Efficacy of pharmacokinetic interactions between piperonyl butoxide and albendazole against gastrointestinal nematodiasis in goats.

To test the hypothesis that modulation of hepatic microsomal sulphoxidation and sulphonation by the cytochrome P450 inhibitor piperonyl butoxide could increase bioavailability of albendazole, the present study was undertaken to understand the pharmacokinetics of albendazole in goats at a dose of 7.5 mg kg- 1 body weight with and without co-administration with piperonyl butoxide at 63.0 mg kg- 1 body weight. Plasma albendazole sulphoxide metabolite, the anthelmintically active moiety, reached its maximum concentration of 0.322 ± 0.045 µg ml- 1 and 0.384 ± 0.013 µg ml- 1 at 18 h and 24 h after administration of albendazole alone and co-administration of albendazole with piperonyl butoxide, respectively. Analysis of the data revealed statistically increased albendazole sulphoxide levels at 24 (P 0.05) in values of maximum concentration (normal and calculated) could be observed between groups of goats. However, values of time to reach the concentration maximum (normal and calculated), area under the concentration-time curve (0-∞ and calculated), minimum residence time, distribution half-life, elimination half-life and total area under the first movement of plasma drug concentration-time curve were significantly higher (P <  0.05) in plasma levels of albendazole sulphoxide in goats following single oral co-administration of albendazole with piperonyl butoxide. The faecal egg count reduction and lower 95% confidence limit for the group treated with albendazole alone were 97 and 68%, while for co-administration of albendazole and piperonyl butoxide the values were 99 and 97%, respectively. The ED50 for egg hatch was 0.196, indicating suspected resistance to benzimidazole anthelmintics. The drug combination proved efficacious against an albendazole-resistant nematode parasite population in goats.

Diet-induced modulation of pharmacokinetics of albendazole in Sahiwal cattle.

The influence of diet type and pre-treatment fasting on the kinetic disposition of albendazole was evaluated in Sahiwal heifers following oral and intra-ruminal administration of the drug. The anthelmintically active moiety albendazole sulphoxide appeared early and was eliminated early in cattle offered green fodder, with decreased maximum concentration (C max) and area under concentration-time curve (AUC) when the drug was administered both through oral and intra-ruminal routes. Further, the elimination half-life (t ½ß) revealed significantly increased values for albendazole sulphoxide in cattle administered albendazole through the intra-ruminal route. An increased AUC and t ½ß is reflective of increased bioavailability of albendazole in animals offered dry fodder. Increased values (P <  0.05) of C max, time to C max (T max), AUC and t ½ß for albendazole sulphoxide occurred in cattle with a pre-treatment 24-h fast, resulting in its increased bioavailability. Extrapolation of data of the active metabolite albendazole sulphoxide levels in terms of drug-parasite contact revealed increased exposure of parasites to the drug in cattle administered albendazole through the intra-ruminal route and with 24-h pre-treatment fasting.

Fungus-benzimidazole interactions: a prerequisite to deploying egg-parasitic fungi Paecilomyces lilacinus and Verticillium chlamydosporium as biocontrol agents against fascioliasis and amphistomiasis in ruminant livestock.

In vitro trials investigating the effects of albendazole and triclabendazole anthelmintics on the growth profiles of the egg-parasitic fungi Paecilomyces lilacinus and Verticillium chlamydosporium were undertaken. In addition, in vivo trials were conducted in goats fed on millet grain cultures of each fungus and administered albendazole and triclabendazole anthelmintics. In vitro growth revealed V. chlamydosporium to be more sensitive to albendazole compared to P. lilacinus. In contrast, triclabendazole had the least inhibitory effect on in vitro growth of both P. lilacinus and V. chlamydosporium. Similar to albendazole, growth of P. lilacinus was more vigorous at 0.5 ppm concentration of triclabendazole. Efforts to re-isolate these egg-parasitic fungi from faeces of goats fed on fungal millet grain cultures before and following single intraruminal administration of albendazole and triclabendazole showed that P. lilacinus was not able to be re-isolated from the faeces at any sampling period. In contrast, V. chlamydosporium was able to be re-isolated from the faeces at all of the sampling periods except for the samples taken at 8-18 h and 18-24 h after administration of albendazole and triclabendazole, respectively. Lack of fungal activity at these times coincided with peak plasma availability of anthelmintics and suggests faecal levels of drugs were also high at these times and impacted negatively on fungal viability.

Screening for Indian isolates of egg-parasitic fungi for use in biological control of fascioliasis and amphistomiasis in ruminant livestock.

