Characterization and purification of cytosolic and membrane-bound protease(s) in adults of Haemonchus contortus

The activity of protease(s) was observed separately in cytosolic and membrane-bound protease(s) from male and female Hae monchus contortus (Nematoda: Trichostrongylidae). Different fractions showed optimum protease activity at 37 degrees C, pH 8.5 and 8.0 mg casein concentration. The female fractions had a particularly high activity of protease(s) in comparison with the male fractions, especially of membrane-bound enzymes in the anterior half. Inhibition, activation studies revealed the presence of four kinds of protease(s) in cytosolic and membrane-bound fractions. Protease(s) in different fractions are purified to a greater extent by higher concentrations of saturated ammonium sulphate solution, i.e. ranging from 50 to 65%. The purification study revealed the presence of multiple forms of protease(s) in cytosolic and membrane-bound extracts of H. contortus.     

Cholinesterase and Phosphatase Activities in Adults and Infective-stage Larvae of Levamisole-resistant and Levamisole-susceptible Isolates of Haemonchus contortus

Cholinesterase (ChE) and acid phosphatase (AP) activities, but not alkaline phosphatase activities, were detected in cytosolic and membrane-bound fractions of adult and infective-stage larvae of levamisole-resistant and levamisole-susceptible Haemonchus contortus. In contrast to other gastrointestinal nematodes, the ChE activity was higher in L₃ than in adults and, in both cases, was mainly associated with membranes. ChE activity was inhibited by Triton X-100 and was only detected in membrane-bound fractions when the detergent was removed. Differences between resistant and susceptible L₃ were observed in the response to inhibitors (cytosolic fraction) and in the enzymatic content (membrane-bound fraction). Phosphatase activity was detected at acidic pH in all fractions, being higher in the adult than in the L₃ stage. In the former, most of the enzyme was localized in the membrane-bound fractions, whereas in the latter it was mainly in cytosolic fractions. This difference could be correlated with the activity in the gut. In inhibition assays, a difference between cytosolic fractions from resistant and susceptible adults was observed in their response to 1 mmol/L tartaric acid.     

Anthelmintic screening of fractions of Elephantorrhiza elephantina root extract against Haemonchus contortus

In the previous work conducted, it was found that the root extract of Elephantorrhiza elephantina (Bruch.) Skeels. exhibited good anthelmintic efficacy against eggs and larvae of Haemonchus contortus. This study was therefore undertaken to screen fractions of the extracts of E. elephantina for their anthelmintic activity against adult H. contortus using a bioactivity-guided assay. The adult worm bioassay was conducted using aqueous, hexane and ethyl acetate fractions of the plant extracts at concentrations of 0.312, 0.625, 1.25, 2.5, 5 and 10 mg/ml in vitro. Albendazole and distilled water were used as positive and negative control, respectively. In vitro treatment of adult worms revealed concentration and time-dependent efficacy of the fractions of E. elephantina. The aqueous and ethyl acetate fractions showed a highly significant (P < 0.05) motility inhibition at concentrations of 2.5 mg/ml and above after 6 h of exposure, while the hexane fraction showed a significant (P < 0.05) motility inhibition at concentrations of 5 mg/ml and above. After 30 h of exposure, all the fractions showed insignificant (P > 0.05) inhibition of motility. The fractions of E. elephantina also showed mortality indexes that were not significantly different to each other, as well as to the commercial drug (albendazole). Overall, the ethyl acetate and the aqueous fractions were found to possess the highest anthelmintic activity. We therefore recommend future studies to further investigate effective dosages of these fractions in vivo, as well as a full investigation into the compounds responsible for the biological activity.     

