Multiple anthelmintic resistance in gastrointestinal nematodes of small ruminants in Bangladesh.

Anthelmintic resistance (AR) against gastrointestinal nematodes (GINs) of sheep and goats is a global concern. To address the problem, this study assessed the status of AR in different government and private sheep and goat farms in Bangladesh. We conducted fecal egg count reduction test (FECRT) and Egg hatch assay (EHA) experiments. For the detection of resistant larvae, pooled fecal samples from treated and non-treated groups were subjected to coproculture. Furthermore, 195 adult Haemonchus parasites were genotyped to ascertain benzimidazole (BZ) resistance allele from seven topographic zones of Bangladesh using allele specific PCR (AS-PCR). In FECRT, the percentage reduction along with 95% confidence intervals indicated that GINs were resistant to albendazole (ABZ), levamisole (LEV) and ivermectin (IVM). Coproculture revealed that Haemonchus spp., Oesophagostomum spp. and Trichostrongylus spp. were resistant to anthelmintics. ABZ resistance was also confirmed by in vitro EHA in all the farms except the private goat farm in Mymensingh. The genotype frequencies were 6% for homozygous resistant (rr), 59% for heterozygous (rS) and 35% for homozygous susceptible (SS) among different topographic zones. The allelic frequency of the mutation conferring resistance (r) ranged from 25% to 47% signifying resistance to BZ in nematodes of sheep/goats. The genotype frequencies (rr, rS and SS) and allelic frequencies (r and S) varied significantly (p˂0.05) in different zones in Bangladesh. Overall, the data suggest an alarming condition created by multiple AR in Bangladesh.

First detection of ivermectin resistance in oesophagostomum dentatum in pigs.

In 2016 suspected reduced ivermectin (IVM) efficacy in Oesophagostomum species in pigs was reported in England. Following this initial report, APHA raised awareness amongst private pig veterinary practitioners of the need to monitor the efficacy of the worm control on pig units. In 2017 another veterinary practitioner highlighted a potential in-field lack of IVM efficacy in treating Oesophagostomum species in sows on another breeder-finisher unit. In this trial, the efficacy of IVM against Oesophagostomum species worms has been investigated to determine whether suspected reduced efficacy (52% reduction in mean faecal egg count 14 days post ivermectin administration) on a mixed indoor and outdoor breeder-finisher pig farm in England reflected true IVM resistance under controlled experimental conditions. On days 0 and 40 of the trial, twenty helminth-naive pigs were artificially infected per os with 5000 Oesophagostomum L3 obtained from the farm under investigation. The pigs were allocated to treatment or control groups (n = 10 per group). Treatment group pigs received IVM (0.3 mg kg body weight) by sub-cutaneous injection as per manufacturer's instructions on day 44. Control group animals were left untreated. Faecal worm egg counts were monitored throughout the trial from day 15 post infection to determine time to patency. On day 50 all pigs were euthanased to assess the worm burdens. Resistance to IVM was confirmed in Oesophagostomum dentatum based on the results of a faecal egg count reduction test (FECRT) and a controlled efficacy test (CET). Efficacy based on mean reduction in faecal egg count of IVM-treated pigs compared to untreated control pigs was 86%. Mean reduction in IVM-treated pig worm burdens was 5% against an adult worm population and 94% against an L3/L4 population. The apparent discrepancy between FECRT and CET efficacy results appears to be due to egg development and/or oviposition suppression in IVM-treated female worms. The detection of IVM resistance in Oesophagostomum species worms for the first time in UK pigs is particularly important considering the global situation where resistance to pyrantel, levamisole and benzimidazole anthelmintics in Oesophagostomum species in pigs have already been reported. The results also provide an opportunity to discuss the wider issue of anthelmintic usage and efficacy on pig farms and highlight the need for wider surveillance for the occurrence of anthelmintic resistance in pigs.

Menthol acts as a positive allosteric modulator on nematode levamisole sensitive nicotinic acetylcholine receptors.