Wild isolates of the egg-parasitic fungi Paecilomyces lilacinus and Verticillium chlamydosporium, obtained from the organic environment of Durg, Chhattisgarh, India, were subjected to screening for in vitro growth using different media types, range of incubation temperature and pH, and their predatory activity to the eggs of Fasciola gigantica and Gigantocotyle explanatum. Maximum growth of P. lilacinus was obtained in corn-meal agar compared to any other media types. The preferred medium for growth of V. chlamydosporium was corn-meal agar, followed by potato-dextrose agar. After initial growth for 16 h of incubation, no growth was observed in water agar for both the fungi. Six different temperatures--4 degrees C, 10 degrees C, 18 degrees C, 26 degrees C, 34 degrees C and 40 degrees C--were used to observe growth profiles of the fungi in corn-meal agar medium. While no and very little growth of P. lilacinus and V. chlamydosporium was observed at 4 degrees C and 10 degrees C, respectively, growth profiles of both the fungi were optimal at 26-40 degrees C. A range of pH (pH 4-8) supported growth of both P. lilacinus and V. chlamydosporium. Full-grown plates of the fungi baited with viable eggs of F. gigantica and G. explanatum revealed that V. chlamydosporium was more vigorous in its egg-parasitic ability compared to P. lilacinus. Distortion of the eggs started on day 2-3 of egg baiting in culture plates of V. chlamydosporium, with complete distortion by day 7. On the contrary, P. lilacinus exhibited very limited egg-parasitic ability and some of the baited eggs even showed development of miracidia.

Formulation of a strategy for the application of Duddingtonia flagrans to control caprine parasitic gastroenteritis.

Experiments on the influence of egg density and varying quantities of chlamydospores on the nematode-trapping ability of Duddingtonia flagrans, influence of D. flagrans on the larval translation of gastrointestinal nematodes, doses of chlamydospores required for the effective control of gastrointestinal nematodosis and the epidemiology of nematode parasites were conducted in goats, which generated baseline data required for strategic application of the biocontrol agent. The nematode-trapping ability of D. flagrans, measured by numerical enumeration of infective third-stage larvae developed in the faecal culture, revealed that the efficacy is dependent on both nematode egg and chlamydospore density. Pasture plot studies revealed that D. flagrans, if deposited at the same time as nematode eggs, prevents translation of third-stage larvae of caprine nematodes from the faecal pats onto the grass blades. Feeding of 1 x 106 chlamydospores per kg body weight and above to goats virtually eliminated larvae from the pasture. Application of as few as 1 x 104 and 1 x 105 chlamydospores per kg body weight had a profound impact on larval recovery. The effect persisted as long as the chlamydospores were fed. Monthly faecal worm egg counts of adult goats maintained under a semi-intensive management system on the Chhattisgarh plain and pasture larval burden revealed that June to August were the months of high risk for nematodosis. Haemonchus was the dominant species recorded throughout the year. The present data can best be utilized by formulating a strategic control measure when the larval challenge to the animal is maximum (June to August), so as to prevent establishment of patent infection. The observations reinforced the strategy to be adopted for nematode parasite control in goats by applying the biocontrol option at the onset of the monsoon.

Kinetic disposition of albendazole in goats subclinically infected with gastrointestinal nematodes vis-à-vis naive animals following oral and intraruminal administration.

The influence of subclinical nematodosis on the kinetic disposition of albendazole was evaluated in goats following oral and intraruminal administration. The disposition curves of its metabolites indicated increased uptake of the drug in parasitized goats following intraruminal compared to oral dosing (P < 0.05). The midpoint for the pharmacologically active metabolite, albendazole sulphoxide, in the circulatory compartment was around 0.6 mug ml- 1 both in parasitized and naïve goats. The period of exposure to this concentration was around 14 h (oral route), 18 h (intraruminal route) and 16 h (oral route), 17 h (intraruminal route) in parasitized and naïve goats, respectively. As the duration of exposure of parasites to the toxic concentration of the anthelmintically active metabolite was prolonged, it could be assumed that intraruminal delivery of the drug would improve the efficacy of albendazole in parasitized goats.

The nematode-trapping efficacy of two chlamydospore-forming fungi against Haemonchus contortus in sheep.