Anthelminthic activity of acetone extract and fractions of Vernonia amygdalina against Haemonchus contortus eggs and larvae

The current control of parasitic nematodes in small ruminants relies on the use of chemical anthelminthics, but the development of resistance and the problem of drug residues require research into alternatives. Acetone extract and solvent–solvent fractions of Vernonia amygdalina Del. (Compositae) were evaluated in vitro for potential anti-parasitic effects against the eggs and larvae of Haemonchus contortus. Significant effects were obtained with the extract and fractions but differences were observed depending on the parasitic stage. The acetone extract and fractions inhibited egg hatching and inhibited larval development and killed larvae of H. contortus in a concentration-dependent manner. Best-fit 50% lethal concentration (LC₅₀) values were 957.0, 76.0, 524.0, 309.0 and 224.0 μg/ml for the acetone extract, and the butanol, hexane, chloroform and 35% water in methanol fractions, respectively, when tested against nematode eggs. Best-fit LC₅₀ values for the larval viability test were 508.2, 485.5, 569.3, 348.9 and 196.6 μg/ml for the acetone extract, and the butanol, hexane, chloroform and 35% water in methanol fractions, respectively. The butanol fraction was most active against nematode eggs while the 35% water in methanol fraction was the most active on nematode larvae, although differences in activity between fractions were not significant (p > 0.05). Overall, these in vitro results suggest that V. amygdalina, traditionally used by small farmers in Western Africa, does possess anti-parasitic properties.     

An AC-5 cathepsin B-like protease purified from Haemonchus contortus excretory secretory products shows protective antigen potential for lambs

The immunogenic properties of cysteine proteases obtained from excretory/secretory products (ES) of Haemonchus contortus were investigated with a fraction purified with a recombinant H. contortus cystatin affinity column. The enrichment of H. contortus ES for cysteine protease was confirmed with substrate SDS-PAGE gels since the cystatin-binding fraction activity was three times higher than total ES, despite representing only 3% of total ES. This activity was inhibited by a specific cysteine protease inhibitor (E64) and by recombinant cystatin. The one-dimensional profile of the cystatin-binding fraction displayed a single band with a molecular mass of 43 kDa. Mass spectrometry showed this to be AC-5, a cathepsin B-like cysteine protease which had not been identified in ES products of H. contortus before. The cystatin binding fraction was tested as an immunogen in lambs which were vaccinated three times (week 0, 2.5 and 5), challenged with 10 000 L3 H. contortus (week 6) before necropsy and compared to unvaccinated challenge controls and another group given total ES (n = 10 per group). The group vaccinated with cystatin-binding proteins showed 36% and 32% mean worm burden and eggs per gram of faeces (EPG) reductions, respectively, compared to the controls but total ES was almost without effect. After challenge the cystatin-binding proteins induced significantly higher local and systemic ES specific IgA and IgG responses.     

Effects of age and immune suppression of sheep on fecundity, hatching and larval feeding of different strains of Haemonchus contortus

The effects of host age and immune suppression on abomasal parasitic infection in sheep were investigated following single experimental oral infections with MHco3 (ISE), MHco4 (WRS) and MHco10 (CAVR) strains of Haemonchus contortus in naïve 5-month-old crossbred lambs (n = 1 per group) and 15-month-old Greyface sheep treated with methyl prednisolone acetate (n = 2 per group) or without corticosteroid treatment (n = 2 per group). Adult female H. contortus in 5-month-old lambs (n = 1 per group) shed on average 6.5, 3.1 and 8.0 times more eggs than in 15-month-old sheep (n = 4 per group) following infection with MHco3 (ISE), MHco4 (WRS) and MHco10 (CAVR) strains of H. contortus, respectively, over a period of 28 days following the commencement of patency. There was no obvious effect of age of sheep or corticosteroid treatment on the abomasal establishment of H. contortus or on in vitro assays for egg hatching or larval feeding at different concentrations of anthelmintics, although statistical analysis could not be performed due to the small group sizes.     

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

Abstract

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.     

