Ecology of the free-living stages of cattle nematodes in the dry season in the Lerma Valley, Salta province, Argentina.

The objective of this work was to describe the dynamics of development and survival of the free-living stages of cattle gastrointestinal nematodes (GIN) in fecal matter (FM) and pasture during the dry season in the Lerma Valley, Salta province, northwestern Argentina (NWA) to contribute to GIN management. The climate in the region is characterized by a rainy summer followed by a dry season from middle autumn to early spring. Fecal matter from calves naturally infected with GIN was deposited on three experimental field plots in April, July and October 2019, corresponding to the beginning, middle and end of the dry season, respectively. Each experimental unit consisted of 7 stools of about 800 g and had four repetitions. To determine the development from egg to infective larvae (L3), the first sampling (5 g fecal matter) was performed from the 10th day post-contamination and continued every 3 days until L3 were found. Subsequently, a monthly sampling was made until two consecutive negative results were obtained. Sampling of pasture began three days after the L3 recovery from FM, and continued monthly until two negative results were obtained. The following parameters were evaluated: development time and development rate from egg to L3; permanence time of L3 in feces; time of appearance on pasture; migration rate; and permanence time of L3 on pasture. The main genera of parasites present were Cooperia and Haemonchus. Significant differences were observed in the development time among contamination months (p < 0.001); development time was highest in the July contamination (28 days), with October and April contamination averaging 9 and 10 days, respectively. Development time also showed significant differences (p < 0.01) among contamination months, being highest in October (31.48%). The highest permanence time in fecal matter values were recorded in the July contamination (183 days) and migration rate was highest in the October contamination (42.49%). The highest time of appearance on pasture value was recorded in the July contamination (117 days). Finally, the highest permanence time of L3 in feces values were detected in the October contamination (148 days). The results of this work show that fecal contamination in the NWA region in the dry season would play an epidemiological role in the GIN cycle as a source of infection for the next productive cycle in the rainy season.

Eficacia antiparasitaria in vitro del extracto de quebracho (Schinopsis balansae) sobre larvas infectantes de Haemonchus contortus de ovinos / In vitro antiparasitic efficacy of the quebracho extract (Schinopsis balansae) on infecting larvae of Haemonchus contortus of sheep

RESUMEN El objetivo de este trabajo fue evaluar in vitro la eficacia del extracto de quebracho (Schinopsis spp.), rico en taninos condensados, en el control de H. contortus de ovinos, ya que existen evidencias de que estos taninos pueden reducir la excreción de huevos, la fecundidad de las hembras y la carga de parásitos adultos. Para evaluar el efecto antihelmíntico in vitro sobre larvas infectantes de H. contortus susceptibles a todos los grupos químicos, se utilizó el test de inhibición de migración larval (IML) a 3 concentraciones diferentes (5 mg/ml, 15 mg/ml y 30 mg/ml). El efecto de los tratamientos fue analizado mediante un análisis de varianza y la estimación de las diferencias entre grupos se realizó por medio de la prueba LSD Fisher. Los resultados del test in vitro demostraron una reducción de la migración larval que varió entre el 74% y el 80%, a las concentraciones de entre 5 mg/ml y 30 mg/ml. Del análisis de varianza surgen diferencias significativas entre tratamientos (p = 0,0494). Al realizar la prueba de comparación de medias se evidenciaron diferencias significativas (p < 0,05) entre los promedios de migración a las diluciones de 5 mg/ml y 15 mg/ml, y de 5 mg/ml y 30 mg/ml, mientras que no se detectaron diferencias significativas entre la dilución de 15 mg/ml y 30 mg/ml. Estos resultados señalaron que el extracto de quebracho, a las diluciones evaluadas in vitro, presentó actividad antihelmíntica sobre larvas L3 susceptibles de H. contortus. Sin embargo, se requiere ampliar los estudios in vivo para demostrar un efecto antihelmíntico en ovinos.

