced a decrease in RR50 to almost one third of the RR50 reported in absence of the synergist. Amitraz plus PBO lethal activity was similarly increased in pyrethroid susceptible and resistant nymphs. Our data indicate that deltamethrin synergism by amitraz was higher against resistant than to susceptible nymphs (Synergist ratio (SR50) of: 7.2- and 4.1-fold, respectively). In pyrethroid resistant nymphs, the highest level of synergism was obtained combining deltamethrin with amitraz and PBO (SR50: 26.7-fold). These results indicate that this combination could be considered an effective alternative for the control of T. infestans.

Prevalence, Abundance and Intensity of Eggs and Oocysts of Gastrointestinal Parasites in the Opossum Didelphis Virginiana Kerr, 1792 in Yucatan, Mexico.

Virginia opossum, Didelphis virginiana, is a synanthropic mammal associated with peridomestic areas of Yucatán, However, little is known about the gastrointestinal parasite infections of this species. The infection prevalence, mean abundance and mean intensity of eggs and oocysts of gastrointestinal parasites, in opossums captured in the peridomestic areas were estimated in six rural localities of Yucatán, Mexico. Eighty-four faecal samples were processed by flotation technique. McMaster test was used to estimate the number of helminth eggs and protozoa oocysts per gram of feces. Seven genera of gastrointestinal parasites were identified, and then infection prevalence was estimated as follows: Protozoa Eimeria sp. (51.9 %) and Sarcocystis sp. (1 %); nematodes Ancylostoma sp. (80.56 %), Cruzia sp. (62.04 %), Trichuris sp. (60.19 %), Capillaria sp. (29.63 %), Turgida sp. (23.15 %), Toxocara sp. (11.11 %), and Ascaris sp. (1.85 %); and one acanthocephalan: Oligacanthorhynchus sp. (14.81 %). This is the first study on the diversity of gastrointestinal parasites in Virginia opossums, and first evidence about the potential role of opossums in the transmission of zoonotic gastrointestinal parasites in peridomestic areas of Yucatán, Mexico.

First report of amitraz and cypermethrin resistance in Rhipicephalus sanguineus sensu lato infesting dogs in Mexico.

Engorged female Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) were collected from dogs in the state of Yucatán, Mexico. Fourteen tick populations were collected from dogs at seven veterinary clinics, four residential homes and three cattle farms. The larval immersion test was used in the progeny of collected adult females to test susceptibility to amitraz and cypermethrin. Dose-mortality regressions, 50% lethal concentrations (LC50 ), confidence intervals and slope were estimated by probit analysis. For amitraz, 12 tick populations (85.7%) were classified as resistant and low inter-population variation in the phenotypic level of resistance was evident [resistance ratios (RRs) at LC50 : 1.0-13.0]. For cypermethrin, 12 tick populations (85.7%) were classified as resistant and substantial inter-population variation in the phenotypic level of resistance was evident (RRs at LC50 : 1.0-104.0). Thus, amitraz resistance in R. sanguineus s.l. is common, but generally occurs at low levels; however, alarmingly high levels of cypermethrin resistance are present in R. sanguineus s.l. populations in dogs in Yucatán, Mexico. The intensive use of both acaricides to control ectoparasites on dogs is likely to lead to more serious resistance problems that may cause high levels of control failure in the future.

Putrescine: Essential factor for in vitro proliferation of Babesia bovis.

This study reports the effect of putrescine addition, either alone or in combination with insulin, transferrin and selenite (ITS), to serum-free Advanced DMEM/F12 (A-DMEM/F12) medium, on the in vitro culture of Babesia bovis and using a perfusion bioreactor to improve efficiency of the process. A B. bovis strain previously adapted to proliferate in serum-free medium (Bbovis-SF) was evaluated using eight increasing concentrations of putrescine. The percentage of parasitized erythrocytes (PPE) obtained from cultures supplemented with 0.101 mg/L was 6.23% compared with 2.3% for control cultures with M199 with Earle's salts (M199) and 40% serum. The combination of putrescine (0.101 mg/L) and a mixture of ITS (2000, 1100, and 1.34 mg/L, respectively) (Pu-ITS), in A-DMEM/F12 culture medium without serum yielded a maximum PPE of 17.26% compared to 2.58% in the control medium. This new formulation of culture medium, together with the use of a hollow-fiber perfusion bioreactor system (HFPBS), caused a substantial increase in the proliferation of B. bovis, yielding a maximum cumulative PPE of 118.8% after five days, compared to 58.6% in cultures treated with control medium M199 and 40% serum. We concluded that the addition of the ITS mixture and putrescine to the culture medium stimulated the proliferation of B. bovis in vitro. This new medium formulation, used in a HFPBS culture system, can be an effective, automated-prone system that can induce massive proliferation of B. bovis for use as a source of parasite antigens and immunogens.