Changes in hepatic glucose and lipid metabolism‐related parameters in domestic pigeon (Columba livia) during incubation and chick rearing

This study aimed to evaluate the hepatic glucose and lipid metabolism‐related parameters of adult male and female White King pigeons (Columba livia) during incubation and chick rearing. At day 4 (I4), 10 (I10) and 17 (I17) of incubation and day 1 (R1), 7 (R7), 15 (R15) and 25 (R25) of chick rearing, livers were sampled from six pigeons for each sex. Glycogen and fat contents, activities of glycolytic enzymes (hexokinase, HK; 6‐phosphofructokinase, 6‐PFK), and genes expressions of key enzymes involved in glycolysis (pyruvate kinase, PK; glucokinase, GK), gluconeogenesis (phosphoenolpyruvate carboxykinase cytosolic, PCK1; fructose‐1,6‐bisphosphatase, FBP1; glucose‐6‐phosphatase, G6Pase), fatty acid synthesis (fatty acid synthase, FAS; acetyl‐CoA carboxylase, ACC) and fatty acid β‐oxidation (carnitine palmitoyltransferase 1, CPT1; acyl‐CoA 1, ACO) were measured. In male and female pigeon livers, glycogen content and HK activity dramatically increased after I17 and after R1, respectively; expressions of FBP1 and G6Pase genes were maximized at R15; activity of 6‐PFK and expressions of PK and CPT1 genes were highest at R7; fat content and expressions of FAS and ACC genes steeply increased from I10 to R1. In females, hepatic expressions of GK and PCK1 genes were greatest at R7 and I17, respectively; however, in males, both of them were maximized at R15. Hepatic expression of ACO gene was significantly enhanced at R1 compared to I17 and R7 in males, whereas it was notably up‐regulated at I17 and R7 in females. Furthermore, expressions of PCK1, GK, FAS and ACC genes were in significant relation to fat content in the livers of female pigeons, while fat content in male pigeons was highly correlated with expression of PCK1, ACC, CPT1 and ACO genes. In conclusion, regulations of glucose and lipid metabolic processes were enhanced in parent pigeon livers from terminal phases of incubation to mid phase of chick rearing with sexual effects.     

Use of disophenol for the control ofHaemonchus contortus in sheep in west java,Indonesia

Summary The result of a field trial investiguting the anthelmintic effect of disophenol againstH. contortus in sheep raised under Asian small farming practice is reported. It was clearly shown that a single injection of disophenol suppressed the numbers ofH. contortus to very low levels for periods up to 3 months despite the presence of large populations ofH. contortus infective larvae available on pasture and herbage grasses.

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Uso Del Disofenol Para El Control DeHaemonchus Contortus En Ovejas En El Oeste De Java, Indonesia

Resumen Se describen los resultados de un trabajo de campo, en donde se investigó el efecto anti helmíntico del disofenol contra elH. contortus, en ovejas criadas en minifundios.Se demostró claramente que, una sola inyección de disofenol, redujo sensiblemente el número deH. contortus por períodso de 3 meses, a peser del gran número de larves infectivas presentes en el pasto.

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Le Disophenol Dans La Lutte ContreHaemonchus Contrortus Chez Le Mouton A L'est De Java, En Indonesie

Résumé Les résultats obtenus lors d'essais expérimentaux sur l'effet anthelminthique du Disophenol contreH. contortus chez des moutons élevés suivant les formules des petits élevages asiatiques sont rapportés.Il a été clairement démontré qu'une seule injection de Disophenol ramène le nombre deH. contortus à de très bas niveaux pendant une période de 3 mois en dépit de la présence de nombreuses populations de larves infectieuses deH. contortus présentes dans les pâturages et plantations fourragères.

