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 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.

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.

Plant products and secondary metabolites with acaricide activity against ticks.

The present review documents the results of studies evaluating the acaricidal activity of different plant products and secondary metabolites against ticks that are resistant and susceptible to conventional acaricides. Studies published from 1998 to 2016 were included. The acaricidal activity of plant extracts, essential oils and secondary compounds from plants have been evaluated using bioassays with ticks in the larval and adult stages. There is variable effectiveness according to the species of plant and the concentrations used, with observed mortalities ranging from 5 to 100% against the Rhipicephalus (Boophilus), Amblyomma, Dermacentor, Hyalomma, and Argas genera. A number of plants have been reported to cause high mortalities and/or affect the reproductive capacity of ticks in the adult phase. In the majority of these trials, the main species of plants evaluated correspond to the families Lamiaceae, Fabaceae, Asteraceae, Piperaceae, Verbenaceae, and Poaceae. Different secondary metabolites such as thymol, carvacrol, 1,8-cineol and n-hexanal, have been found to be primarily responsible for the acaricidal activity of different essential oils against different species of ticks, while nicotine, dibenzyldisulfide and dibenzyltrisulfide have been evaluated for plant extracts. Only thymol, carvacrol and 1,8-cineol have been evaluated for acaricidal activity under in vivo conditions. The information in the present review allows the conclusion that the secondary metabolites contained in plant products could be used as an alternative for the control of ticks that are susceptible or resistant to commercial acaricides.

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.

First documentation of ivermectin resistance in Rhipicephalus sanguineus sensu lato (Acari: Ixodidae).

The brown dog tick, Rhipicephalus sanguineus sensu lato (Latreille, 1806), is an ectoparasite and disease vector of significant veterinary and public health importance that is distributed widely around the world. The intensive use of synthetic acaricides for tick control exerts a strong selective pressure for brown dog ticks to become resistant to them. Here, we investigated claims from the field regarding treatment failure associated with the use of veterinary products containing ivermectin (IVM) to control brown dog ticks infesting dogs in Yucatan state, Mexico. Dogs in six state municipalities were inspected to sample 15 R. sanguineus s.l. POPULATIONS: Interviews were conducted with dog owners to gather information on the history of dog treatment with conventional acaricides and IVM. The larval immersion test was used on the progeny of adult female ticks infesting dogs to test for susceptibility to IVM. Dose-mortality regressions, lethal concentrations (LC), their confidence intervals, and slope were estimated by probit analysis. Ten tick populations (66.7%) were classified as resistant compared with the most susceptible population. A high inter-population variation in the phenotypic level of IVM resistance was evident (resistance ratio at LC50% and LC99% ranged from 1.0 to 30.5, and from 1.0 to 458.8, respectively). Tick populations classified as resistant were collected from dogs known to have been treated with IVM. To our knowledge, this is the first report of IVM resistance in R. sanguineus s.l. worldwide. Veterinary and pet owner education on integrated tick management practices is required to avoid widespread resistance and increased treatment failure with products containing IVM and other macrocyclic lactones used to control endo- and ectoparasites affecting dogs. Integrated tick management will also help mitigate the burden of brown dog tick-borne diseases on human and animal populations.

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.

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.

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.

Synergistic action of fatty acids, sulphides and stilbene against acaricide-resistant Rhipicephalus microplus ticks.

Six compounds in a methanolic extract of Petiveria alliacea stem (cis-stilbene; benzyl disulphide; benzyl trisulphide; and methyl esters of hexadecanoic acid, octadecadienoic acid and octadecenoic acid) are known to exercise acaricide activity against cattle tick Rhipicephalus microplus larvae and adults. The synergistic effect of 57 combinations of these six compounds on acaricide activity against R. microplus was evaluated. Larvae immersion tests produced the lethal concentrations needed to kill 50% (LC50) and 99% (LC99) of the population. Adult immersion tests produced rates (%) for mortality, oviposition inhibition and eclosion inhibition. Individually, none of the compounds (1% concentration) exhibited acaricide activity (mortality ≤2.3%). When combined, however, nine mixtures exhibited a synergistic increase in activity, with high mortality rates (≥92%) in larvae. Values for LC50 ranged from 0.07 to 0.51% and those for LC99 from 0.66 to 5.16%. Thirty six compound mixtures had no significant activity (mortality ≤30%) against larvae. Two mixtures exhibited synergism against adults, with high rates (≥92%) of oviposition inhibition. The mixtures based on the benzyl disulphide+benzyl trisulphide pairing produced a synergistic effect against acaricide-resistant R. microplus larva and adults, and are therefore the most promising combination for controlling this ubiquitous ectoparasite.

Ticks collected from humans, domestic animals, and wildlife in Yucatan, Mexico.