The ongoing and widespread emergence of resistance to the existing anti-nematodal pharmacopeia has made it imperative to develop new anthelminthic agents. Historically, plants have been important sources of therapeutic compounds and offer an alternative to synthetic drugs. Monoterpenoids are phytochemicals that have been shown to produce acute toxic effects in insects and nematodes. Previous studies have shown nicotinic acetylcholine receptors (nAChRs) to be possible targets for naturally occurring plant metabolites such as carvacrol and carveol. In this study we examined the effects of monoterpenoid compounds on a levamisole sensitive nAChR from Oesophagostomum dentatum and a nicotine sensitive nAChR from Ascaris suum. We expressed the receptors in Xenopus laevis oocytes and used two-electrode voltage-clamp to characterize the effect of various compounds on these cys-loop receptors. At 100 µM the majority of these compounds acted as antagonists. Interestingly, further experiments revealed that both 0.1 µM and 10 µM menthol potentiated acetylcholine and levamisole responses in the levamisole sensitive receptor but not the nicotine sensitive receptor. We also investigated the effects of 0.1 µM menthol on the contractility of A. suum somatic muscle strips. Menthol produced significant potentiation of peak contractions at each concentration of acetylcholine. The positive allosteric modulatory effects of menthol in both in vivo and in vitro experiments suggests menthol as a promising candidate for combination therapy with cholinergic anthelmintics.

Economic viability of anthelmintic treatment in naturally infected beef cattle under different nutritional strategies after weaning.

The aim of the present study was to evaluate the effects of treatment with different anthelmintic compounds on the productivity of naturally infected calves and the economic viability of these treatments within extensive breeding systems employing different nutritional strategies after weaning. For this purpose, 4 farms with 42-60 calves naturally infected with gastrointestinal nematodes were selected. The calves were distributed into 6 groups (7-10 animals each) per farm and treated with ivermectin 1%, ivermectin 3.15%, eprinomectin 5%, levamisole 7.5%, albendazole 15%, and control group (no treatment). These animals were evaluated over an experimental period of 150 days. Levamisole 7.5% presented the best capacity for the reduction of eggs per gram (EPG) of feces in all herds evaluated, followed by albendazole 15% and eprinomectin 5%. Parasite resistance to multiple drugs was found in all herds, especially those of Cooperia, Haemonchus, Oesophagostomum, and Trichostrongylus. For farm 1, differences in weight gain and EPG reduction percentages led to a difference of US$285.06 between the levamisole and ivermectin 3.15% groups. Similar findings were noted for the levamisole and ivermectin 1% groups of farm 3, with a difference of US$399.37 because of the final weight gain in these groups. For farms 2 and 4, the ivermectin 3.15% and control groups, respectively, were the most profitable; these unexpected results were possibly influenced by variables not measured during the experimental period. This study suggested that anthelmintic treatments should always precede an efficacy test, once they are demonstrated to be most profitable under adequate breeding conditions, to ensure adequate control of gastrointestinal nematode infection.

Lack of efficacy of monepantel against trichostrongyle nematodes in a UK sheep flock.

Monepantel resistance was diagnosed during routine monitoring of the effectiveness of a farm's roundworm control strategy. Weaned lambs had become ill thrifty and developed diarrhoea, despite the routine use of monepantel. This clinical presentation was caused by trichostrongylosis. The faecal egg count reduction was 76.7% (95% CI: 55.1-82.2%) following treatment with 2.5 mg/kg monepantel. Predominantly Trichostrongylus vitrinus along with small proportions of Oesophagostomum venulosum and Trichostrongylus vitrinus were identified by deep amplicon sequencing of pools of larvae recovered from pre and post monepantel treatment coprocultures and on postmortem examinations. The undifferentiated FECRT showed resistance to monepantel, but not to levamisole, ivermectin, or moxidectin. Examination of farm anthelmintic treatment and animal movement records suggested that treatments before movement onto silage aftermaths, putatively with low numbers of susceptible nematodes in refugia, may have placed a high selection pressure on monepantel resistance. Effective control of parasitic gastroenteritis using anthelmintic drugs is a prerequisite for sustainable sheep production. This case reiterates the need for care when combining anthelmintic treatments with movements to safe grazing, and the value of monitoring of anthelmintic efficacy as part of iterative planned animal health management.

Monepantel is a non-competitive antagonist of nicotinic acetylcholine receptors from Ascaris suum and Oesophagostomum dentatum.

