Phytochemicals in Gastrointestinal Nematode Control: Pharmacokinetic-Pharmacodynamic Evaluation of the Ivermectin plus Carvone Combination.

A wide variety of plant-derived phytochemicals with anthelmintic effects have been described. Most of them have shown activity against parasites in vitro but have not been extensively explored in vivo. The aim of the current work was to study the pharmacokinetic-pharmacodynamic relationship of the combined administration of carvone (R-CNE) and ivermectin (IVM) to lambs. Three trials were conducted to evaluate the pharmacological interaction between R-CNE and IVM in lambs infected with resistant nematodes. Drug concentrations were measured in plasma, target tissues, and H. contortus by HPLC with fluorescent (IVM) and ultraviolet (R-CNE) detection. The effect of both compounds on parasites was estimated by the fecal egg count reduction. Coadministration with R-CNE significantly increased the plasma bioavailability of IVM. R-CNE showed a moderate anthelmintic effect, which was greater on the susceptible isolate of H. contortus. After the combination of R-CNE and IVM as an oral emulsion, both compounds were quantified in H. contortus recovered from infected lambs. However, R-CNE concentrations were much lower than those reported to achieve anthelmintic effects in the in vitro assays. Optimization of the pharmaceutical formulation, dose rate, and administration schedule is needed to take advantage of the intrinsic anthelmintic activity of phytochemicals.

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.

Combination of bioactive phytochemicals and synthetic anthelmintics: In vivo and in vitro assessment of the albendazole-thymol association.

The search of novel strategies for anthelmintic control is a crucial need considering the widespread increase in resistant parasitic populations in livestock. Bioactive phytochemicals may contribute to improve parasite control by enhancing the effect of existing anthelmintic drugs. The aim of the current work was to evaluate the in vivo and in vitro pharmaco-chemical interaction and the in vivo efficacy of the combination of albendazole (ABZ) with thymol (TML) in lambs naturally infected with resistant gastrointestinal nematodes. Thirty (30) lambs were allocated into three experimental groups. Each group was treated orally with either ABZ (5 mg/kg), TML (150 mg/kg, twice every 24 h) or the co-administration of both compounds. Blood samples were collected between 0 and 51 h post-treatment and TML, ABZ and its metabolites were measured by HPLC. Individual faecal samples were collected at days -1 and 14 post-treatment to perform the faecal egg count reduction test. Additionally, the effect of TML on the sulphoreduction and sulphonation of ABZ sulphoxide was assessed in vitro using ruminal content and liver microsomes, respectively. The metabolism of TML in the ruminal content was very low and the monoterpene exhibited a low degree of association with the particulate phase of the ruminal content. No changes in the pharmacokinetic behavior of ABZ sulphoxide were observed in the presence of the natural product (TML). In contrast, the ABZ sulphone Cmax and AUC were lower (P 0.002 and 0.001 respectively) in the co-administered animals (0.16 ±â€¯0.07 µg/mL and 3.63 ±â€¯1.21 µg.h/mL) compared with those that received ABZ alone (0.45 ±â€¯0.15 µg/mL and 9.50 ±â€¯2.84 µg.h/mL). TML was detected in the bloodstream between 1 and 48 h post-treatment, which indicates the time of target nematodes being exposed to the bioactive monoterpene. However, the in vivo efficacy of TML was 0% and the presence of this terpene did not increase the efficacy of ABZ. The presence of TML significantly inhibited the ruminal sulphoreduction (P 0.001) and the hepatic sulphonation (P 0.001) of ABZ sulphoxide. These observations point out that in vivo pharmaco-parasitological studies are relevant to corroborate the adverse kinetic/metabolic interactions and the efficacy of bioactive natural products combined with synthetic anthelmintics.

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.

Anthelmintic resistance in grazing beef cattle in central and northeastern areas of Argentina - An update.

The presence of anthelmintic resistance in Argentina has experienced a marked increase in cattle, with numerous reports showing levels of resistance of different parasite genera to different chemical groups. The aim of this study is to update comprehensively the situation of anthelmintic resistance to the different chemical groups in the most important areas of cattle production in Argentina. The study involved the determination of anthelmintic resistance in 62 cattle farms in 7 provinces using the faecal egg count reductions test. The results showed a marked increase of anthelmintic resistance compared to previous reports; the main resistant genera were Cooperia and Haemonchus to ivermectin, Ostertagia and Cooperia to ricobendazole, and Haemonchus to fenbendazole. There was also a distinct difference in clinical efficacies between subcutaneous ricobendazole and oral fenbendazole in favour of the latter, probably attributed to the administration route. Levamisole has showed high efficacy and broad antiparasitic spectrum. Anthelmintic resistance is widely and firmly established in grazing cattle production systems in the country; the diagnosis of resistance must be done in every particular farm in order to design a sustainable parasite control based on anthelmintics use.

Comparative tissue pharmacokinetics and efficacy of moxidectin, abamectin and ivermectin in lambs infected with resistant nematodes: Impact of drug treatments on parasite P-glycoprotein expression.

The high level of resistance to the macrocyclic lactones has encouraged the search for strategies to optimize their potential as antiparasitic agents. There is a need for pharmaco-parasitological studies addressing the kinetic-dynamic differences between various macrocyclic lactones under standardized in vivo conditions. The current work evaluated the relationship among systemic drug exposure, target tissue availabilities and the pattern of drug accumulation within resistant Haemonchus contortus for moxidectin, abamectin and ivermectin. Drug concentrations in plasma, target tissues and parasites were measured by high performance liquid chromatography. Additionally, the efficacy of the three molecules was evaluated in lambs infected with resistant nematodes by classical parasitological methods. Furthermore, the comparative determination of the level of expression of P-glycoprotein (P-gp2) in H. contortus recovered from lambs treated with each drug was performed by real time PCR. A longer persistence of moxidectin (P < 0.05) concentrations in plasma was observed. The concentrations of the three compounds in the mucosal tissue and digestive contents were significant higher than those measured in plasma. Drug concentrations were in a range between 452 ng/g (0.5 day post-treatment) and 32 ng/g (2 days post-treatment) in the gastrointestinal (GI) contents (abomasal and intestinal). Concentrations of the three compounds in H. contortus were in a similar range to those observed in the abomasal contents (positive correlation P = 0.0002). Lower moxidectin concentrations were recovered within adult H. contortus compared to abamectin and ivermectin at day 2 post-treatment. However, the efficacy against H. contortus was 20.1% (ivermectin), 39.7% (abamectin) and 89.6% (moxidectin). Only the ivermectin treatment induced an enhancement on the expression of P-gp2 in the recovered adult H. contortus, reaching higher values at 12 and 24 h post-administration compared to control (untreated) worms. This comparative pharmacological evaluation of three of the most used macrocyclic lactones compounds provides new insights into the action of these drugs.

Design and evaluation of a specific primer pair for the diagnosis and identification of Acanthamoeba polyphaga.

Random amplified polymorphism DNA (RAPD) is a useful tool for species identification. The obtained band patterns can be used for specific primer pair design that may be useful for species diagnosis. In this study, a distinctive a 962-bp band in A. polyphaga band patterns was found, by using the OPC20 primer (ACTTCGCCAC). The DNA fragment was used to design a specific primer pair that was useful for the identification of different isolates as A. polyphaga species. A case of A. polyphaga in disseminated acanthamoebiasis affecting mesenteric nodes is also reported.