An in vitro study was carried out to determine efficacy of Indian isolates of the nematode-trapping fungi Arthrobotrys musiformis and Duddingtonia flagrans to capture infective larvae of Haemonchus contortus. These fungi have previously been screened and selected for their survival in the gastrointestinal tract of sheep without losing growth and nematode capturing potential. Following the feeding of chlamydospores of these two fungi alone or in combination in sheep experimentally infected with Haemonchus contortus, coprocultures were set up to enumerate the infective third stage larvae. The number of larvae captured from faeces of fungus-fed sheep was significantly higher compared with fungus-unfed controls irrespective of the fungus used. The fungal combination produced no antagonistic effect and thus can be used as efficiently as the fungi alone in the biological control of animal parasitic nematodes.

Implications of fungicidal effects of benzimidazole compounds on Duddingtonia flagrans in integrated nematode parasite management in livestock.

An in vitro trial with carbendazim fungicide on the growth profile of the predatory fungus Duddingtonia flagrans was undertaken and in vivo trials in sheep and buffaloes, fed on chlamydospores of D. flagrans and administered albendazole anthelmintic, were conducted. Although no growth inhibition was detected at a carbendazim concentration of 0.05 ppm, growth inhibition was recorded of 50% and above at concentrations of 0.25 and 1.00 ppm (p < 0.001) and of around 90% at concentrations of 2.00 to 5.00 ppm (p <0.0001). Scanty recovery of the fungus was made from faecal culture 48 h following a single dose of albendazole both in sheep and buffaloes. However, profuse fungal recovery was made from 96 h post dosing onwards. When the drug was used as an intraruminal slow-release capsule, no faecal fungal recovery could be made from day 3 after administration of the capsule, when the albendazole sulphoxide concentration was around 1.0 microg/ml. However, profuse and scanty fungal recovery could be made on days 1 and 2, respectively, after administration of the capsule, when the plasma albendazole sulphoxide concentration was around 0.4 and 0.9 microg/ml, respectively. The implications for use of a combination of anthelmintics and biological control in sustainable parasite control programmes are discussed.

Dense molecular marking reveals genetic diversity in morphologically similar isolates of the nematophagous fungus Duddingtonia flagrans.

Three morphologically similar isolates of Duddingtonia flagrans [(Duddington) R. C. Cooke] viz., Df-2550, Df-2507 and Df-BJ were subjected to RAPD (Randomly Amplified Polymorphic DNA) and SRFA (Selective Fragment Length Amplification) mode of DNA fingerprinting analysis to generate 233 different anonymous DNA markers. Mean number of alleles per primer/primer pair for RAPD and SRFA primers was 13.75 and 29.66 respectively. Phylogenetic analysis through bootstrapping of 1000 simulated replicates of the data set demonstrated that Df-2550 was ancestral in the group of three and did not align with Df-2507 and Df-BJ, which appeared to diversify recently and therefore remained at the end of the phylogenetic tree. Genomic islands were also identified by three SRFA primer pairs, where Df-2550 aligned with Df-BJ, which is reverse to the master consensus-grouping pattern. Scanning image of the amplicon profiles when represented graphically generated unique molecular signature for the isolates.

Top dressing of feed with desiccated chlamydospores of Duddingtonia flagrans for biological control of the pre-parasitic stages of ovine Haemonchus contortus.

Feeding trials were conducted with stall-fed sheep parasitized with Haemonchus contortus. For 10 days they were offered 250 g of a concentrate feed that had been top-dressed with desiccated chlamydospores of Duddingtonia flagrans at 1 x 10(5), 5 x 10(5), 1 x 10(6) or 2 x 10(6) chlamydospores/kg body weight. Pooled faeces from each group on day 7 of spore feeding were spread on different pasture plots. On day 28 after the start of spore feeding, further pooled faeces from each group were spread on the same plots. The larval burdens on the plots were monitored for 2 months and the larval harvest from in vitro faecal cultures were monitored regularly. The application of 1 x 10(6) or more spores/kg body weight virtually eliminated larvae from both the pasture and the faecal cultures. The application of as few as 1 x 10(5) spores/kg body weight had a profound impact on larval recovery. The effect persisted while the spores were being fed but not for more than 4 days following discontinuation of spore feeding. Top dressing supplementary feed with dried chlamydospores offers a potential way of using D. flagrans for biological control of the pre-parasitic stages of H. contortus.

Anthelmintic resistance on an organized sheep farm in India.

Monitoring anthelmintic resistance in strongyle nematodes by the faecal egg count reduction test and a commercial larval development assay on an organized sheep farm in the semi-arid area of Rajasthan revealed the emergence of resistance to benzimidazoles and rafoxanide and a potential risk of the development of levamisole/tetramisole resistance. A benzimidazole/levamisole combination, avermectins and closantel were each found to be efficacious.