ABSTRACT The objective of this work was to evaluate in vitro efficacy of the quebracho extract (Schinopsis spp.), rich in condensed tannins, against H. contortus in sheep, since there is evidence that this tanninsthese tannins can reduce egg excretion, fecundity of females and the burden of adult parasites. A larval migration inhibition (IML) test with 3 different concentration (5 mg/ml, 15 mg/ml, and 30 mg/ml) was used to evaluate the in vitro anthelmintic effect upon iInfective H. contortus larvae,from a susceptible strain to all chemical groupswere utilized with 3 diferentconcentration (5mg/ml, 15mg/ml, and 30mg/ml). The effect of the treatments was submitted to a variance analysis and the estimation of the differences between groups was evaluated using LSD Fisher test. Results from the in vitro test, revealed a reduction of the larval migration that varies from 74% to 80%, at the concentrations between 5 mg/ml to 30 mg/ml. From the analysis of variance, significant differences appear between treatments (p = 0,0494). After When performing the mean comparison test were performed, significant differences (p < 0,05) were found between the migration averages at dilutions of 5 mg/ml and 15 mg/ml, and between 5 mg/ml and 30 mg/ml, while were no't detected significant differences between the dilution of 15 mg/ml and 30 mg/ml. These results indicated that quebracho extract at the dilutions evaluated in vitro showed anthelmintic activity on L3 susceptible to H. contortus. However, it is necessary to conduct further studies in vivo to demonstrate an anthelmintic effect in sheep.

Combined moxidectin-levamisole treatment against multidrug-resistant gastrointestinal nematodes: A four-year efficacy monitoring in lambs.

Nematicide combinations may be a valid strategy to achieve effective nematode control in the presence of drug resistance. The goal of the current trial was to evaluate the pharmaco-parasitological performance of the moxidectin (MOX) and levamisole (LEV) combination after four years of continuous use in lambs naturally parasitized with multi-resistant gastrointestinal nematodes. At the beginning of the trial, 40 lambs were divided into four groups (n = 10), which were untreated (control) or subcutaneously treated with MOX (0.2 mg/kg), LEV (8 mg/kg) or with the combination MOX + LEV (administered separately at 0.2 and 8 mg/kg, respectively). Blood samples were collected at different times post-treatment and LEV and MOX plasma concentrations were measured by HPLC. The clinical efficacy of the continuous use of MOX + LEV combination was assessed with the controlled efficacy test (CET), performed at the beginning and end of the study, and with the faecal egg count reduction (FECR) test, performed over the four-year study period. No significant adverse pharmacokinetic changes were observed either for MOX or LEV after their co-administration to infected lambs. The CET (first year) showed efficacies of 84.3 % (Haemonchus contortus), 100 % (Teladorsagia circumcincta and Trichostrongylus axei), and 97.4 % (T. colubriformis). After the repetitive use of the combined treatment for four years, those efficacies remained high (100 %) and only decreased to 58 % against T. colubriformis. The evaluation of the FECR over the study period showed fluctuations in the performance of the combined administration. The initial FECR (2014) was 99 % (MOX), 85 % (LEV) and 100 % (MOX + LEV). The co-administration of MOX + LEV during the four-year experimental period resulted in a significantly higher anthelmintic effect (87 %) than that of MOX (42 %) or LEV (69 %) given alone. The combined use of MOX + LEV to control resistant gastrointestinal nematodes appears to be a valid strategy under specific management conditions. A high initial therapeutic response to the combination would be a relevant feature for the success of this tool.

Plant-Derived Compounds as a Tool for the Control of Gastrointestinal Nematodes: Modulation of Abamectin Pharmacological Action by Carvone.

