Effects of ivermectin on development of Calliphora vicina, Robineau-Desvoidy 1830 (Diptera, Calliphoridae).

Ivermectin is one of the most widely used drugs for parasite control. Previous studies have shown a reduction in the abundance and diversity of "non-target" coprophilous organisms due to the presence of ivermectin (IVM) in bovine faecal matter (FM). Due to its breadth of behavioural habits, Calliphora vicina is a suitable dipteran species to evaluate the effects of IVM in FM. The aim of this work was to evaluate the effect of five concentrations of IVM in FM (3000, 300, 100, 30, and 3 ng/g) on the development of C. vicina. The following endpoints were evaluated: survival (between the first larval stage and emergence of new adults), larval development times to pupation and pupation times to adult, and adult emergence (% sex) and LC50. Sampling was performed from larval hatching at 60 and 120 min and at 3, 4, 5, and 12 h, and every 24 h specimens were weighed until pupae were observed. Data were analysed by ANOVA using a non-parametric Kruskal-Wallis test and as a function of elapsed development time and accumulated degree hours (ADH). Mortality at 3000 and 300 ng/g was 100% and 97%, respectively. There were statistically significant delays in adult emergence time (p = 0.0216) and in the ADH (p = 0.0431) between the control group (C) and 100 ng/g. The LC50 was determined at 5.6 ng/g. These results demonstrate the lethal and sub-lethal effects of IVM on C. vicina, while highlighting the usefulness of this species as a bioindicator for ecotoxicological studies.

In vitro and in vivo effects of chlorpyrifos and cypermethrin on blood cholinesterases in sheep.

The combination of the organophosphate (OP) chlorpyrifos (CPF) and the pyrethroid cypermethrin (CPM) is commonly marketed as pour-on formulations for the control of sheep lice, ked, and blowflies. CPF irreversibly inhibits acetylcholinesterases (AChE), while pyrethroids are not AChE inhibitors. However, combinations of pyrethroids with OPs showed a highly synergistic effect on AChE inhibition. Thus, the aim of the current work was to evaluate in vitro and in vivo the inhibitory potency of both pesticides, alone and in combination with AChE and butyrylcholinesterase (BChE) activities in sheep blood. In vitro, IC50 values were similar after CPF or CPF plus CPM incubations. The pour-on coadministration of recommended doses of CPF and CPM did not cause a significant inhibition of AChE and BChE in sheep blood. Only slight percentages of inhibition of their catalytic activities were observed when both drugs were given at 4-fold higher dose rates. The lower systemic availability of topical administration of OPs in sheep may help to explain the lower degree of inhibition of blood AChE and BChE in vivo. The results emerged from this research are a further contribution to the knowledge of the risks of implementing higher dosage regimens of OPs-containing antiparasitic formulations.

Assessment of liver slices for research on metabolic drug-drug interactions in cattle.

1. Precision-cut liver slices (PCLS) from food-producing animals have not been extensively used to study xenobiotic metabolism, and thus information on this field of research is sparse. 2. The aims of the present work were to further validate the technique of production and culture of bovine PCLS and to characterize the metabolic interaction between the anthelmintic albendazole (ABZ) and the flavin-monooxygenase (FMO) inhibitor methimazole (MTZ). 3. Nine steers were used as donors. PCLS were produced and incubated under two methods: a dynamic organ culture (DOC) incubator and a well-plate (WP) system. 4. Tissue viability, assessed through both structural and functional markers, was preserved throughout 12 h of incubation. ABZ was metabolized to its (+) and (-) albendazole sulfoxide stereoisomers (ABZSO) in bovine PCLS. The interaction between ABZ and MTZ resulted in a reduction (p < 0.001) in the rates of appearance of (+) ABZSO. Conversely, in presence of MTZ, the rates of appearance of (-) ABZSO increased under both systems (p < 0.05). 5. Both culture systems were suitable for assessing the interaction between ABZ and MTZ. 6. Overall, the results presented herein show that PCLS are a useful and reliable tool for short-term studies on metabolic drug-drug interactions in the bovine species.

The ABCG2 Efflux Transporter in the Mammary Gland Mediates Veterinary Drug Secretion across the Blood-Milk Barrier into Milk of Dairy Cows.

