Spread of Mycobacterium avium subsp. paratuberculosis through soil and grass on a mouflon (Ovis aries) pasture.

The aims of this study were to describe spatial contamination of the environment on a mouflon pasture, as well as to assess the contamination of grass and roots after surface contamination and in depth contamination with feces and buried tissues from animals infected with Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis). Samples of soil, roots, and aerial parts of plants were collected from different locations inside the mouflon pasture, and one control sample site was chosen outside the area where the animals are living. M. a. paratuberculosis DNA was present in all the examined sites and was more often detected in roots than in soil. DNA was detected at up to 80 cm of depth and was spatially more widespread than the initial hypothesis of M. a. paratuberculosis leaching vertically into deeper layers of soil. This study broadens our knowledge of the spread and persistence of M. a. paratuberculosis in an environment with highly infected animals.

Factors affecting pharmacokinetics of benzimidazole anthelmintics in food-producing animals: the consequences and potential risks.

Benzimidazoles are frequently and widely used veterinary anthelmintics. Unfortunately, an administration of these anthelmintics does not always result in the expected therapeutic success. Many host-related factors modify pharmacokinetic behavior and efficacy of a chosen anthelmintic. Pharmacokinetics of anthelmintics varies among animals of different species, sex and age. Also diseases, medication, feed and environmental conditions can significantly affect behavior of anthelmintics and resultant drug efficacy in animals. The presented review gathers information, gained in last 20 years, on factors which bring about the variability in performance of benzimidazole anthelmintics in food-producing animals. It is focused particularly on differences in absorption and metabolism of these anthelmintics as these stages of the pharmacokinetic process seem to be the most important for the overall anthelmintic efficacy. The consequences of abnormalities and alterations in pharmacokinetics of benzimidazole anthelmintics are summarized and discussed.

Flubendazole metabolism and biotransformation enzymes activities in healthy sheep and sheep with haemonchosis.

The aim of this project was to study the influence of haemonchosis, a common parasitic infection of small ruminants caused by Haemonchus contortus, on the activity of biotransformation enzymes and on in vitro flubendazole (FLU) biotransformation in liver and small intestine of lambs (Ovis aries). Twelve lambs were divided into three groups: non-infected animals, animals orally infected with larvae of H. contortus ISE strain for 7 weeks and for 11 weeks. At the end of the experiment, hepatic and intestinal subcellular fractions were prepared and used for assays of biotransformation enzymes activities and FLU metabolism testing. The activities of hepatic cytochromes P450, flavine monooxygenases and carbonyl-reducing enzymes were decreased in infected animals. UDP-glucuronosyl transferase activity was significantly lower (by 35%) in 11 weeks infected animals than that in control animals. When in vitro metabolism of FLU was compared in control and infected animals, significantly lower velocity of FLU reduction was found in infected animals. Slower FLU reduction may be beneficial for the haemonchosis treatment using FLU, because FLU will remain longer in the organism and could cause longer contact of parasites with FLU.

Phase I biotransformation of albendazole in lancet fluke (Dicrocoelium dendriticum).

Dicroceliosis, a lancet fluke infection, is a frequent parasitosis of small ruminants and the anthelmintic drug albendazole (ABZ) is effective in control of this parasitosis. The aim of our project was to study the metabolism of ABZ and ABZ sulphoxide (ABZ.SO) in lancet fluke. Both invitro (subcellular fractions of fluke homogenates) and exvivo experiments (adult flukes cultivated in medium) were performed for this purpose. ABZ was metabolised invitro by lancet fluke NADPH-dependent enzymes by two oxidative steps (sulphoxidation and sulphonation). The apparent kinetic parameters of these reactions have been determined. In the exvivo experiments, only ABZ sulphoxidation was observed. The stereospecificity in ABZ sulphoxidation invitro was slight, with preferential formation of (+)-ABZ.SO enantiomer. In contrast (-)-ABZ.SO formation predominated in exvivo experiments. Sulphoreduction of ABZ.SO occurred neither invivo nor exvivo. The detection of ABZ oxidative metabolites indicates the presence of drug metabolising oxidases in lancet fluke.

Pharmacokinetics of flubendazole and its metabolites in lambs and adult sheep (Ovis aries).