     

The In Vitro Secretion of Acetylcholinesterase by Adult Stages of Heligmosomoides polygyrus: the Effects of Broad‐Spectrum Anthelmintics

The secretion of acetylcholinesterase (AChE) by female and male Heligmosomoides polygyrus was studied in different in vitro culture media. AChE secretion was increased in the presence of fetal calf serum or bovine serum albumin (BSA). In the absence of crowding effects, specific AChE activity in excretion/secretion products was higher for male (2.41 ± 0.07 µmol min^?1 l^?1 mg^?1) than for female (0.56 ± 0.04 µmol min^?1 mg^?1) worms but on a per nematode basis both sexes showed comparable rates of secretion. Acetylthiocholine iodide was the favoured substrate of the enzyme. When the nematodes were incubated in vitro with albendazole (ABZ), ricobendazole (RBZ), mebendazole (MBZ), levamisole (LVM), morantel (MRT) or ivermectin (IVM), at concentrations from 1 mM to 10 nM, in RPMI medium for 2 or 6 h and then transferred to a drug‐free medium (RPMI medium supplemented with 0.5 % BSA) for 24 h or continuously exposed to the drugs in supplement‐free medium (24 h), the concentration‐ and time‐dependent inhibitory effects on AChE secretion were observed. The continued exposure to the drugs for all incubation periods (with a single exception for LVM 1 mM) produced the highest levels of inhibition. Under these conditions, the concentrations inhibiting the secretion of AChE by 50 % (IC₅₀) relative to drug‐free controls were estimated. The IC₅₀ values ranged from 0.012 µM (IVM) to 2.96 µM (MRT). The potential of this bioassay for the selective primary evaluation of new compounds with broad‐spectrum anti‐nematodal activity is discussed.     

The effect of sex on the quality of meat from farmed pheasants (Phasianus colchicus)

The present study aimed to investigate the influence of sex on the quality of meat from farmed pheasants. Male (roosters, n = 18) and female (hens, n = 18) pheasants were slaughtered at 25 weeks of age. The breast muscles (Pectoralis major) were cut out from chilled carcasses and analyzed to determine the proximate chemical composition, fatty acid profile, and physicochemical and sensory properties of meat. Chemical analyses revealed that the meat of male had higher (p ≤ .05) content of collagen, Na, Cu, and Zn, whereas meat from females had higher (p ≤ .05) concentrations of total nitrogen of water‐soluble compounds, nitrogen of water‐soluble non‐protein compounds, K, and Mn. The meat of male was characterized by greater (p ≤ .05) cooking loss. A sensory evaluation revealed that meat from females had lower (p ≤ .05) taste intensity and aroma desirability. Meat from male and female pheasants revealed differences in the content of minerals as well as low‐molecular‐weight water‐soluble nitrogen compounds, which may affect the flavor of cooked meat. Irrespective of these differences, the analyzed pheasant meat was characterized by high quality, which indicates that it could offer an attractive alternative to other types of meat.     

Effect of selenium and vitamin E on oxidative stress in lambs experimentally infected with <Emphasis Type="Italic">Haemonchus contortus</Emphasis>

The objective of this study was to evaluate oxidative stress in lambs experimentally infected with Haemonchus contortus and supplemented with selenium and vitamin E. Twenty male Corriedale lambs were divided into four experimental groups with five animals each: G1 consisted of animals infected and supplemented with 0.2 mg/kg of live weight (LW) sodium selenite by intramuscular injection (IM); G2 consisted of animals infected with larvae and supplemented with 0.2 mg/kg LW sodium selenite IM and 2000 IU per animal of Vitamin E IM; G3 consisted of animals infected with larvae and supplemented with 2000 IU per animal of Vitamin E IM; and G4 consisted of animals infected with larvae. The animals were infected orally with 500 H. contortus larvae (L3) every 48 hours for 20 days. For biochemical analyses and eggs per grams of feces (EPG) evaluation, blood and feces were both collected at zero (T0), 20 (T1), 40 (T2) and 60 (T3) days. The weight of the animals was also measured at these times. Lower TBARS values were observed in the supplemented animals compared to the control group. The groups supplemented with Selenium exhibited blood GSH-Px activity higher than that of non-supplemented animals. Supplementation with selenium provided greater antioxidant protection against oxidative stress generated from experimental infection of lambs with H. contortus.     