Zolvix® is a recently introduced anthelmintic drench containing monepantel as the active ingredient. Monepantel is a positive allosteric modulator of DEG-3/DES-2 type nicotinic acetylcholine receptors (nAChRs) in several nematode species. The drug has been reported to produce hypercontraction of Caenorhabditis elegans and Haemonchus contortus somatic muscle. We investigated the effects of monepantel on nAChRs from Ascaris suum and Oesophagostomum dentatum heterologously expressed in Xenopus laevis oocytes. Using two-electrode voltage-clamp electrophysiology, we studied the effects of monepantel on a nicotine preferring homomeric nAChR subtype from A. suum comprising of ACR-16; a pyrantel/tribendimidine preferring heteromeric subtype from O. dentatum comprising UNC-29, UNC-38 and UNC-63 subunits; and a levamisole preferring subtype (O. dentatum) comprising UNC-29, UNC-38, UNC-63 and ACR-8 subunits. For each subtype tested, monepantel applied in isolation produced no measurable currents thereby ruling out an agonist action. When monepantel was continuously applied, it reduced the amplitude of acetylcholine induced currents in a concentration-dependent manner. In all three subtypes, monepantel acted as a non-competitive antagonist on the expressed receptors. ACR-16 from A. suum was particularly sensitive to monepantel inhibition (IC50 values: 1.6 ±â€¯3.1 nM and 0.2 ±â€¯2.3 µM). We also investigated the effects of monepantel on muscle flaps isolated from adult A. suum. The drug did not significantly increase baseline tension when applied on its own. As with acetylcholine induced currents in the heterologously expressed receptors, contractions induced by acetylcholine were antagonized by monepantel. Further investigation revealed that the inhibition was a mixture of competitive and non-competitive antagonism. Our findings suggest that monepantel is active on multiple nAChR subtypes.

Anthelmintic activity of chicory (Cichorium intybus): in vitro effects on swine nematodes and relationship to sesquiterpene lactone composition.

Chicory is a perennial crop that has been investigated as a forage source for outdoor-reared ruminants and pigs, and has been reported to have anthelmintic properties. Here, we investigated in vitro anthelmintic effects of forage chicory-extracts against the highly prevalent swine parasites Ascaris suum and Oesophagostomum dentatum. Methanol extracts were prepared and purified from two different cultivars of chicory (Spadona and Puna II). Marked differences were observed between the anthelmintic activity of extracts from the two cultivars. Spadona extracts had potent activity against A. suum third (L3) and fourth (L4) - stage larvae, as well as O. dentatum L4 and adults, whereas Puna II extracts had less activity against A. suum and no activity towards O. dentatum L4. Transmission-electron microscopy of A. suum L4 exposed to Spadona extracts revealed only subtle changes, perhaps indicative of a specific anthelmintic effect rather than generalized toxicity. Ultra-high liquid chromatography-mass spectrometry analysis revealed that the purified extracts were rich in sesquiterpene lactones (SL), and that the SL profile differed significantly between cultivars. This is the first report of anthelmintic activity of forage chicory towards swine nematodes. Our results indicate a significant anthelmintic effect, which may possibly be related to SL composition.

Lack of efficacy of monepantel against Trichostrongylus colubriformis in sheep in Brazil.

Multiple drug resistance of nematodes against anthelmintics has become one of the most important economic problems in sheep production worldwide. The aim of this experiment was to evaluate the efficacy of monepantel (2.5mg/kg) against gastrointestinal nematodes in fecal egg count reduction test (FECRT) and controlled efficacy test (CT) in naturally infected sheep. We used 30 sheep for the FECRT and 20 sheep for the CT, equally divided into control and treated groups. In the FECRT, the reduction was 98%. Larval identification of pre-treatment coprocultures revealed 100% Haemonchus spp. for both control and treated groups. Post-treatment culture of treated sheep was 100% Oesophagostomum spp., but only few larvae were recovered. In the control group, they were 99% Haemonchus spp and 1% Oesophagostomum spp. larvae. Based on the FECRT, Haemonchus spp. was considered susceptible to monepantel. The efficacy of monepantel in the CT against Haemonchus contortus and Trichostrongylus axei was 100% and against Cooperia curticei was 99.7%. For Trichostrongylus colubriformis, the efficacy was -21.5%. In both treated and untreated animals, Oesophagostomum columbianum was recovered from the large intestines. Based on FECRT and CT and in accordance with WAAVP standards, monepantel was ineffective against T. colubriformis and O. columbianum, but effective against H. contortus, T. axei and C. curticei in the studied flock.

Tribendimidine: mode of action and nAChR subtype selectivity in Ascaris and Oesophagostomum.