Survival and reproduction of Onthophagus landolti (Coleoptera: Scarabaeidae) exposed to ivermectin residues in cattle dung.

Two bioassays were conducted in parallel to assess the effects of cattle treated with either 1% ivermectin (IVM) or 3.15% IVM (dosed at 0.2 and 0.63 mg kg-1, respectively) on reproduction and survival of Onthophagus landolti Harold. Adult beetles were exposed 10 days to faeces of treated cattle starting at: one day before treatment (controls), 3, 6, 14, 28 and 35 days post-treatment. Adult survival of O. landolti was not affected by either of the two treatments. Faecal residues of 1% IVM almost completely suppressed fecundity of beetles at 3, 6 and 14 days post-treatment (dPT), and reduced fecundity of O. landolti at 28 dPT ( 38.3%), relative to controls. Meanwhile, IVM residues after treatment with 3.15% IVM almost completely suppressed fecundity of beetles at 3, 6, 14 and 28 dPT, and reduced fecundity of O. landolti at 35 dPT (80.9%), relative to controls. Larval survival was significantly reduced only at 3 dPT with 1% IVM. Meanwhile, treatment with 3.15% IVM significantly reduced larval survival at 6, 14 and 28 dPT. Larval mortality was recorded only in L-I and L-II instars. Moreover, in both bioassays, most of the L-I and L-II specimens that survived showed signs of toxicity. In conclusion, residual IVM in cattle faeces after treatment with injectable IVM has a detrimental effect on the fecundity of adult O. landolti up to 4 weeks post-treatment and on the subsequent larval survival.

In vitro culture of Babesia bovis in a bovine serum-free culture medium supplemented with insulin, transferrin, and selenite.

Bovine serum is an important factor for the optimal growth of Babesia bovis in vitro. This protozoan can be cultured in M-199 with Earle's salts medium (M-199) supplemented with 40% bovine serum (BS). In the present study, four media were assessed along with the control medium M-199. The effect on the proliferation of B. bovis in vitro was tested when these media were combined with insulin (Ins), transferrin (Trans) and selenite (Sel) in the absence of bovine serum. Treatment with Advanced DMEM/F12 medium (A-DMEM/F12) achieved the highest percentage of parasitized erythrocytes (PPE), reaching a maximum value of 9.59%. A-DMEM/F12 medium supplemented with a mixture of Ins (2000 mg/L), Trans (1100 mg/L), and Sel (1.34 mg/L) allowed for the adaptation and proliferation of B. bovis without bovine serum, showed a constant increase in PPE, and reached a maximum value of 9.7% during seven cycles of in vitro culture. It was concluded that continuous proliferation of B. bovis in vitro could be achieved using A-DMEM/F12 medium supplemented with Ins-Trans-Sel, without bovine serum. After adaptation for proliferation in serum-free medium, the B. bovis strain of parasites could have future use in the study of this economically important protozoan species that affects cattle.

Use of Ivermectin as Endoparasiticide in Tropical Cattle Herds Generates Resistance in Gastrointestinal Nematodes and the Tick Rhipicephalus microplus (Acari: Ixodidae).