Flubendazole (FLU) is indicated for control of helminthoses in pig and avian species (monogastric animals) and its corresponding pharmacokinetics are well known. The information on FLU's pharmacokinetic behavior in animal species with forestomach (ruminants) has been limited although the use of FLU in these species could be beneficial. The aim of this study was to investigate the pharmacokinetics of FLU and its main metabolites in sheep. The effects of animal age (sexually immature and mature ones) and gender were also studied. FLU was orally administered in a single experimental dose (30 mg/kg of body weight) in the form of oral suspension. Treated immature animals (aged 3 months) and 5 months later the same mature individuals (aged 8 months) were kept under the same conditions (food, water and management) and treated with FLU. Within 72 h after FLU administration, plasmatic samples were collected and FLU and its Phase I metabolites were quantified using high-performance liquid chromatography. FLU was detected in very low concentrations only, reduced FLU (FLU-R) was identified as the main metabolite, and hydrolyzed FLU (FLU-H) as the minor one. Formation of FLU-R was stereospecific with (+)-FLU-R domination. The plasmatic concentrations of (+)-FLU-R reached 10-15 times higher values than those of FLU, (-)-FLU-R and FLU-H. A significant gender effect on pharmacokinetics of FLU or (+)-FLU-R metabolite in the mature animals was found and a wide significant difference between lambs and adult sheep in FLU including both metabolites has been proved.

Biotransformation of flubendazole and selected model xenobiotics in Haemonchus contortus.

Haemonchus contortus is one of the most pathogenic parasites of small ruminants (e.g., sheep and goat). The treatment of haemonchosis is complicated because of frequent resistance of H. contortus to common anthelmintics. The development of resistance can be facilitated by the action of drug metabolizing enzymes of parasites that can deactivate anthelmintics and thus protect parasites against the toxic effect of the drug. The aim of this project was to investigate the Phase I biotransformation of benzimidazole anthelmintic flubendazole in H. contortus and to determine the biotransformation of other model xenobiotics. For this purpose, in vitro (subcellular fractions of H. contortus homogenate) as well as ex vivo (live nematodes cultivated in flasks with medium) experiments were used. The results showed that cytosolic NADPH-dependent enzymes of H. contortus metabolize flubendazole via reduction of its carbonyl group. The apparent kinetic parameters of this reaction were determined (V'max=39.8+/-2.1 nM min(-1), K'm=1.5+/-0.3 microM). The reduction of flubendazole in H. contortus is stereospecific, the ratio of (-):(+) enantiomers of reduced flubendazole formed was 90:10. Reduced flubendazole was the only Phase I metabolite found. Effective reduction of other xenobiotics with carbonyl group (metyrapon, daunorubicin, and oracin) was also found. Significant activity of carbonyl-reducing enzymes may be important for H. contortus to survive the attacks of anthelmintics or other xenobiotics with carbonyl group.

Mycobacterium avium subsp. avium distribution studied in a naturally infected hen flock and in the environment by culture, serotyping and IS901 RFLP methods.

Mycobacterium avium subsp. avium (MAA) of serotype 2 and genotype IS901+ and IS1245+ was cultured from 21 naturally infected hens (Gallus domesticus) from one smallholder aviary. From a total of 330 samples taken from hens, 124 mycobacteria were detected. Out of which MAA was detected in 103 (35.7%) of 288 tissues, in 4 (19.0%) of 21 swabs of cloacae and in 9 (42.9%) of 21 faeces samples, 8 other conditionally pathogenic mycobacterial species were also isolated. Tuberculous (TB) lesions were found in the liver, spleen and intestinal organs of seven hens. The isolates of MAA (n=58) from 16 infected hens (7 with TB lesions and 9 without TB lesions) were found to be of 3 IS901 RFLP types AE (n=48), AD (n=4) and E (n=6), where these MAA isolates are highly virulent to hens. Mixed infections with IS901 RFLP types (AE and AD) and (AE and E) were also evident in seven hens. From a total of 35 examined environmental samples, 23 mycobacterial isolates were detected. Out of which four (17.4%) MAA isolates of IS901 RFLP type AE and 19 (82.6%) other isolates of conditionally pathogenic mycobacteria were detected. The finding of identical IS901 RFLP types from both tissues and faecal isolates confirms that infected domestic hens are the principal source of infection for other susceptible hosts and lead to the contamination of the surrounding environment. The presence of different IS901 RFLP types in tissue isolates may indicate the repeated incidence of MAA infection and the occurrence of polyclonal infection.