The cholinergic class of anthelmintic drugs is used for the control of parasitic nematodes. One of this class of drugs, tribendimidine (a symmetrical diamidine derivative, of amidantel), was developed in China for use in humans in the mid-1980s. It has a broader-spectrum anthelmintic action against soil-transmitted helminthiasis than other cholinergic anthelmintics, and is effective against hookworm, pinworms, roundworms, and Strongyloides and flatworm of humans. Although molecular studies on C. elegans suggest that tribendimidine is a cholinergic agonist that is selective for the same nematode muscle nAChR as levamisole, no direct electrophysiological observations in nematode parasites have been made to test this hypothesis. Also the hypothesis that levamisole and tribendimine act on the same receptor, does not explain why tribendimidine is effective against some nematode parasites when levamisole is not. Here we examine the effects of tribendimidine on the electrophysiology and contraction of Ascaris suum body muscle and show that tribendimidine produces depolarization antagonized by the nicotinic antagonist mecamylamine, and that tribendimidine is an agonist of muscle nAChRs of parasitic nematodes. Further pharmacological characterization of the nAChRs activated by tribendimidine in our Ascaris muscle contraction assay shows that tribendimidine is not selective for the same receptor subtypes as levamisole, and that tribendimidine is more selective for the B-subtype than the L-subtype of nAChR. In addition, larval migration inhibition assays with levamisole-resistant Oesophagostomum dentatum isolates show that tribendimidine is as active on a levamisole-resistant isolate as on a levamisole-sensitive isolate, suggesting that the selectivity for levamisole and tribendimidine is not the same. It is concluded that tribendimidine can activate a different population of nematode parasite nAChRs than levamisole, and is more like bephenium. The different nAChR subtype selectivity of tribendimidine may explain why the spectrum of action of tribendimidine is different to that of other cholinergic anthelmintics like levamisole.

Assessment of the anthelmintic activity of medicinal plant extracts and purified condensed tannins against free-living and parasitic stages of Oesophagostomum dentatum.

BACKGROUND: Plant-derived condensed tannins (CT) show promise as a complementary option to treat gastrointestinal helminth infections, thus reducing reliance on synthetic anthelmintic drugs. Most studies on the anthelmintic effects of CT have been conducted on parasites of ruminant livestock. Oesophagostomum dentatum is an economically important parasite of pigs, as well as serving as a useful laboratory model of helminth parasites due to the ability to culture it in vitro for long periods through several life-cycle stages. Here, we investigated the anthelmintic effects of CT on multiple life cycle stages of O. dentatum. METHODS: Extracts and purified fractions were prepared from five plants containing CT and analysed by HPLC-MS. Anthelmintic activity was assessed at five different stages of the O. dentatum life cycle; the development of eggs to infective third-stage larvae (L3), the parasitic L3 stage, the moult from L3 to fourth-stage larvae (L4), the L4 stage and the adult stage. RESULTS: Free-living larvae of O. dentatum were highly susceptible to all five plant extracts. In contrast, only two of the five extracts had activity against L3, as evidenced by migration inhibition assays, whilst three of the five extracts inhibited the moulting of L3 to L4. All five extracts reduced the motility of L4, and the motility of adult worms exposed to a CT-rich extract derived from hazelnut skins was strongly inhibited, with electron microscopy demonstrating direct damage to the worm cuticle and hypodermis. Purified CT fractions retained anthelmintic activity, and depletion of CT from extracts by pre-incubation in polyvinylpolypyrrolidone removed anthelmintic effects, strongly suggesting CT as the active molecules. CONCLUSIONS: These results suggest that CT may have promise as an alternative parasite control option for O. dentatum in pigs, particularly against adult stages. Moreover, our results demonstrate a varied susceptibility of different life-cycle stages of the same parasite to CT, which may offer an insight into the anthelmintic mechanisms of these commonly found plant compounds.

Trichuris suis and Oesophagostomum dentatum show different sensitivity and accumulation of fenbendazole, albendazole and levamisole in vitro.