The combination of synthetic anthelmintics and bioactive phytochemicals may be a pharmacological tool for improving nematode control in livestock. Carvone (R-CNE) has shown in vitro activity against gastrointestinal nematodes; however, the anthelmintic effect of bioactive phytochemicals either alone or combined with synthetic drugs has been little explored in vivo. Here, the pharmacological interaction of abamectin (ABM) and R-CNE was assessed in vitro and in vivo. The efficacy of this combination was evaluated in lambs naturally infected with resistant gastrointestinal nematodes. Additionally, the ligand and molecular docking of both molecules to P-glycoprotein (P-gp) was studied in silico. The presence of R-CNE produced a significant (p < 0.05) increase of Rho123 and ABM accumulation in the intestinal explants. After 60 min of incubation, Rho123 incubated with R-CNE had a 67 ± 21% higher concentration (p < 0.01) than when it was incubated alone. In the case of ABM, a significant increase in the intestinal concentrations was observed at 15 and 30 min after incubation with R-CNE. In the in vivo assay, no undesirable effects were observed after the oral administration of R-CNE. The coadministration of the natural compound prolonged ABM absorption in lambs. ABM T ½ absorption was 1.57-fold longer (p < 0.05) in the coadministered group. Concentrations of R-CNE between 420 and 2,593 ng/mL were detected in the bloodstream between 1 and 48 h posttreatment. The in vivo efficacy of ABM against gastrointestinal nematodes increased from 94.9 to 99.8% in the presence of R-CNE, with the lower confidence interval limit being >90%. In vitro/in vivo pharmacoparasitological studies are relevant for the knowledge of the interactions and the efficacy of bioactive natural products combined with synthetic anthelmintics. While ADMET (absorption, distribution, metabolism, excretion, and toxicity) predictions and the molecular docking study showed a good interaction between ABM and P-gp, R-CNE does not appear to modulate this efflux protein. Therefore, the pharmacokinetic-pharmacodynamic effect of R-CNE on ABM should be attributed to its effect on membrane permeability. The development of pharmacology-based information is critical for the design of successful strategies for the parasite control.

Pharmacologic interaction between oxfendazole and triclabendazole: In vitro biotransformation and systemic exposure in sheep.

The aim of the current work was to evaluate a potential pharmacokinetic interaction between the flukicide triclabendazole (TCBZ) and the broad-spectrum benzimidazole (BZD) anthelmintic oxfendazole (OFZ) in sheep. To this end, both an in vitro assay in microsomal fractions and an in vivo trial in lambs parasitized with Haemonchus contortus resistant to OFZ and its reduced derivative fenbendazole (FBZ) were carried out. Sheep microsomal fractions were incubated together with OFZ, FBZ, TCBZ, or a combination of either FBZ and TCBZ or OFZ and TCBZ. OFZ production was significantly diminished upon coincubation of FBZ and TCBZ, whereas neither FBZ nor OFZ affected the S-oxidation of TCBZ towards its sulfoxide and sulfone metabolites. For the in vivo trial, lambs were treated with OFZ (Vermox® oral drench at a single dose of 5 mg/kg PO), TCBZ (Fasinex® oral drench at a single dose of 12 mg/kg PO) or both compounds at a single dose of 5 (Vermox®) and 12 mg/kg (Fasinex®) PO. Blood samples were taken to quantify drug and metabolite concentrations, and pharmacokinetic parameters were calculated by means of non-compartmental analysis. Results showed that the pharmacokinetic parameters of active molecules and metabolites were not significantly altered upon coadministration. The sole exception was the increase in the mean residence time (MRT) of OFZ and FBZ sulfone upon coadministration, with no significant changes in the remaining pharmacokinetic parameters. This research is a further contribution to the study of metabolic drug-drug interactions that may affect anthelmintic efficacies in ruminants.

An attempt to replace an ivermectin-resistant Cooperia spp. population by a susceptible one on grazing pastures based on epidemiological principles and refugia management.

The maintenance of anthelmintic-susceptible parasite refugia to delay the onset of anthelmintic resistance is an almost impossible effort in many grazing livestock production countries given that current refugia consist of already resistant parasites. Rather, efforts could be focused on replacing the resistant parasite refugia by susceptible parasite ones and implementing sustainable parasite control measures from then on. To this purpose, a trial was conducted to attempt to establish a new population of ivermectin-susceptible Cooperia sp. on a beef cattle farm with proven problems of ivermectin-resistant Cooperia. During two consecutive years, 82 (Year 1) and 100 (Year 2) recently weaned and parasite-free heifers were inoculated with 40,000 or 30,000 susceptible Cooperia L3, respectively, at a time when levels of resistant parasite refugia were normally low. The animals were subsequently allowed to graze on the problem pastures during autumn until the end of spring. Levels of parasitism in the animals and on pasture were monitored monthly and animals were treated with levamisole when needed. The combination of parasitological monitoring and local epidemiological knowledge was essential to determine when treatments were to be administered. No clinical signs of gastrointestinal parasitosis in the herd were observed throughout the study and unnecessary treatments were avoided. Faecal egg counts reduction tests (FECRT) and controlled efficacy tests (CET) employing worm counts were carried out at different times throughout the study to determine the clinical efficacy (FECRT) and the absolute efficacy (CET) of ivermectin, respectively. The clinical efficacy of ivermectin increased from an initial 73% to 99.4%, while the absolute efficacy increased from 54.1% to 87.5% after just two animal production cycles. The switch from a resistant parasite population to a susceptible one requires knowledge of parasitological epidemiology, especially in relation to seasonal variations of parasite populations in both the host and in refugia.