In human and mice ATP-binding cassette efflux transporter ABCG2 represents the main route for active drug transport into milk. However, there is no detailed information on the role of ABCG2 in drug secretion and accumulation in milk of dairy animals. We therefore examined ABCG2-mediated drug transport in the bovine mammary gland by parallel pharmacokinetic studies in lactating Jersey cows and in vitro flux studies using the anthelmintic drug monepantel (MNP) as representative bovine ABCG2 (bABCG2) drug substrate. Animals received MNP (Zolvix, Novartis Animal Health Inc.) once (2.5 mg/kg per os) and the concentrations of MNP and the active MNP metabolite MNPSO2 were assessed by high-performance liquid chromatography. Compared with the parent drug MNP, we detected higher MNPSO2 plasma concentrations (expressed as area under the concentration-versus-time curve). Moreover, we observed MNPSO2 excretion into milk of dairy cows with a high milk-to-plasma ratio of 6.75. In mechanistic flux assays, we determined a preferential time-dependent basolateral-to-apical (B > A) MNPSO2 transport across polarized Madin-Darby canine kidney II cells-bABCG2 monolayers using liquid chromatography coupled with tandem mass spectrometry analysis. The B > A MNPSO2 transport was significantly inhibited by the ABCG2 inhibitor fumitremorgin C in bABCG2- but not in mock-transduced MDCKII cells. Additionally, the antibiotic drug enrofloxacin, the benzimidazole anthelmintic oxfendazole and the macrocyclic lactone anthelmintic moxidectin caused a reduction in the MNPSO2(B > A) net efflux. Altogether, this study indicated that therapeutically relevant drugs like the anthelmintic MNP represent substrates of the bovine mammary ABCG2 transporter and may thereby be actively concentrated in dairy milk.

Environmental monitoring of ivermectin excreted in spring climatic conditions by treated cattle on dung fauna and degradation of faeces on pasture.

The effect of ivermectin excreted in faeces of cattle treated in late winter on the arthropods and the degradation of faeces on pasture were evaluated. Four calves of similar age and weight were allocated to two groups, one group was treated subcutaneously with ivermectin and the other group remained as untreated control. From faeces collected from both groups at 3, 7, 14, 21 and 28 days post-treatment (dpt), three faecal pats of 1 kg each were made and deposited on a mixed paddock. One quarter of each faecal pat was removed at 10, 20, 30 and 60 days postdeposition (dpd) to determine the concentration of ivermectin, the organic matter content, and to collect colonising dung arthropods. Concentrations at days 3 and 7 pt were significantly higher than at the other dpt (p<0.05). The highest ivermectin concentrations were found in samples from 3 dpt (p<0.05). The organic matter percentage was not significantly different between treatments. An edaphic fauna characterised the colonisation of the faeces by organisms. Although arthropods' abundance differences were not significant except for the 28 dpt at 30 dpd (p<0.0003), fewer organisms were collected from the ivermectin group at all times.

Combination of synthetic anthelmintics and monoterpenes: Assessment of efficacy, and ultrastructural and biophysical properties of Haemonchus contortus using atomic force microscopy.