Domestic animals and wildlife play important roles as reservoirs of zoonotic pathogens that are transmitted to humans by ticks. Besides their role as vectors of several classes of microorganisms of veterinary and public health relevance, ticks also burden human and animal populations through their obligate blood-feeding habit. It is estimated that in Mexico there are around 100 tick species belonging to the Ixodidae and Argasidae families. Information is lacking on tick species that affect humans, domestic animals, and wildlife through their life cycle. This study was conducted to bridge that knowledge gap by inventorying tick species that infest humans, domestic animals and wildlife in the State of Yucatan, Mexico. Amblyomma ticks were observed as euryxenous vertebrate parasites because they were found parasitizing 17 animal species and human. Amblyomma mixtum was the most eryxenous species found in 11 different animal species and humans. Both A. mixtum and A. parvum were found parasitizing humans. Ixodes near affinis was the second most abundant species parasitizing six animal species (dogs, cats, horses, white-nosed coati, white-tail deer and black vulture) and was found widely across the State of Yucatan. Ixodid tick populations may increase in the State of Yucatan with time due to animal production intensification, an increasing wildlife population near rural communities because of natural habitat reduction and fragmentation. The diversity of ticks across host taxa documented here highlights the relevance of ecological information to understand tick-host dynamics. This knowledge is critical to inform public health and veterinary programs for the sustainable control of ticks and tick-borne diseases.

Lethal and Sublethal Effects of Ivermectin on Onthophagus landolti (Coleoptera: Scarabaeidae).

Previous work has documented toxic effects of ivermectin (IVM) on dung beetles from the Old World, but very little is known about this drug's effect on Neotropical dung beetles. Accordingly, we conducted a bioassay with dung spiked with IVM to assess its lethal and sublethal effects on the Neotropical dung beetle Onthophagus landolti Harold. The experimental design consisted of five treated groups G1, G2, G3, G4, and G5 receiving 0.001, 0.01, 0.1, 1.0, and 10 mg IVM/kg dung fresh weight, respectively, and two control groups (solvent control [CGA] and untreated control [CGU]). Adult survival and fecundity were measured throughout a 10-d period, and subsequent egg development and survival were monitored. Adult survival was only affected for treatment groups G4 and G5 groups (70 and 30%, respectively); groups G1, G2, G3 and both controls exhibited 100% survival. Fecundity was completely suppressed under treatment groups G4 and G5. Group G3 only had 1.7 and 2.1% brood mass production relative to CGA and CGU, respectively. Additionally, for groups G1 and G2 the proportion of adults emerging from brood masses was lower relative to CGA. Furthermore, development time for the second generation in groups G1 and G2 was 12.5% slower relative to control groups. Finally, dung removal by beetles from groups G3, G4, and G5 was significantly lower relative to control groups. In conclusion, toxic effects of IVM on O. landolti are associated mainly with reduced fecundity and lower dung-removal by adult beetles as well as reduced survival and slower development of offspring.

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.

Frequency of cattle farms with ivermectin resistant gastrointestinal nematodes in Veracruz, Mexico.

This study reports the percentage of cattle farms with ivermectin (IVM) resistant gastrointestinal nematodes (GINs) in Veracruz, Mexico, and identifies the GIN genera involved in the resistances. It also describes management practices of anthelmintic (AH) use on the surveyed farms. Twenty-one farms were assessed by means of the faecal egg count reduction test using the McMaster technique. Only two farms had GIN populations susceptible to IVM (9.5%). The proportion of farms with IVM resistant GIN was 71.4% (15/21). Seven of these farms had less than 80% egg count reductions. Haemonchus and Cooperia were the genera most commonly found in the resistant populations, followed by Oesophagostomum. Inappropriate AH treatment practices were identified from the completed questionnaires. Further management practices such as selective treatment and quarantine treatments are proposed to further reduce the spread of IVM resistance between farms.

Efficacy of entomopathogenic fungi (Ascomycetes: Hypocreales) against adult Haematobia irritans (Diptera: Muscidae) under stable conditions in the Mexican dry tropics.

The purpose of this paper is to evaluate the effect of five strains of Metarhizium anisopliae (Ma) and three strains of Isaria fumosorosea (Ifr) at a concentration of 1×10(8)colony-forming units/ml applied by spraying onto bovines with controlled infestation of Haematobia irritans under stable conditions in the Mexican dry tropics. Four experiments were performed, in each of which three treatments (two fungal strains and one control) were evaluated with eight repetitions for each one, by carrying out a single application of the aqueous suspension of each strain. The animals were isolated in individual cages and direct counts of the infestation were carried out for 13 days. It was observed that strains Ma2, Ma6, Ma10, Ma14, and Ma34 caused 94-100% reduction in infestation between days 12 and 13 post-treatment, while strains Ifr19, Ifr11, and Ifr12 reduced infestation from 90% to 98% up to day 13 post-application. There was an effect in the generation of horn flies from the excrement of bovines that were treated with different strains, reducing the reproduction of subsequent generations. It was concluded that the strains of M. anisopliae and I. fumosorosea evaluated in this study can be used as biocontrol agents in infestations of H. irritans in stabled bovines.

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.

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.