The effect of long-term feeding of fresh and ensiled cassava (Manihot esculenta) foliage on gastrointestinal nematode infections in goats

The benefit of long-term feeding of fresh or ensiled cassava foliage on gastrointestinal parasite in goats was evaluated. Eighteen male goats (15.15 ± 2.83kg and between 4–6months) were randomly allocated into three treatments supplemented with 200g of wheat bran head⁻¹ day⁻¹. All groups were fed ad-libitum on either grass (CO), fresh cassava (CaF) or ensiled cassava foliage (CaS). At the beginning of the trial, each goat was inoculated with 3000 L3 containing ≈50% Haemonchus contortus. Individual LWt, FEC and PCV were measured at weekly intervals for 10weeks. At the termination of the experiment all goats were slaughtered for worm recovery and enumeration. The goats in CaF and CaS had similar weight gains while those in CO lost weight (p < 0.05) through the trial. FEC in CaF and CaS were lower (p < 0.05) than CO during the patency of parasite infections, but there was no difference between CaF and CaS goats. PCV of all groups decreased from above 30% to around 25% at the end of the trial. The compositions of established worm burdens were mainly H. contortus (19–40%) and Trichostrongylus colubriformis (55–76%). TWB did not differ among the groups, however, CaS significantly reduced H. contortus burdens, as compared to CaF and CO (p ≤ 0.005). Thus, ensiled cassava foliage reduced the H. contortus population while the fresh foliage only reduced worm fecundity.     

Protein kinase-C activity in phorbol myristate acetate-stimulated neutrophils from newborn and adult cattle.

Neutrophils from newborn calves have been shown to be deficient in ability to generate superoxide anion (O2-) after stimulation of the respiratory burst enzyme with the phorbol ester, phorbol 12-myristate 13-acetate (PMA). This compound activates the O(2-)-generating enzyme of bovine neutrophils through a pathway involving protein kinase C (PKC). To investigate the biochemical basis underlying this functional difference between neutrophils from newborn and adult cattle, we measured and compared the activity of the enzyme PKC in nonstimulated and PMA-stimulated bovine neutrophils. Neutrophils from newborn calves (n = 5) and adult cows (n = 5) were stimulated with various concentrations of PMA (0, 10, 100, and 500 ng/ml) for 3 minutes, and PKC activity was assayed in the cytosolic and the membrane fractions. In nonstimulated cells, most PKC activity was detected in the cytosolic fraction of neutrophils from newborn and adult cattle. Activity of PKC in the cytosol was dependent on the presence of added calcium and phospholipids, whereas membrane-associated PKC in nonstimulated cells did not have such dependence. Significant differences in PKC activity were not observed between newborn and adult cattle in either the cytosolic or the membrane fractions from nonstimulated cells. Stimulation with PMA caused redistribution of PKC activity in the cell (translocation) in newborns and adults, consisting of decrease in cytosolic PKC activity and increase in membrane-associated PKC activity. Similar to that in nonstimulated cells, PKC activity in cytosolic fractions from PMA-stimulated neutrophils was dependent on the presence of cofactors (calcium and phospholipids), whereas PKC activity in the membrane did not have such requirement.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of xenobiotic metabolizing enzymes in bovine small intestinal mucosa

