Population replacement of benzimidazole-resistant Haemonchus contortus with susceptible strains: evidence of changes in the resistance status.

The spread of anthelmintic resistance (AR) in nematode populations threatens the viability of sheep production systems worldwide, and warrants the adoption of sensitive, practical, and standardized tests to detect AR. The aim of this study was to characterize the replacement of an Haemonchus contortus population resistant to benzimidazoles (BZDs) by a susceptible one, by means of both phenotypic and genotypic techniques. Phenotypic methods to assess BZD resistance included in vivo tests, such as the fecal egg count reduction test (FECRT), and in vitro tests, such as the egg hatch assay (EHA). Additionally, genotypification of polymorphisms associated with BZD resistance by sequencing a fragment of the isotype 1 ß-tubulin gene was carried out. The initial, BZD-resistant population (initial Balcarce population) exhibited an egg count reduction (ECR) of 59.3%. Following refugium replacement, the final population (final Balcarce population) exhibited an ECR of 95.2%. For the initial Balcarce population, the median effective dose (ED50) for the EHA was 0.607 µg thiabendazole (TBZ)/mL, with a rate of eclosion at a discriminating dose (EDD) of 0.1 µg TBZ/mL of 76.73%. For the final Balcarce population, ED50 was 0.02 µg TBZ/mL, and EDD was 1.97%. In the initial population, 93% of the analyzed individuals exhibited genotypic combinations associated with BZD resistance (53% Phe/Phe167-Tyr/Tyr200, 37% Phe/Tyr167-Phe/Tyr200, and 3% Phe/Tyr167-Glu/Leu198). Conversely, no combination associated with resistance was found in individuals from the final population. All of the tests were useful for detecting AR to BZDs. The results from the genetic and phenotypical studies were consistent, and the resulting information greatly aided in interpreting the outcomes of the population replacement and the potential impact of this strategy on management of AR.

Influence of faecal culture media and incubation time on the yield of infective larvae of Haemonchus contortus (Rudolphi 1803).

The aim of this experiment was to determine the yield of Haemonchus contortus third-stage larvae (L3) in faecal cultures in different conditions, including incubation time (7 or 14 days), the addition of inert additives (polystyrene pellets, vermiculite or no additive) and physical condition of the incubated faeces (ground or whole pellets). Twelve groups of 10 cultures each were arranged and incubated at 24 °C to evaluate the interaction of the above-mentioned conditions. Significantly, more L3 (p=0.0019 to p=0.0200) were recovered from cultures incubated for 7 days than for 14 days, except for the groups containing whole pellets with no additives (p=0.53) or with vermiculite (p=0.41). Larval yields from 7-day incubated cultures did not differ between groups (p=0.47), but for the whole pellets with vermiculite group, which yielded significantly less L3 (p<0.0001) than the rest of the cultures. Incubation for 14 days showed that cultures containing whole pellets with no additives yielded significantly more L3 (p<0.05) than the rest. Culturing faeces with H. contortus seems not to require inert additives or extra manipulation to obtain good L3 yields.

Recovery of fenbendazole efficacy on resistant Haemonchus contortus by management of parasite refugia and population replacement.

The recovery of fenbendazole efficacy against Haemonchus contortus was attempted in a sheep intensive production system, using a strategy of population replacement in which the initial absolute efficacy of fenbendazole was 0%. The strategy was based on managing the parasite populations in refugia. Firstly, the resistant parasite population was reduced by means of anthelmintic treatments with efficacious drugs (Phase I), then a new, susceptible population was introduced in summer by way of artificially infected lambs at weaning, which were left to graze on the experimental pasture for eleven months (Phase II). Lastly, the impact of the replacement strategy, in terms of benzimidazole efficacy, was measured (Phase III). Faecal egg counts from permanent lambs and worm burdens as a measure of pasture infectivity from tracer lambs were determined throughout the study. During Phase I, faecal egg counts diminished from a peak of 2968 (300-7740) epg to 0 epg at the end, while adult worm burdens of H. contortus were reduced from 2625 (800-5100) to 0, which showed that the treatment strategy used in Phase I was effective in reducing the resistant population. These parameters also showed that good levels of pasture contamination and infectivity were achieved in Phase II, as faecal egg counts of up to 7275 (3240-13080) epg and adult worm burdens of 500 (200-800) H. contortus were reached. The absolute benzimidazole efficacy on H. contortus estimated at 16 months post-population replacement (Phase III) was 97.58%. The results lead to the conclusion that the recovery of anthelmintic efficacy of fenbendazole against a resistant population of H. contortus may be achieved by means of a strategy based on management of refugia and a subsequent introduction of a susceptible population. This strategy might be translatable to other resistant nematode genera.