Mouflon (Ovis musimon) dicrocoeliosis: effects of parasitosis on the activities of biotransformation enzymes and albendazole metabolism in liver.

Parasitic infections can modify the host's ability to metabolize drugs and other xenobiotics by altering the biotransformation enzymes; these changes may have various pharmacological, toxicological or physiological consequences. In our study, several activities of liver biotransformation enzymes and in vitro metabolism of albendazole (ABZ) were tested and compared in non-infected mouflons (Ovis musimon) and in mouflons infected by lancet fluke (Dicrocoelium dendriticum). Subcellular fractions of liver homogenates were isolated from 5+5 mouflon rams (1-year-old) parasitologically negative or naturally infected by fluke. From the eight enzyme activities that were assayed, only two activities significantly differ in the case of Dicrocoelium-infected versus non-infected animals. In infected mouflons, a significant increase (53%) of thiobenzamide-S-oxidase (TBSO) activity, corresponding mainly to the activity of flavine monooxygenase (FMO), and significant decrease (60%) of glutathione-S-transferase (GST) activity was observed. In addition, dicrocoeliosis caused the enhancement of ABZ hepatic biotransformation. The velocity of the formation of (+)-ABZ sulfoxide and ABZ sulfone was significantly increased. However, the shifts in ABZ biotransformation were very mild that undesirable alterations in ABZ pharmacokinetic are not expected. From this point of view, the use of ABZ in the therapy of mouflon dicrocoeliosis in young animals can be recommended. The treatment of the same mouflons by other drugs that are mainly conjugated with glutathione, seems to be more problematic; hence, all consequences of documented reduced GST activity should be accounted.

Activities of biotransformation enzymes in pheasant (Phasianus colchicus) and their modulation by in vivo administration of mebendazole and flubendazole.

Basal activities of certain pheasant hepatic and intestinal biotransformation enzymes and modulation of their activities by anthelmintics flubendazole (FLBZ) and mebendazole (MBZ) were investigated in subcellular fractions that were prepared from liver and small intestine of control and FLBZ or MBZ treated birds. Several oxidation, reduction and conjugation enzyme activities were assessed. In the liver, treatment of pheasants by FLBZ or MBZ caused very slight or no changes in monooxygenase activities and conjugation enzymes. More significative changes were detected in small intestine. Metyrapone and daunorubicin reductase activities were increased by both substances in the liver. This is the first evidence that certain benzimidazoles modulate reductases of carbonyl group. With respect to the relatively slight extent of the changes caused by FLBZ or MBZ we can assume that repeated administration of therapeutic doses of both FLBZ and MBZ has probably no serious influence on pheasant biotransformation enzyme system.

Detection of Lawsonia intracellularis in wild boar and fallow deer bred in one game enclosure in the Czech Republic.

The prevalence of Lawsonia intracellularis between wild boar (Sus scrofa) and fallow deer (Dama dama) bred in one game reserve was investigated using the nested PCR method. In the study, 88 clinically healthy wild boars of different age categories and two fallow deer bagged in the game reserve were examined. Lawsonia intracellularis was demonstrated in the mucous membrane of the intestine of eight (9.1%) wild boars and one fallow deer. Of the nine wild boar whose tissues of corresponding lymph nodes were examined in addition to the mucous membrane of the ileum, one tested positive for the microorganism. A relationship between the occurrence of L. intracellularis and age of wild boar was demonstrated. Because wild boar and fallow deer are bred together in one game reserve, the possibility of inter-species transfer of L. intracellularis should be borne in mind.

Modulation of porcine biotransformation enzymes by anthelmintic therapy with fenbendazole and flubendazole.