The objective of the present study was to determine simultaneously the status of resistance against ivermectin (IVM) in gastrointestinal nematodes (GIN) and Rhipicephalus microplus (Canestrini, 1888) ticks in 12 cattle farms where IVM was used for the control of GIN in the Mexican tropics. Six farms had frequent use of IVM (≥ 4 times per year) and six farms had low frequency of IVM use (1-2 times per year). The fecal egg count reduction test and the larval immersion test were used to determine the resistant status of GIN and R. microplus against IVM, respectively. The results indicated that 100% of the surveyed farms had IVM-resistant GIN (reduction % from 0 to 67%). The genera involved were Haemonchus, Cooperia, Ostertagia, Trichostrongylus, and Oesophagostomum. Although the IVM was never used for the control of ticks, 50% of the surveyed farms presented GIN and R. microplus simultaneously resistant to IVM. Furthermore, two R. microplus populations showed high resistance ratio (RR) to IVM (farm TAT: RR50% = 7 and RR99% = 40.1; and farm SLS: RR50% = 2.4; RR99% = 11.0). A high frequency of IVM use (≥ 4 times per year) seemed to promote IVM resistance amongst R. microplus ticks compared with the farms with low frequency of IVM use (1-2 times per year; 66.6 vs. 25.0%, respectively). However, the number of surveyed farms was insufficient to show clear statistical inferences (odds ratio = 6.00; 95% CI = 0.341-105.5). The use of IVM for the control of GIN promoted simultaneously the development of IVM resistance in the GIN and R. microplus populations of the cattle herds surveyed.

Reduction of benzimidazole resistance in established Haemonchus contortus populations in goats using a single infection with a benzimidazole-susceptible isolate.

An in vivo study in goats evaluated the effect of superimposing a single artificial infection with a benzimidazole (BZ)-susceptible Haemonchus contortus isolate upon established H. contortus populations of known BZ resistance by measuring the phenotypic BZ resistance of eggs collected from faeces before and after re-infection. Two H. contortus isolates, one benzimidazole resistant (BZR) and the other susceptible (BZS), were used to infect worm-free goats. Eight goats were initially infected with 2000 third-stage larvae (L3). In each case the inoculum contained a pre-determined proportion of the two isolates: 100% BZS (one goat), 75% BZS/25% BZR (two goats), 50% BZS/50% BZR (two goats), 25%BZS/75% BZR (two goats) and, finally, 100% BZR (one goat). The phenotypic BZ susceptibility of the H. contortus population formed in each goat after the first infection was determined on days 30 and 36 post-infection using an egg-hatch assay (EHA) that estimated the concentration of thiabendazole (TBZ) required for 95% inhibition of larval hatching (EC(95)) with a 95% confidence interval (95% CI). On day 49 post-infection, goats were re-infected with 2000 L3 of the BZS isolate alone. A second set of EHA bioassays was performed 28 days and 34 days after re-infection. The first infection protocol produced three populations classified as BZS (EC(95) 0.055-0.065 µg TBZ/ml) while four were categorized as BZR (EC(95) 0.122-0.344 µg TBZ/ml). The status of one other population could not be determined. After re-infection with BZS L3, the number of susceptible populations increased to six (EC(95) 0.043-0.074 µg TBZ/ml) while the remaining two were deemed resistant (EC(95) 0.114-119 µg TBZ/ml). Re-infection with BZS L3 thereby reduced the resistance status of most H. contortus populations.

Infection levels of intestinal helminths in two commensal rodent species from rural households in Yucatan, Mexico.

The aim of the present study was to calculate the prevalence and intensity of intestinal helminths in the house mouse (Mus musculus) and the black rat (Rattus rattus) trapped in rural households of Yucatan, Mexico. Sampling was conducted during the rainy season from October to December 2011 and the dry season from January to March 2012. A total of 154 M. musculus and 46 R. rattus were examined, with 84.2% of M. musculus being infected with helminths compared with a significantly lower prevalence of 52.2% in R. rattus (P< 0.01). Adult M. musculus were more likely to be infected with helminths (89%) than subadults (63%) (P< 0.01). Four helminth species were identified: Taenia taeniaeformis larvae, Nippostrongylus brasiliensis, Syphacia muris and Trichuris muris. Nippostrongylus brasiliensis was present more frequently in M. musculus than in R. rattus (P< 0.01) and in adult mice compared to subadults (P< 0.01). Trichuris muris was present only in adult mice. This is the first report of N. brasiliensis, S. muris and T. muris in Yucatan, Mexico, as well as the first to report the presence of N. brasiliensis in M. musculus from Mexico. The helminth fauna of commensal rodents present in households appears to constitute a low potential health risk to local inhabitants; however, it would be advisable to conduct further studies to better understand the public health risk posed by these rodent intestinal helminths.

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.

Red deer (Cervus elaphus) as a host for the cattle tick Rhipicephalus microplus (Acari: Ixodidae) in Yucatan, Mexico.