BACKGROUND: The single-dose benzimidazoles used against Trichuris trichiura infections in humans are not satisfactory. Likewise, the benzimidazole, fenbendazole, has varied efficacy against Trichuris suis whereas Oesophagostomum dentatum is highly sensitive to the drug. The reasons for low treatment efficacy of Trichuris spp. infections are not known. METHODOLOGY: We studied the effect of fenbendazole, albendazole and levamisole on the motility of T. suis and O. dentatum and measured concentrations of the parent drug compounds and metabolites of the benzimidazoles within worms in vitro. The motility and concentrations of drug compounds within worms were compared between species and the maximum specific binding capacity (Bmax) of T. suis and O. dentatum towards the benzimidazoles was estimated. Comparisons of drug uptake in living and killed worms were made for both species. PRINCIPAL FINDINGS: The motility of T. suis was generally less decreased than the motility of O. dentatum when incubated in benzimidazoles, but was more decreased when incubated in levamisole. The Bmax were significantly lower for T. suis (106.6, and 612.7 pmol/mg dry worm tissue) than O. dentatum (395.2, 958.1 pmol/mg dry worm tissue) when incubated for 72 hours in fenbendazole and albendazole respectively. The total drug concentrations (pmol/mg dry worm tissue) were significantly lower within T. suis than O. dentatum whether killed or alive when incubated in all tested drugs (except in living worms exposed to fenbendazole). Relatively high proportions of the anthelmintic inactive metabolite fenbendazole sulphone was measured within T. suis (6-17.2%) as compared to O. dentatum (0.8-0.9%). CONCLUSION/SIGNIFICANCE: The general lower sensitivity of T. suis towards BZs in vitro seems to be related to a lower drug uptake. Furthermore, the relatively high occurrence of fenbendazole sulphone suggests a higher detoxifying capacity of T. suis as compared to O. dentatum.

Transcriptomic evaluation of the nicotinic acetylcholine receptor pathway in levamisole-resistant and -sensitive Oesophagostomum dentatum.

Nematode anthelminthic resistance is widespread for the 3 major drug classes commonly used in agriculture: benzamidazoles, macrocyclic lactones, and nicotinic agonists e.g. levamisole. In parasitic nematodes the genetics of resistance is unknown other than to the benzimidazoles which primarily involve a single gene. In previous work with a levamisole resistant Oesophagostomum dentatum isolate, the nicotinic acetylcholine receptor (nAChR) exhibited decreased levamisole sensitivity. Here, using a transcriptomic approach on the same isolate, we investigate whether that decreased nAChR sensitivity is achieved via a 1-gene mechanism involving 1 of 27 nAChR pathway genes. 3 nAChR receptor subunit genes exhibited ≥2-fold change in transcript abundance: acr-21 and acr-25 increased, and unc-63 decreased. 4 SNPs having a ≥2-fold change in frequency were also identified. These data suggest that resistance is likely polygenic, involving modulated abundance of multiple subunits comprising the heteropentameric nAChR, and is not due to a simple 1-gene mechanism.

Computational cloning of drug target genes of a parasitic nematode, Oesophagostomum dentatum.

BACKGROUND: Gene identification and sequence determination are critical requirements for many biological, genomic, and bioinformatic studies. With the advent of next generation sequencing (NGS) technologies, such determinations are predominantly accomplished in silico for organisms for which the genome is known or for which there exists substantial gene sequence information. Without detailed genomic/gene information, in silico sequence determination is not straightforward, and full coding sequence determination typically involves time- and labor-intensive PCR-based amplification and cloning methods. RESULTS: An improved method was developed with which to determine full length gene coding sequences in silico using de novo assembly of RNA-Seq data. The scheme improves upon initial contigs through contig-to-gene identification by BLAST nearest-neighbor comparison, and through single-contig refinement by iterative-binning and -assembly of reads. Application of the iterative method produced the gene identification and full coding sequence for 9 of 12 genes and improved the sequence of 3 of the 12 genes targeted by benzimidazole, macrocyclic lactone, and nicotinic agonist classes of anthelminthic drugs in the swine nodular parasite Oesophagostomum dentatum. The approach improved upon the initial optimized assembly with Velvet that only identified full coding sequences for 2 genes. CONCLUSIONS: Our reiterative methodology represents a simplified pipeline with which to determine longer gene sequences in silico from next generation sequence data for any nematode for which detailed genetic/gene information is lacking. The method significantly improved upon an initial Velvet assembly of RNA-Seq data that yielded only 2 full length sequences. The identified coding sequences for the 11 target genes enables further future examinations including: (i) the use of recombinant target protein in functional assays seeking a better understanding of the mechanism of drug resistance, and (ii) seeking comparative genomic and transcriptomic assessments between parasite isolates that exhibit varied drug sensitivities.

Proteomic analysis of Oesophagostomum dentatum (Nematoda) during larval transition, and the effects of hydrolase inhibitors on development.