Fenbendazole (FEN) and flubendazole (FLU) are benzimidazole anthelmintics often used in pig management for the control of nematodoses. The in vivo study presented here was designed to test the influence of FLU and FEN on cytochrome P4501A and other cytochrome P450 (CYP) isoforms, UDP-glucuronosyl transferase and several carbonyl reducing enzymes. The results indicated that FEN (in a single therapeutic dose as well as in repeated therapeutic doses) caused significant induction of pig CYP1A, while FLU did not show an inductive effect towards this isoform. Some of the other hepatic and intestinal biotransformation enzymes that were assayed were moderately influenced by FEN or FLU. Strong CYP1A induction following FEN therapy in pigs may negatively affect the efficacy and pharmacokinetics of FEN itself or other simultaneously or consecutively administered drugs. From the perspective of biotransformation enzyme modulation, FLU would appear to be a more convenient anthelmintic therapy of pigs than FEN.

Liver microsomal biotransformation of albendazole in deer, cattle, sheep and pig and some related wild breeds.

Albendazole (ABZ) biotransformation was studied in vitro in liver microsomes of adult noncastrated male farm animals (ram, buck, bull and boar), castrated adult males (wether, billy and hog), and free living males (fallow buck, red deer stag, mouflon ram, roe buck and wild boar). Liver microsomal fractions were incubated with either ABZ or racemic albendazole sulphoxide (ABZSO). ABZ was extensively metabolized to the (+) and (-) enantiomers of ABZSO, whereas ABZSO underwent a slow oxidation to albendazole sulphone (ABZSO2) in all species. In all species both ABZSO enantiomers were detected. The chiral ratio, (+)-ABZSO/(-)-ABZSO, was greater than one in farm animals, mouflon and wild boar, and less than one in three species of deer. For total ABZ sulphoxidation, deer like species had lower values compared to the other species. Mouflon ram and ram had lower total sulphoxidation rates compared to wethers, as well as ABZ suphoxidation towards (+)-ABZSO. No significant difference occurred comparing ABZSO formation in mouflon ram and ram, but ABZSO2 formation rate in mouflon ram was higher than in rams and wethers. Roe deer stag, fallow buck and red deer stag did not differ in both total-ABZSO and (-)-ABZSO synthesis rates and roe deer stag and fallow buck did not differ in synthesis rates of (+)-ABZSO and ABZSO2. The bull differed from other species in all metabolites studied, except for red deer stag and boar in (-)-ABZSO synthesis rate. The extent of ABZSO sulphonation to ABZSO2 in bull microsomes was more than twice that of other species.

The effects of flubendazole and mebendazole on cytochromes P4501A in pheasant hepatocytes.

Many benzimidazoles are known inducers of cytochromes P4501A (CYP1A) in laboratory animals and cell lines. As flubendazole and mebendazole are benzimidazole anthelmintics often used in a pheasant, in the present study an effect of these drugs in primary cultures of pheasant (Phasianus colchicus) hepatocytes was investigated. After 48 h incubation of the hepatocytes with the benzimidazoles (0.2-5 microM), CYP1A activities -- ethoxyresorufin O-deethylation (EROD) and methoxyresorufin O-demethylation (MROD) activities were measured and the CYP1A protein levels were determined by Western blotting. None of the tested benzimidazoles influenced the CYP1A protein content. No pharmacologically significant enhancement of CYP1A after exposure of the hepatocytes to flubendazole and mebendazole was found. Inhibition of the EROD/MROD activities caused by both tested substances was observed only at the highest concentration (5 microM). From a point of view of CYP1A induction or inhibition, the treatment of pheasants by both anthelmintics tested seems to be safe. Our study demonstrates the inter-species differences in CYP1A inducibility and the importance of induction/inhibition studies on target animals.

Albendazole repeated administration induces cytochromes P4501A and accelerates albendazole deactivation in mouflon (Ovis musimon).