Comparative pharmacokinetic and pharmacodynamic response of single and double intraruminal doses of ivermectin and moxidectin in nematode-infected lambs.

AIMS: To compare the pharmacokinetics, distribution and efficacy (pharmacodynamic response) of intraruminal ivermectin (IVM) and moxidectin (MXD) administered at 0.2 and 0.4 mg/kg to naturally nematode-infected lambs, and to determine the ex vivo accumulation of these anthelmintics by Haemonchus contortus. METHODS: Romney Marsh lambs, naturally infected with IVM-resistant H. contortus, were allocated to treatment groups based on faecal nematode egg counts. They received 0.2 or 0.4 mg/kg IVM or MXD (n=10 per group), or no treatment (Control; n=6), on Day 0. Samples from four animals from each treatment group, including abomasal parasites, were obtained on Day 1. Plasma samples were also collected from Day 0 to 14, and a faecal egg count reduction test (FECRT) and a controlled efficacy trial were carried out on Day 14. Concentrations of IVM and MXD in plasma, in abomasal and intestinal tissues and in H. contortus were evaluated by high-performance liquid chromatography. Additionally, the ex vivo drug accumulation of IVM and MXD by H. contortus was determined. RESULTS: Peak plasma concentrations and the area under the concentration vs. time curve for both IVM and MXD were higher for 0.4 than 0.2 mg/kg treatments (p<0.05), but there were no differences for other parameters. Concentrations of IVM and MXD in the gastrointestinal target tissues and in H. contortus were higher compared to those measured in plasma. Concentrations of both drugs in H. contortus were correlated with those observed in the abomasal content (r=0.86; p<0.0001). The exposure of H. contortus to IVM and MXD was related to the administered dose. Mean FECRT and efficacy for removal of adult H. contortus was 0% for IVM at 0.2 and 0.4 mg/kg. For MXD, FECRT were >95% for both treatments, and efficacy against H. contortus was 85.1% and 98.1% for 0.2 and 0.4 mg/kg, respectively. The ex vivo accumulation of IVM and MXD in H. contortus was directly related to the drug concentration present in the environment and was influenced by the duration of exposure. CONCLUSION: Administration of IVM and MXD at 0.4 compared with 0.2 mg/kg accounted for enhanced drug exposure in the target tissues, as well as higher drug concentrations within resistant nematodes. The current work is a further contribution to the evaluation of the relationship between drug efficacy and basic pharmacological issues in the presence of resistant parasite populations.

Closantel plasma and milk disposition in dairy goats: assessment of drug residues in cheese and ricotta.

Closantel (CLS) is currently used in programs for the strategic control of gastrointestinal nematodes. CLS is extralabel used in different dairy goat production systems. From available data in dairy cows, it can be concluded that residues of CLS persist in milk. The current work evaluated the concentration profiles of CLS in plasma and milk from lactating orally treated dairy goats to assess the residues pattern in dairy products such as cheese and ricotta. Six (6) female Saanen dairy goats were treated orally with CLS administered at 10 mg/kg. Blood and milk samples were collected between 0 and 36 days post-treatment. The whole milk production was collected at 1, 4, 7, and 10 days post-treatment to produce soft cheese and ricotta. CLS concentrations in plasma, milk, cheese, whey, and ricotta were determined by HPLC. The concentrations of CLS measured in plasma were higher than those measured in milk at all sampling times. However, the calculated withdrawal time for CLS in milk was between 39 and 43 days postadministration to dairy goats. CLS residual concentrations in cheese (between 0.93 and 1.8 µg/g) were higher than those measured in the milk used for its production. CLS concentrations in ricotta were sixfold higher than those in the milk and 20-fold higher than those in the whey used for its production. The persistent and high residual concentrations of CLS in the milk and in the cheese and ricotta should be seriously considered before issuing any recommendation on the extralabel use of CLS in dairy goat farms.