Virkel, G., Carletti, M., Cantiello, M., Della Donna, L., Gardini, G., Girolami, F., Nebbia, C. Characterization of xenobiotic metabolizing enzymes in bovine small intestinal mucosa. J. vet. Pharmacol. Therap. 33 , 295–303. The intestinal mucosa plays a capital role in dictating the bioavailability of a large array of orally ingested drugs and toxicants. The activity and the expression of several xenobiotic metabolizing enzymes were measured in subcellular fractions from the duodenal mucosa of male veal calves and beef cattle displaying a functional rumen but differing in both age (about 8 months vs. 18 to 24 months) and dietary regimens (i.e., milk replacer plus hay and straw vs. corn and concentrated meal). Intestinal microsomes showed cytochrome P450 (CYP) 2B, 2C‐ and 3A‐mediated activities and the presence of the corresponding immunorelated proteins, but no proof of CYP1A expression and/or functions could be provided. Intestinal microsomes were also active in performing reactions typically mediated by carboxylesterases (indophenylacetate hydrolysis), flavin‐containing monooxygenases (methimazole S‐oxidation), and uridindiphosphoglucuronyltransferases (1‐naphthol glucuronidation), respectively. Cytosolic fractions displayed the glutathione S‐transferase (GST)‐dependent conjugation of 1‐chloro‐2,4‐dinitrobenzene; besides, the GST‐mediated conjugation of ethacrinic acid (GSTπ) or cumene hydroperoxide (GSTα) was matched by the presence of the corresponding immunorelated proteins. Conversely, despite the lack of measurable activity with 3,4‐dichloronitrobenzene, a protein cross reacting with anti‐rat GSTμ antibodies could be clearly detected. Although, as detected by densitometry, CYPs and GST isoenzymes tended to be more expressed in beef cattle than in veal calf preparations, there was a general poor correlation with the rate of the in vitro metabolism of the selected diagnostic probes.     

Genotyping of benzimidazole susceptible and resistant alleles in different populations of <Emphasis Type="Italic">Haemonchus contortus</Emphasis> from Himalayan and sub-Himalayan regions of North-West India

An allele-specific PCR was standardized to diagnose the mutation (Phe to Try) at residue 200 of the isotype 1 beta tubulin gene responsible for benzimidazole resistance in Haemonchus contortus adult and infective larvae. Adult and infective larvae of Haemonchus contortus were collected from sheep under different managemental practices (intensive and extensive) in temperate Himalayan regions (Mukteshwar and Kedarkhatta), sub-himalayan region (Pantnagar) of Uttarakhand state and subtropical region of Uttar Pradesh (Bareilly) in north-west India. Genotyping of adult H. contortus, collected from abomasi of slaughtered sheep reared under extensive management, by AS-PCR revealed that the frequency of resistant (r) alleles was significantly higher (P < 0.001) at Pantnagar (0.57) as compared to Bareilly (0.25) and Mukteshwar (0.08). Also, genotyping of infective larvae of the parasite from intensively managed sheep farms indicated that the frequency of resistant (r) alleles was significantly higher (P < 0.001) at Pantnagar (0.85) as compared to Kedarkattha (0.70) and Bareilly (0.62). The results revealed that managemental practices followed in the areas under study have a direct bearing on the spread of benzimidazole resistant alleles.     

Ocular dirofilariosis by Dirofilaria immitis in a dog: first case report from Europe

A five‐year‐old, entire female mixed‐breed dog was presented with corneal oedema and episcleral hyperaemia in the left eye. The ophthalmological examination revealed the presence of a free‐swimming nematode in the anterior chamber. Circulating microfilariae were not observed by a modified Knott test nor were adult antigens detected in serum by a commercial ELISA. The parasite was surgically removed from the dog's eye, but its anterior end was damaged during the surgery. Based on the morphology of the posterior end, the nematode was preliminarily identified as a male Dirofilaria immitis. The species identification was confirmed by PCR amplification and sequencing of the mitochondrial coxI and 12S rDNA genes, using a DNA barcoding approach. Although other cases of ocular dirofilariosis by D. immitis have been previously recorded in Australia and the United States, the case reported herein is the first in a dog from Europ.     