Studies on vertical transmission of Trichinella spiralis in experimentally infected guinea pigs (Cavia porcellus).

An experimental study to enhance knowledge on the capability of Trichenella spiralis to pass from guinea pigs to progeny at different periods of pregnancy or lactation was performed. For this purpose, 18 female adult guinea pigs were inoculated with 100 or 1000 T. spiralis muscle larvae (ML) during early, late gestation and during lactation period. The presence of T. spiralis (ML) in mothers and newborns was studied through enzymatic digestion from muscle samples. ML were observed in 9 of 42 newborn guinea pigs and levels of infection were significantly higher when infections of mothers were done during late gestation (p = 0.0046) with the high infective dose (p = 0.0043). T. spiralis ML were not recovered from any of the newborns from mothers infected in the lactation period. Ten out of 18 infected mothers presented larvae 1 in their mammary glands. Muscle samples from the tongue and the masseter showed the highest larval burdens. These observations confirm previous reports on that ML of T. spiralis are capable to pass through placental tissues to reach and encyst in striated muscle groups of newborn guinea pigs. This study may also reinforce the importance of preventive programs to control trichinellosis in those endemic areas where pregnant women would have high risk of infection.

Resistant nematodes in cattle: Pharmaco-therapeutic assessment of the ivermectin- ricobendazole combination.

Nematodicidal combinations have been proposed as a valid strategy to achieve effective nematode control in the presence of drug resistance. The goals of this study were: (1) to compare the clinical efficacy (therapeutic response) of ivermectin (IVM) and ricobendazole (RBZ) given subcutaneously either by separate or combined administration to calves naturally infected with gastrointestinal nematodes resistant to IVM, and (2) to evaluate the potential pharmacokinetic (PK) and/or pharmacodynamic (PD) interactions occurring after the co-administration of both anthelmintics. Sixty male calves naturally infected with gastrointestinal nematodes resistant to IVM were randomly allocated into four groups (n=15). Untreated control: animals not receiving anthelmintic treatment; IVM alone: animals treated with IVM by subcutaneous (SC) injection (0.2mg/kg); RBZ alone: animals received RBZ by the SC route (3.75mg/kg); IVM+RBZ: animals treated with IVM and RBZ (0.2 and 3.75mg/kg, respectively), by SC injection in two separates sites. Eight animals of each treated group were randomly selected to perform the PK study. Plasma samples were taken from those animals up to 28days post-treatment. IVM and RBZ plasma concentrations were quantified by HPLC. The therapeutic response was determined by faecal egg count reduction test (FECRT). The proportions of third-stage larvae (L3) recovered from coprocultures were used to calculate the efficacy against the main parasite genera. The daily total egg deposition for each experimental group was estimated. Similar pharmacokinetic trends were obtained for both IVM and RBZ allying the single-drug and the combined treatments, which indicates the absence of PK interactions between both anthelmintics. The observed overall clinical drug efficacies were 48% (IVM alone), 94% (RBZ alone) and 98% (IVM+RBZ). Haemonchus spp. and Cooperia spp. were recovered in the coproculture after IVM treatment, suggesting that resistance to IVM includes both genera. In fact, the efficacy against Cooperia spp. was 83% (IVM), 98% (RBZ) and 98% (IVM+RBZ), while the efficacy against Haemonchus spp. was 0% (IVM), 97% (RBZ) and 100% (IVM+RBZ). The combination was the only treatment that achieved 100% clinical efficacy against IVM-resistant Haemonchus spp. The total egg excretion was reduced to 49.9% (IVM alone group), 6.3% (RBZ alone group) and 1.8% (IVM+RBZ combined group) compared to the untreated control. Although the combined treatment did not significantly increase the overall clinical efficacy in the current natural field conditions, an additive effect was achieved against IVM-resistant nematodes. In fact, the combination obtained significantly higher efficacy against IVM-resistant Haemonchus spp. than RBZ alone. Additionally, the epidemiological relevance of the reduction in the number of eggs excreted following the combined treatment is not negligible and should be taken into account in future studies. Further work is required to understand the advantages of nematodicidal combinations in different natural anthelmintic resistance scenarios.