Characterization of rpo-B mutant of a strain of Escherichia coli to confirm its suitability for routine work related to recombinant DNA technology.

Rifampicin resistant spontaneous mutant of a popular laboratory strain of Escherichia coli (DH5 alpha) was isolated and found to resist high level of the drug in growth medium. The growth of the isolate was found to be slower than its wild-type counterpart. Its ability to get transformed into drug-resistant state through transformation by chemical means as tested using plasmid DNA from three different size categories, was found to be at par with the wild type. Other properties, viz., alpha-complementation and ability to express foreign gene remained unaltered. The utility of the rifampicin-resistant phenotype as a potential chromosomal genetic marker was demonstrated in a typical conjugation experiment to establish the ability of the mutant to act as recipient strain for a recombinant, mobilizable plasmid DNA molecule with the advantage of drug-mediated, high efficiency selection. Substitution of the wild strain with the mutant for routine experimentations related to recombinant DNA technology was concluded to be appropriate and of advantage.

Screening for Indian isolates of predacious fungi for use in biological control against nematode parasites of ruminants.

Four isolates of predacious fungi, two each of Arthrobotrys oligospora isolated from a sheep and a male crossbred calf and of Duddingtonia flagrans isolated from a sheep and a female buffalo in western India, were studied for their suitability as biocontrol agents against parasitic nematodes of ruminants, using growth assay, predatory activity, germination potential and ability to survive passing through the ruminants gut as criteria. The study showed that isolates of D. flagrans grew well in artificial media, had encouraging predatory activity, produced profuse chlamydospores that germinated easily at 25 degrees C and could survive passage through the ruminant gut. The ovine isolate of D. flagrans was superior in all respects to the isolate from buffalo and was the most promising candidate for biological control of nematode parasites of ruminants.

Comparative anthelmintic activity of strategic sustained low-level administration of albendazole in feed pellets compared to single doses of closantel and tetramisole against natural ovine parasitic gastroenteritis.

The strategic use of single therapeutic doses of closantel, tetramisole or sustained low-level administration of albendazole in feed pellets in controlling naturally acquired parasitic gastroenteritis in sheep was investigated on a farm in semi-arid Rajasthan, India. A total of 303 5- to 6-month-old sheep were divided into three groups. Two groups were dosed with single therapeutic doses of closantel and tetramisole and the third group was given a low-level medication with albendazole through feed pellets for 30 days. Faecal egg counts revealed significantly lower counts (p<0.001) in the group treated with closantel compared to the other two groups. The faecal egg counts in the group receiving sustained low-level albendazole rose after withdrawal of the medication but remained significantly lower than those in the group treated with tetramisole up to 7 weeks after treatment (p<0.05). On the other hand, in the group treated with tetramisole, the mean faecal egg count rose from 3 weeks after treatment and remained continuously higher than those in any other group up to 12 weeks after treatment. The closantel-treated group gained more body weight but the first six-monthly greasy fleece yield was greater in the group treated with medicated pellets. During the first 3 months of the experiment, three animals in the group treated with tetramisole died of parasitic gastroenteritis. Following sustained low-level administration of albendazole in feed pellets, the plasma disposition curve of both the sulphoxide and sulphone metabolites reached its plateau level by day 5 and remained almost constant thereafter. The comparative cost-effectiveness of the three treatment regimes during the first 3 months of treatment was best for the group treated with closantel followed by the group treated with medicated feed pellets.

Influence of diet type and pretreatment fasting on the disposition kinetics of albendazole in sheep.

The influence of the quality and quantity of diets on the disposition kinetics of albendazole were studied in sheep in two different experiments. The plasma concentration profiles of albendazole sulphoxide and albendazole sulphone were measured following intraruminal administration of albendazole at 5.0 mg/ kg body weight in weaner sheep offered three different diets: 100% green Sorghum spp., 100% dry mature Cenchrus ciliaris hay and a 50:50 mix of these two diets. The peak plasma concentrations and the availability of the albendazole metabolites, as measured by the area under the concentration time curve, were significantly higher (p < 0.01) in the animals offered exclusively dry fodder compared to other diets. Changing the diet from dry to green fodder resulted in a significantly lower systemic availability of the drug metabolites. It is suggested that a decreased transit time of the digesta in the bowel on the green diet, with its high water content, limited the systemic availability of the drug by reducing the time available for gastrointestinal absorption. An experiment on the influence of different levels of pretreatment fasting on the pharmacokinetics of albendazole revealed significantly higher (p < 0.05) plasma concentrations of the anthelmintically active sulphoxide metabolite from 12 h onwards following administration of the drug in animals subjected to 24 h of pretreatment fasting compared to other groups with pretreatment fasting of 8, 12 or 18 h. The area under the concentration time curve and the minimum residence time of the drug metabolites were significantly greater (p < 0.05) in animals that had been fasted for 24 h. It is suggested that fasting induces a decrease in the flow of digesta through the gastrointestinal tract of ruminants and prolongs the duration of dissolution of the drug, resulting in enhancement of the absorption of albendazole and of the systemic availability of its metabolites.