Rhipicephalus microplus is the most economically important cattle tick in the Mexican tropics. Wild ungulate species, including red deer (Cervus elaphus), are gaining popularity in diversified livestock ranching operations in Mexico. However, there is no information available on the susceptibility of red deer to infestation with the cattle tick, R. microplus, under hot, subhumid tropical conditions in Mexico. Biological data on R. microplus as an ectoparasite of cattle and red deer in a farm in the Mexican tropics are presented here. Ticks collected from red deer were identified as R. microplus (97 %) and Amblyomma cajennense (3 %), and tick species infesting cattle included R. microplus (95 %) and A. cajennense (5 %). Standard counts of R. microplus engorged females on red deer were 11 times higher than on cattle (428 ± 43 vs. 40 ± 18; p < 0.001). The reproductive efficiency index and larval hatching of R. microplus collected from cattle and red deer were similar (p > 0.05). Hemolymph samples of R. microplus collected from cattle were positive for Babesia spp. (10 %, 2/50) and all the samples from ticks infesting red deer were negative. Seventeen and ten percent of the blood samples from cattle and red deer were positive for Anaplasma marginale, respectively. The role of red deer as a host of R. microplus in Yucatan, Mexico and the importance of this host-parasite relationship relative to the epidemiology of R. microplus-borne diseases are discussed.

Leishmania spp. epidemiology of canine leishmaniasis in the Yucatan Peninsula.

Canine Leishmaniasis is widespread in various Mexican states, where different species of Leishmania have been isolated from dogs. In the present study, we describe the detection of L. braziliensis, L. infantum, and L. mexicana in serum of dogs from the states of Yucatan and Quintana Roo in the Yucatan Peninsula (Mexico). A total of 412 sera were analyzed by ELISA using the total extract of the parasite and the iron superoxide dismutase excreted by different trypanosomatids as antigens. We found the prevalence of L. braziliensis to be 7.52%, L. infantum to be 6.07%, and L. mexicana to be 20.63%, in the dog population studied. The results obtained with ELISA using iron superoxide dismutase as the antigen were confirmed by western blot analysis with its greater sensitivity, and the agreement between the two techniques was very high.

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.

The occurrence of the larval cestode Cysticercus fasciolaris in rodent populations from the Cuxtal ecological reserve, Yucatan, Mexico.

Cysticercus fasciolaris is the larval stage of the cestode Taenia taeniaeformis, whose definitive hosts are mainly cats. This larval stage uses a wide variety of small rodents, and occasionally birds and humans, as intermediate hosts. In the Yucatan, there are no reports of the presence of this cestode in animal populations. The aim of this study was to evaluate the occurrence of C. fasciolaris in rodent populations from the Cuxtal ecological reserve, Yucatan, Mexico. Trapping of rodents was conducted from October 2009 to April 2010 in 40 households in Molas, in which Sherman traps were placed both inside and outside backyards. Rodents were dissected to inspect the liver for the presence of the worm. To determine risk factors associated with infection, univariate analysis was performed using sex, age, species, trapping site, and season as independent variables. Variables with a P value <  0.2 were analysed using a logistic regression model. In this study, 411 individuals of six rodent species were trapped; Mus musculus was the most abundant (78%), followed by Rattus rattus (13%) and the wild species Peromyscus yucatanicus, Ototylomys phyllotis, Heteromys gaumeri and Reithrodontomys gracilis (9%). Only 7.5% (n = 31) of M. musculus and R. rattus were infected with C. fasciolaris (demonstrated by the presence of liver cysts) with a prevalence of 9.0% and 3.5%, respectively. Both adults and male mice were 4.33 and 3.46 (OR values) times more likely to have C. fasciolaris than juveniles and females respectively. We can conclude that in the Cuxtal Reserve, Yucatan, Mexico, the prevalence of C. fasciolaris is higher in M. musculus, and that adult males had a higher probability of infection. Wild species, mainly P. yucatanicus, were not found to be infected with the cestode, but its presence in the backyards of households could result in a potential risk of acquiring this infection.

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.

Spatial distribution of acaricide profiles (Boophilus microplus strains susceptible or resistant to acaricides) in southeastern Mexico.