Measurement of ivermectin concentrations in target worms and host gastrointestinal tissues: influence of the route of administration on the activity against resistant Haemonchus contortus in lambs.

The influence of the administration route on the relationship between efficacy and ivermectin concentration profiles achieved in the bloodstream, the gastrointestinal mucosal tissues/fluid contents and within a target abomasal parasite (Haemonchus contortus) was evaluated in lambs. Twenty-six (26) parasitized lambs were assigned into three experimental groups: untreated (control) and ivermectin treated by the subcutaneous and intraruminal route at 0.2mg/kg. Blood samples were collected between 0 and 15 days post-treatment (plasma disposition study). Four animals from each group were sacrificed at day 3 post-treatment. Mucosa and content samples from abomasum and small intestine and adult specimens of H. contortus were collected. Drug concentrations were measured by HPLC. Individual fecal egg counts were evaluated at -1, 3 and 15 days post treatment. Post-mortem examination was done at day 15 post-treatment. Adult nematodes recovered from the digestive tract were counted and identified by species. Ivermectin plasma availability was higher (P<0.05) after the subcutaneous administration (129 ng.d/ml) compared to the intraruminal treatment (58.4 ng.d/ml). However, ivermectin concentrations measured in the gastrointestinal contents were higher in lambs treated by the intraruminal route. The mean ivermectin concentrations achieved (3 days post-treatment) in the abomasal content were 143 ng/g (intraruminal) and 2.53 ng/g (subcutaneous). Ivermectin concentrations were 15-fold higher in H. contortus recovered from intraruminally treated lambs. Whereas the subcutaneous administration reduced the number of adult nematodes from 4376 to 1300, the number of adult nematodes after the treatment with ivermectin given by the intraruminal route was 206 (P<0.05). The higher ivermectin concentrations achieved in the digestive tract shortly after the intraruminal treatment may account for the observed enhanced efficacy compared to the parenteral administration against parasites of reduced susceptibility.

Interference with P-glycoprotein improves ivermectin activity against adult resistant nematodes in sheep.

The in vivo co-administration of ivermectin (IVM) with P-glycoprotein (P-gp) modulator agents has been shown to enhance its systemic availability. However, there is no sufficient evidence on the impact that this type of drug-drug interaction may have on the in vivo efficacy against resistant nematodes in ruminant species. The current work reports on the effects of loperamide (LPM), a P-gp modulating agent, on both IVM kinetic behaviour and anthelmintic activity in infected lambs. Eighteen (18) lambs naturally infected with IVM-resistant gastrointestinal nematodes were allocated into three (3) experimental groups. Group A remained as untreated control. Animals in Groups B and C received IVM (200mug/kg, subcutaneously) either alone or co-administered with LPM (0.2 mg/kg, twice every 12h), respectively. Individual faecal samples were collected from experimental animals at days -1 and 14 post-treatment to perform the faecal eggs count reduction test (FECRT). Blood samples were collected between 0 and 14 days post-treatment and IVM plasma concentrations were determined by HPLC. Additionally, at day 14 post-treatment, lambs from all experimental groups were sacrificed and adult gastrointestinal nematode counts were performed. FECRT values increased from 78.6 (IVM alone) to 96% (IVM+LPM). Haemonchus contortus was highly resistant to IVM. The IVM alone treatment was completely ineffective (0% efficacy) against adult H. contortus. This efficacy value increased up to 72.5% in the presence of LPM. The efficacy against Trichostrongylus colubriformis increased from 77.9% (IVM alone) to 96.3% (IVM+LPM). The described favorable tendency towards improved anthelmintic efficacy was in agreement with the enhanced IVM plasma availability (P<0.05) and prolonged elimination half-life (P<0.05) induced by LPM in infected lambs. A LPM-induced P-gp modulation increases IVM systemic exposure in the host but also it may reduce P-gp efflux transport over-expressed in target resistant nematodes.