Adult mouflon ewes (Ovis musimon) were treated repeatedly with therapeutic doses of albendazole (ABZ, p.o. 7.5 mg/kg of body weight/day, for five consecutive days). Animals (treated or control) were sacrificed 24 h after the fifth dose of ABZ and liver and small intestine were collected to prepare microsomes. The activities of several biotransformation enzymes were measured in both hepatic and intestinal microsomes. A significant increase in the activity and amount of cytochromes P4501A (CYP1A) was observed in both tissues of ABZ treated mouflons compared to control animals. No other biotransformation enzymes tested were affected by five ABZ doses. The in vitro biotransformation of ABZ was studied in hepatic and intestinal microsomes from ABZ treated and control mouflons. Concentrations of two main ABZ metabolites - pharmacologically active ABZ sulfoxide and pharmacologically inactive ABZ sulfone were analysed using HPLC. A significant increase in rate of formation of ABZ sulfone (which is catalysed by CYP1A) was observed in hepatic as well as in intestinal microsomes from ABZ treated animals. The enhancement of ABZ deactivation by its repeated administration may affect the anthelmintic efficacy of this drug and may contribute to the development of parasite resistance.

Paratuberculosis and avian tuberculosis infections in one red deer farm studied by IS900 and IS901 RFLP analysis.

As the attempt to eradicate paratuberculosis in one red deer (Cervus elaphus) farm failed, all 167 red deer of different age groups were slaughtered and examined by culture for mycobacteria, and the farm was closed down. Spleen and hepatic lymph nodes, mediastinal lymph node, ileocecal lymph node, and ileum were collected from each animal and examined (a total of 835 organs). Neither tuberculosis lesions nor pathognomic signs of paratuberculosis were detected. Among all microscopically negative for mycobacteria organs, Mycobacterium avium subsp. paratuberculosis alone was isolated from 165 organs, M. a. avium alone from 41 organs, and both pathogens from four organs. M. a. paratuberculosis alone was detected in 71 red deer, M. a. avium alone in 13 red deer and both pathogens in 18 red deer. Using standardised RFLP methods, three IS900 RFLP types B-C1, B-C16, and B-C32 were identified among 40 M. a. paratuberculosis isolates and four IS901 RFLP types N-B1, N-B3, N-B4, and P-B3 among 17 M. a. avium isolates.

Paratuberculosis in farmed and free-living wild ruminants in the Czech Republic (1999-2001).

Due to the occurrence of the infection of Mycobacterium avium subspecies paratuberculosis among domestic ruminants and the rapid development of farmed deer industry and the market of cloven-hoofed game we have carried surveys of paratuberculosis, beginning in 1997, in the most common four species of wild ruminants in the Czech Republic [Pavlik et al., Vet. Microbiol. 77 (2000) 231-251]. From 1999 the prevalence of paratuberculosis has been slightly reduced in all three types of husbandry of wild ruminants. Nevertheless paratuberculosis has been diagnosed in wild ruminants in three districts, in four game parks and in five farms. M. a. paratuberculosis was isolated from 128 (5.3%) out of 2,403 wild ruminants of four animal species: 106 red deer, 2 roe deer, 4 fallow deer and 16 mouflons. In red deer farms, the highest number of clinical paratuberculosis cases was in yearling deer. RFLP type B-C1 of M. a. paratuberculosis predominated during the second period (1999-2001) in all types of husbandry with no relationship to wild ruminant species. New "cattle" RFLP types B-C5 and B-C16 of M. a. paratuberculosis were described in infected farmed red deer and one "intermediate" RFLP type R-I4 in fallow deer from one game park. The survival of M. a. paratuberculosis was found to be 4 months during winter in the pasture after destocking of all cattle infected with paratuberculosis. We found that non-vertebrates, wild ruminants or non-ruminant wildlife can be vectors and potentially become a risk factor in the spread of M. a. paratuberculosis infection.

The effects of fenbendazole, flubendazole and mebendazole on activities of hepatic cytochromes P450 in pig.

Fenbendazole (FBZ), flubendazole (FLBZ) and mebendazole (MBZ) are benzimidazole anthelmintics widely used in veterinary medicine. The effects of these drugs on cytochromes P450 (CYP) were investigated in primary cultures of swine (Sus scrofa f. domestica) hepatocytes. After 48-h incubation of hepatocytes with benzimidazoles (0.1-2.5 microm), ethoxyresorufin O-deethylation (EROD), benzoxyresorufin O-dearylation (BROD), testosterone hydroxylase (6beta-TOH) and testosterone oxidase (17-TO) activities were measured and the CYP1A and 3A protein levels were determined by Western blotting. FBZ produced a significant, concentration-dependent increase of CYP1A activity (EROD) and protein level. No enhancement of CYP1A was observed after exposure to FLBZ and MBZ. All benzimidazoles tested did not cause any induction of CYP3A (BROD, 6beta-TOH, 17-TO activities and protein content). On the other hand, MBZ produced a significant, concentration-dependent decrease of CYP3A (BROD, 6beta-TOH and 17-TO) activities. Pharmacological and toxicological consequences of CYP1A induction and CYP3A inhibition should be taken into account in treatment of pigs with FBZ and MBZ.