The pharmacokinetics and efficacy of long-term low-level and split-dose administration of albendazole through in-feed formulations against ovine and caprine parasitic gastroenteritis.

Two trials were conducted against natural and experimentally induced parasitic gastroenteritis in sheep and goats using an in-feed formulation of albendazole to evaluate its therapeutic and prophylactic efficacy. In the first trial, albendazole was incorporated in feed pellets to deliver an average daily dose of 0.7 mg/kg body weight in order to evaluate its prophylactic efficacy. In the second trial, feed pellets were offered to deliver an average total dose of 8.0 mg/kg body weight in two equal split doses in order to evaluate its curative efficacy. Sustained plasma concentrations of the active compound, albendazole sulphoxide, and its metabolite albendazole sulphone, sufficient to prevent establishment of infection, were achieved when the animals were allowed to feed on medicated pellets for 10 consecutive days. The bioavailability of the metabolites of albendazole following the administration of a therapeutic dose in two split doses of the in-feed formulation was sufficient to remove established adult nematodes. The concentrate feed pellets could be used for self-medicating small ruminants for therapeutic use as well as for prophylaxis based on their strategic use appropriate to the epidemiology of the parasitic disease.

Pharmacokinetics and efficacy of long-term low-level administration of triclabendazole in urea molasses blocks against induced bovine and bubaline fasciolosis.

The pharmacokinetics and flukicidal efficacy of triclabendazole delivered in low doses on a daily basis through urea molasses blocks were studied in cattle and buffaloes experimentally infected with Fasciola gigantica. The observations were compared with single therapeutic doses at 12.0 and 24.0 mg/kg body weight in cattle and buffaloes, respectively, prior to becoming experimentally infected. After receipt of triclabendazole by cattle and buffaloes at 0.83 and 3.0 mg/kg body weight, respectively, on a daily basis, both the animal species exhibited equilibrium between its absorption and disposition of its metabolites in plasma on day 4 and remained almost unchanged thereafter. The continuous subtherapeutic dosing of triclabendazole through urea molasses block at 0.83 mg/kg body weight in cattle and 3.0 mg/kg body weight in buffalo proved to be efficacious against mature liver flukes.

Effect of single and divided dose administration on the pharmacokinetics of albendazole in sheep and goat.

The pharmacokinetics of albendazole were studied in sheep and goats following single and divided dose administration at nematocidal and flukicidal dose rates. The disposition curves of the metabolites indicated increased uptake of the drug both in sheep and goats at divided dose schedules compared to single dose administration (P < 0.05). The increased bioavailability of benzimidazole anthelmintics in divided dose schedules could improve their efficacy and help in extending their lives.

The disposition kinetics of albendazole following the administration of single and divided doses to cattle and buffalo.

Concentrations of albendazole sulphoxide and its sulphone metabolite in plasma in cattle and buffalo were measured by high-performance liquid chromatography after single and divided intraruminal administration of albendazole at the recommended nematocidal and fasciolicidal dose rates of 7.5 and 15.0 mg/kg body weight, respectively. No significant differences in the plasma concentrations of the metabolites or their pharmacokinetic parameters were observed between cattle or buffalo at either dose rate. Pharmacokinetic analysis and the disposition curve of the metabolites indicated increased uptake of the drug in both cattle and buffalo when the same total amount of the drug was given in divided doses compared to a single dose (p < 0.05). The divided dose schedules of administration could possibly be exploited to extend the life of the available benzimidazole anthelmintics.

Comparative disposition kinetics of albendazole in sheep following oral and intraruminal administration.

The pharmacokinetics of albendazole was studied in sheep following single oral and intraruminal administration at nematocidal dose rates. The disposition curves of its metabolites indicated increased uptake of the drug in sheep following intraruminal as compared to oral dosing (p < 0.05). The increased bioavailability of benzimidazole anthelmintics given by the intraruminal route could be exploited for optimizing the use of anthelmintic for sustained parasite control in small ruminants.