Assessment of the pharmacological interactions between the nematodicidal fenbendazole and the flukicidal triclabendazole: In vitro studies with bovine liver microsomes and slices

Parasitic diseases have a significant impact on livestock production. Nematodicidal drugs, such as fenbendazole (FBZ) or its oxidized metabolite oxfendazole (OFZ), can be used along with the trematodicidal triclabendazole (TCBZ), to broaden the spectrum of anthelmintic activity. However, co‐exposure to these compounds could lead to drug–drug (D‐D) interactions and eventually alter the clinical profile of each active principle. The aim of this study was to assess the presence of such interactions by means of two in vitro models, namely bovine liver microsomal fractions and bovine precision‐cut liver slices (PCLSs). To this end, an in vitro assessment involving incubation of FBZ and TCBZ or a combination of FBZ and TCBZ was carried out. Results with microsomal fractions showed a 78.4% reduction (p = .002) in the rate of OFZ production upon co‐incubation, whereas the sulfoxide metabolite of TCBZ (TCBZSO) exhibited a decreasing tendency. With PCLS, OFZ accumulation in the incubation medium increased 1.8‐fold upon co‐incubation, whereas TCBZSO accumulation decreased by 28%. The accumulation of FBZ and OFZ in the liver tissue increased upon 2‐hr co‐incubation, from 2.1 ± 1.5 to 18.2 ± 6.1 (p = .0009) and from 0.4 ± 0.1 to 1.3 ± 0.3 nmol (p = .0005), respectively. These results confirm the presence of D‐D interactions between FBZ and TCBZ. Further studies are needed to determine the extent of involvement of drug‐metabolizing enzymes and membrane transporters in interactions between compounds largely used in livestock production systems.     

Meat characteristics of Qinghai yak and semi-wild yak

Six domestic yaks aged 6 years, comprising three culling male and three culling female yaks, from the Qinghai Lake area and three male semi‐wild yaks aged 6 months from Datong Yak Farm were used to detect the levels of amino acids, mineral elements, residual heavy metals and pesticides in yak meat. The results showed that there was little difference in amino acid levels for different types of yak by age and sex. The meat of the adult male yak was 7.35 mg/kg higher in calcium (P < 0.05) and 124 mg/kg higher in phosphorus (P < 0.01) than that of the adult female yak. There was an obvious difference in zinc content, and there was also a significant difference for copper and sulfur between adult male and female yaks (P < 0.01). Iron was 8.85 mg/kg higher in semi‐wild yaks than in domestic yaks, but there were no differences for other minerals. The levels of residual heavy metals and residual pesticides were well within those allowed by the National Standard of China. Furthermore, the data showed that introducing wild yak’s blood did not result in a quality change in the domestic yak meat. Yak meat from Qinghai Plateau has the characteristics of being non‐polluted and rich in amino acids in comparison with local yellow cattle meat.     

DNA methylation and expression of imprinted genes are associated with the viability of different sexual cloned buffaloes

The DNA methylation of imprinted genes is an important way to regulate epigenetic reprogramming of donor cells in somatic cell nuclear transfer (SCNT). However, the effects of sexual distinction on the DNA methylation of imprinted genes in cloned animals have seldom been reported. In this study, we analysed the DNA methylation status of three imprinted genes (Xist, IGF2 and H19) from liveborn cloned buffaloes (L group, three female and three male), stillborn cloned buffaloes (S group, three female and three male) and natural reproduction buffaloes (N group, three female and three male), using bisulphite sequencing polymerase chain reaction (BS‐PCR). The expression levels of these imprinted genes were also investigated by quantitative real‐time PCR (QRT‐PCR). The DNA methylation levels of H19 were not significantly different among the groups. However, the Xist in female and IGF2 in male of the S group were found to be significantly hypomethylated in comparison with the same sexual buffaloes in L group and N group (p < .05). Furthermore, the expression levels of Xist, IGF2 and H19 in the stillborn female cloned buffaloes of S group were significantly higher than that of the female buffaloes in the L group and N group (p < .05). The expression levels of IGF2 and H19 in the stillborn male cloned buffaloes in the S group were significantly higher than that of the male buffaloes in the L group and N group (p < .05). These results indicate that Xist may be associated with the viability of female cloned buffaloes, and IGF2 may also be related to the viability of male cloned buffaloes.