The resistance of Haemonchus contortus to synthetic anthelmintics is of increasing concern; and different strategies are being evaluated to improve parasite control. The present study investigated the in vitro effects of combinations of synthetic compounds and monoterpenes. Additionally, the chemical association of the best combinations and their impact on the ultrastructural and biophysical properties of H. contortus eggs was evaluated. We assessed the efficacy of the monoterpenes, carvacrol, thymol, r-carvone, s-carvone, citral, and p-cymene and the anthelmintics, albendazole and levamisole using the egg hatch test (EHT) and the larval migration inhibition test (LMIT), respectively. The minimum effective concentrations of the monoterpenes, according to the EHT (efficacy ranging from 4.4%-11.8%) and LMIT (efficacy ranging from 5.6%-7.4%), were used in combination with different concentrations of synthetic compounds, and the IC50 and synergism rate (SR) were calculated. Fourier-transform infrared spectroscopy (FTIR) was used to analyze the chemical association between the best combinations as revealed by the in vitro tests (albendazole and levamisole with r-carvone or s-carvone). Atomic force microscopy (AFM) was used to assess the ultrastructural and biophysical properties of H. contortus eggs treated with the albendazole and r-carvone combination. Among the monoterpenes, the highest efficacies were exhibited by carvacrol (IC50 = 185.9 µg/mL) and thymol (IC50 = 187.0 µg/mL), according to the EHT, and s-carvone and carvacrol (IC50 = 1526.0 and 1785.3 µg/mL, respectively), according to the LMIT. According to the EHT, albendazole showed a slight statistically significant synergism in combination with r-carvone (SR = 3.8) and s-carvone (SR = 3.0). According to the LMIT, among the monoterpenes, r-carvone (SR = 1.7) and s-carvone (SR = 1.7) showed an increase in efficacy with levamisole; however, this was not statistically significant. The FTIR spectra of albendazole and levamisole, in association with r-carvone and s-carvone, indicated the presence of chemical interactions between the synthetic and natural molecules, contributing to the possible synergistic effects of these associations. Eggs treated with albendazole and r-carvone showed an increase in roughness and a decrease in height, suggesting that the treatment induced damage to the egg surface and an overflow of its internal contents. Overall, the combination of albendazole with r-carvone and s-carvone was efficacious against H. contortus, demonstrating a chemical association between the compounds; the significant changes in the egg ultrastructure justify this efficacy.

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.

Effects of faecal residues of moxidectin and doramectin on the activity of arthropods in cattle dung.

Dung invertebrate colonization and degradation levels of faeces from cattle treated with endectocides were studied. Faeces of control and doramectin (DRM) (subcutaneous) and moxidectin (MXD) (subcutaneous and topical) treated animals were deposited on the field from 3 to 21 days post-treatment (pt). Pats were recovered after 6 to 42 days post-deposition (pd). Faecal weight, dry matter, arthropods number, and drugs concentrations were determined. Total arthropods number was higher in control (P<0.0001) than in the other groups from days 3 to 21 pt. Total number of insects recovered on days 3, 11, and 21 pt from control pats was significantly (P<0.001) higher than in treated-animal pats during all the trial. At day 21 pt, the insects' number in dung voided by DRM-treated cattle was (P<0.05) lower than in the other groups. Comparisons of dung degradation among treatments were inconclusive. A lower adverse effect was observed for MXD compared with DRM. No significant degradation of MXD or DRM was observed during the present trial.

New recommendations for measuring collagen solubility.

The heat-solubility of intramuscular collagen is usually conducted in 1/4 Ringer's solution at pH7.4, despite this ionic strength and pH being inappropriate for post-rigor meat. The current work studied the percentage of soluble collagen and hydrothermal isometric tension characteristics of perimysial strips on bovine semitendinosus muscles in either 1/4 Ringer's solution, distilled water, PBS, or a solution of the same salt concentration as 1/4 Ringer's but at pH5.6. Values of % soluble collagen were lower at pH7.4 than 5.6. Increasing ionic strength reduced % soluble collagen. The maximum perimysial isometric tension was independent of the bathing medium, but the percent relaxation was higher at pH7.4 than at pH5.6, and increased with ionic strength of the media. It is recommended that future measurements of collagen solubility and tests on connective tissue components of post-rigor meat should be carried out in a solution of concentrations NaCl and KCl equivalent to those in 1/4 Ringer's, but at pH5.6, a pH relevant to post-rigor meat.

The herbicide glyphosate is a weak inhibitor of acetylcholinesterase in rats.

The current work evaluated the inhibitory potency of the herbicide glyphosate (GLP) on acetylcholinesterase (AChE) activity in male and female rat tissues. The AChE activity in brain was higher (p<0.05) than those observed in kidney (females: 2.2-fold; males: 1.9-fold), liver (females: 6-fold; males: 6.9-fold) and plasma (females: 14.7-fold; males: 25.3-fold). Enzyme activities were higher in presence of 10mM GLP compared to those measured at an equimolar concentration of the potent AChE inhibitor dichlorvos (DDVP). Moreover, IC50s for GLP resulted between 6×10(4)- and 6.8×10(5)-fold higher than those observed for DDVP. In conclusion, GLP is a weak inhibitor of AChE in rats.