The ability of Boophilus microplus strains to be susceptible (-) or resistant (+) to amidines (Am), synthetic pyrethroids (SP), and/or organo-phosphates (OP) (or acaricide profiles) was investigated in 217 southeastern Mexican cattle ranches (located in the states of Yucatán, Quintana Roo, and Tabasco). Three questions were asked: (1) whether acaricide profiles varied at random and, if not, which one(s) explained more (or less) cases than expected, (2) whether the spatial distribution of acaricide profiles was randomly or non-randomly distributed, and (3) whether acaricide profiles were associated with farm-related covariates (frequency of annual treatments, herd size, and farm size). Three acaricide profiles explained 73.6% of the data, representing at least twice as many cases as expected (P<0.001): (1) Am-SP-, (2) Am+SP+, and (3) (among ranches that dispensed acaricides > or = 6 times/year) Am-OP+SP+. Because ticks collected in Yucatán ranches tended to be susceptible to Am, those of Quintana Roo ranches displayed, predominantly, resistance to OP/SP, and Tabasco ticks tended to be resistant to Am (all with P < or = 0.05), acaricide profiles appeared to be non-randomly disseminated over space. Across states, two farm-related covariates were associated with resistance (P < or = 0.02): (1) high annual frequency of acaricide treatments, and (2) large farm size. Findings supported the hypothesis that spatial acaricide profiles followed neither random nor homogeneous data distributions, being partially explained by agent- and/or farm-specific factors. Some profiles could not be explained by these factors. Further spatially explicit studies (addressing host-related factors) are recommended.

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 of the Dirofilaria immitis infection in dogs from Merida, Yucatan, Mexico.

The aim of the present study was to estimate the prevalence of Dirofilaria immitis infection, and to examine the relationship between host factors (gender, age and breed) and D. immitis infection in dogs. The study was designed as a cross-sectional study. A total of 676 dogs were examined for D. immitis infection. Adult worms (necropsy) and blood samples were collected from all animals for diagnosis of D. immitis. Blood samples were examined using a modified Knott's and thick drop techniques. Fifty-six of 676 dogs were diagnosed infected with D. immitis. The odds of infection were 2.85 times higher in female dogs, compared to male dogs (OR = 2.85, 95% CI = 1.5-5.5, P = 0.01). In addition, the odds of infection were 2.11 times higher in > or =3 years old dogs, compared with younger dogs (OR = 2.11, 95% CI = 1.1-4.2, P = 0.03). This information is important for a better understanding of the epidemiology of D. immitis in dogs in Yucatan, Mexico.

Anthelminthic activity of methanol extracts of Diospyros anisandra and Petiveria alliacea on cyathostomin (Nematoda: Cyathostominae) larval development and egg hatching.

Methanol extracts of plant structures are promising alternatives to traditional pharmaceutical anthelminthic treatments. An in vitro evaluation was done of how methanol extracts of Diospyros anisandra bark and leaves, and Petiveria alliacea stems and leaves, collected during the rainy and dry seasons, effected cyathostomin larval development and egg hatching. Seven concentrations (600, 300, 150, 75, 37.5, 18.7 and 9.3µg/ml) were tested using the egg hatch assay. An ANOVA was applied to identify differences between the concentrations and the controls. Fifty percent lethal concentration (LC50) and the 95% confidence interval were calculated with a probit analysis. At and above 37.5µg/ml, the D. anisandra bark extracts from both seasons exhibited ≥95% egg hatch inhibition (EHI), while the D. anisandra leaf extracts had >90% EHI at and above 75µg/ml. For P. alliacea, the extracts from leaves and stems from either season exhibited >97% EHI at and above 300µg/ml, although similar efficacy was also observed at lower concentrations with the rainy season stems (75µg/ml) and leaves (150µg/ml). Values for LC50 were lowest for the rainy season D. anisandra bark (10.2µg/ml) and leaf extracts (18.4µg/ml), followed by the rainy season P. alliacea stems extract (28.2µg/ml). In the D. anisandra extracts, EHI was largely due to its ovicidal activity (≥96% beginning at 37.5µg/ml), whereas in the P. alliacea extracts it was due to L1 larval hatch failure (≥90% beginning at 75µg/ml). Overall, the rainy season D. anisandra bark extracts had a strong in vitro anthelminthic effect against cyathostomins by inhibiting larval development, and the rainy season P. alliacea stem extracts had a strong effect by preventing egg hatching. Both are possible control alternatives for these nematodes.