Benzimidazole drugs and modulation of biotransformation enzymes.

Benzimidazole drugs (e.g., anthelmintics albendazole, fenbendazole, oxfenbendazole, thiabendazole, mebendazole; inhibitors of proton pump omeprazole, lansoprasole, pantoprasole) represent substances used in both human and veterinary medicine; however, from the point of view of induction and inhibition of biotransformation enzymes, research has been carried out mainly due to the initiative of human pharmacologists. The purpose of the present review is to inform about inductive and inhibitive effects of benzimidazole drugs in man, animals and cell cultures. Pharmacological and toxicological consequences of modulation of biotransformation enzymes are discussed and the significance of studies in the field of modulation of biotransformation enzymes in food-producing animals is explained. Since the modulating effect of benzimidazoles strongly varies depending on structure of the individual substances, the particular attention is paid to structure-modulation relationships.

Comparison of in vitro activities of biotransformation enzymes in pig, cattle, goat and sheep.

In vitro activities of cytochromes P450 (7-alkyl/aryloxyresorufin dealkyl(aryl)ases, testosterone hydroxylase/oxidase, 6-chlorzoxazone hydroxylase, 7-methoxy-4-trifluoromethyl-coumarin demethylase, and lauric acid hydroxylases), reductases of carbonyl group (toward metyrapone, daunorubicin, glyceraldehyde, and 4-pyridine-carboxaldehyde) and conjugation enzymes (p-nitrophenol-UDP-glucuronosyl transferase, 1-chloro-2,4-dinitrobenzene glutathione-S-tranferase) in young adults, males, non-castrated (N=6) farm animals were studied and compared. Presence of proteins cross-reacting with anti-human CYP3A4, CYP2C9, and CYP2E1 IgG was detected in all farm species. Bovine microsomes differed from other microsomes of farm species in very high 7-ethoxyresorufin-O-deethylase activity (CYP1A1/2). Significantly higher 7-methoxy-4-trifluoromethyl-coumarin demethylase (2-3 times) and 12-lauric acid hydroxylases (4-10 times) activities (probably corresponding to CYP2C and CYP4A, respectively) were found in ovine microsomes. The highest 6beta-testosterone hydroxylase activity, which is usually considered to be a CYP3A activity marker, was found in pig. Reductases of all farm animals display considerable ability to reduce carbonyl group of xenobiotics. Significant differences in level and activity of many biotransformation enzymes tested suggest that extrapolation of pharmacokinetic data obtained in one species to another (even related) could be misleading.

The effects of albendazole and its metabolites on hepatic cytochromes P450 activities in mouflon and rat.

Albendazole (ABZ) is a benzimidazole anthelmintic widely used in veterinary medicine. The effects of ABZ on cytochromes P450 were investigated in primary cultures of mouflon (Ovis musimon) and rat (Rattus norvegicus) hepatocytes. Besides ABZ, its two main metabolites (albendazole-sulphoxide, ABZSO and albendazole-sulphone, ABZSOO) were tested to clarify which compound is responsible for the induction potency of this benzimidazole drug. After 48 h incubation of hepatocytes with benzimidazoles (0.2-25 microM), ethoxyresorufin O-deethylation (EROD) and benzoxyresorufin O-dearylation (BROD) were measured and the P4501A and 3A protein levels were determined by Western blotting. All benzimidazoles provoked a significant increase of EROD and BROD activities in rat hepatocytes. ABZSO and ABZSOO seemed to be responsible for the induction effect of ABZ on P450s in rat. In mouflon, no pharmacologically significant induction of EROD and BROD activities by benzimidazoles tested was observed. From this point of view, anthelmintic therapy of mouflons with ABZ seems to be safe.