Disposition of doramectin milk residues in lactating dairy sheep.

Doramectin (DRM) is a broad spectrum macrocyclic lactone antiparasitic drug not approved for use in dairy animals. However, DRM and other endectocide compounds are widely used extra-label to control endo- and ectoparasites in dairy sheep. The plasma disposition kinetics and the pattern of DRM excretion in milk were characterized following its subcutaneous administration to lactating dairy sheep. DRM concentration profiles were measured in plasma and milk samples after validation of a specific HPLC-based methodology. DRM was detected between 1 h and 30 days post-treatment. DRM concentrations of 0.48 ng.mL(-1) (plasma) and 1.03 ng.mL(-1) (milk) were measured at 30 days post-treatment. DRM was extensively distributed from the bloodstream to the mammary gland, and large concentrations were excreted in milk. The peak concentrations and total amount of DRM recovered in milk (expressed as area under the concentration versus time curve) were 3-fold higher than those measured in plasma; 2.44% of the total DRM dose was excreted in milk. The long persistence of DRM milk residues should be seriously considered before its extra-label use in dairy animals is recommended.

Loperamide modifies the tissue disposition kinetics of ivermectin in rats.

Ivermectin (IVM) is a broad-spectrum antiparasitic drug extensively used in human and veterinary medicine that is largely excreted in bile and faeces. Loperamide (LPM) is an opioid derivative that reduces gastrointestinal secretions and motility. Both IVM and LPM have been reported to act as P-glycoprotein substrates (P-GP). The goal of the present work was to study the LPM-induced modifications to the pattern of tissue distribution for IVM. Thirty-six Wistar male rats were randomly allocated to two groups (n = 18) and treated subcutaneously with IVM alone or co-administered with LPM. Rats were killed at different times post-treatment and samples (blood and tissues) were collected and analyzed by HPLC. The presence of LPM induced a marked enhancement in the IVM plasma concentrations, resulting in a significantly higher area under concentration time curve (AUC) value (P < 0.01) than that obtained after the administration of IVM alone. Significantly higher IVM availabilities in the liver tissue and small intestine wall (P < 0.05) were obtained in the presence of LPM. There were no statistically significant differences in drug availability in the large intestinal wall after both treatments. However, LPM induced a marked decrease in the amount of IVM recovered in the large intestinal lumen content. The ratio between IVM concentrations in the large intestinal luminal content and plasma at day 1 post-treatment was 4.64-fold higher in the absence of LPM. The delayed intestinal transit time caused by LPM accounting for an extended plasma-intestine recycling time, and a potential competition between IVM and LPM for the P-GP-mediated bile-intestinal secretion processes, may account for the enhanced IVM systemic availability reported in the current study.

Is the metabolism of 25-hydroxyvitamin D3 age-dependent in dairy cows?

It has recently been demonstrated that prepartum administered 25-hydroxyvitamin D3 (25-OHD3) is a promising candidate to assist the maintenance of peripartal calcium homeostasis in dairy cows. Since the incidence of peripartal hypocalcemia and the reported beneficial effects of the treatment are both associated with the lactation number, we investigated pharmacokinetic aspects of 25-OHD3 related to the age of dairy cows. The daily oral administration of 3mg 25-OHD3 in rapeseed oil as well as a treatment with 4 and 6mg included in the feed during the last eight to ten days of gestation resulted in linear dosage- and age-dependent increases in plasma 25-OHD3. After parturition the administration was stopped and blood samples were taken to calculate the plasma half-life. Irrespective of the supplemented dosage, cows starting the 2nd lactation showed a significantly longer plasma half-life of 25-OHD3 than cows starting the 3rd or higher lactation. Age-dependent differences in the increase of plasma 25-OHD3 could already be found before parturition when calcium homeostasis was not yet significantly challenged. Additionally, no correlations between plasma half-life of 25-OHD3 and 1,25-dihydroxyvitamin D3, PTH or the bone resorption marker CrossLaps were observed after parturition. Thus we conclude that the influence of the lactation number on the pharmacokinetics of 25-OHD3 is related directly to the age of the cows. This article is part of a Special Issue entitled 'Vitamin D Workshop'.