In this study, in vitro drug testing was combined with proteomic and bioinformatic analyses to identify and characterize proteins involved in larval development of Oesophagostomum dentatum, an economically important parasitic nematode. Four hydrolase inhibitors ο-phenanthroline, sodium fluoride, iodoacetamide and 1,2-epoxy-3-(pnitrophenoxy)-propane (EPNP) significantly inhibited (≥90%) larval development. Comparison of the proteomic profiles of the development-inhibited larvae with those of uninhibited control larvae using two-dimensional gel electrophoresis, and subsequent MALDI-TOF mass spectrometric analysis identified a down-regulation of 12 proteins inferred to be involved in various larval developmental processes, including post-embryonic development and growth. Furthermore, three proteins (i.e. intermediate filament protein B, tropomyosin and peptidyl-prolyl cis-trans isomerase) inferred to be involved in the moulting process were down-regulated in moulting- and development-inhibited O. dentatum larvae. This first proteomic map of O. dentatum larvae provides insights in the protein profile of larval development in this parasitic nematode, and significantly improves our understanding of the fundamental biology of its development. The results and the approach used might assist in developing new interventions against parasitic nematodes by blocking or disrupting their key biological pathways.

Oesophagostomum dentatum: effect of glutathione S-transferase (GST) inhibitors on GST activity and larval development.

Sulphobromophthalein (SBP) inhibits isolated glutathione S-transferase of the porcine nodule worm Oesophagostomum dentatum (Od-GST) and reduces larval development in vitro. In this study possible inhibitory effects of various inhibitors were evaluated in an enzymatic (CDNB) assay with isolated Od-GST and in a larval development assay (LDA). Reversibility was tested in the LDA by removing the inhibitor from culture halfway through the cultivation period. SBP, indomethacin and ethacrynic acid inhibited both enzyme activity and larval development in a dose-dependent and reversible manner. HQL-79 also reduced larval development but had only a minor effect on the isolated enzyme. The phospholipase A(2) inhibitors dexamethasone and hydrocortisone had no major effect. High thermal stability of Od-GST was demonstrated with increasing activity between 4 and 50°C. Differences between Od-GST and GST of other organisms indicate structural and possibly functional peculiarities and highlight the potential of such enzymes as targets of intervention.

Microfluidic bioassay to characterize parasitic nematode phenotype and anthelmintic resistance.

With increasing resistance to anti-parasitic drugs, it has become more important to detect and recognize phenotypes of resistant isolates. Molecular methods of detecting resistant isolates are limited at present. Here, we introduce a microfluidic bioassay to measure phenotype using parameters of nematode locomotion. We illustrate the technique on larvae of an animal parasite Oesophagostomum dentatum. Parameters of sinusoidal motion such as propagation velocity, wavelength, wave amplitude, and oscillation frequency depended on the levamisole-sensitivity of the isolate of parasitic nematode. The levamisole-sensitive isolate (SENS) had a mean wave amplitude of 135 µm, which was larger than 123 µm of the levamisole-resistant isolate (LEVR). SENS had a mean wavelength of 373 µm, which was less than 393 µm of LEVR. The mean propagation velocity of SENS, 149 µm s-1, was similar to LEVR, 143 µm s-1. The propagation velocity of the isolates was inhibited by levamisole in a concentration-dependent manner above 0.5 µm. The EC50 for SENS was 3 µm and the EC50 for LEVR was 10 µm. This microfluidic technology advances present-day nematode migration assays and provides a better quantification and increased drug sensitivity. It is anticipated that the bioassay will facilitate study of resistance to other anthelmintic drugs that affect locomotion.

Prostaglandin D(2) synthesis in Oesophagostomum dentatum is mediated by cytosolic glutathione S-transferase.

Glutathione S-transferases (GSTs) of Oesophagostomum dentatum possess considerable similarity to synthetic prostaglandin D synthase (PGDS), and therefore their ability to convert prostaglandin (PG) H(2) to PGD(2)in vitro was investigated with a commercial Prostaglandin D Synthase Inhibitor Screening Assay Kit. Fractioned homogenates of O. dentatum third-stage larvae only displayed cytosolic but not microsomal GST. Both total larval homogenate and isolated GST could metabolise PGH(2) to PGD(2), which could be inhibited by the GST inhibitor sulfobromophthalein (SBP) in a dose-dependent manner, whereas reactions to the specific PGDS inhibitor HQL-79 were not dose-dependent. Inhibition of larval development by SBP in vitro was abolished by the addition of PGD(2) but not by PGH(2), supporting the assumption that GST acts as PGDS and is important for nematode development. Since motility and viability of O. dentatum larvae are reduced in vitro by various inhibitors of eicosanoid metabolism, enzymes of this pathway, including GST, constitute putative intervention targets.

Some aspects of nematode infection in pigs from small herds.

Two traditionally maintained, small herds from southern Poland, with 8 and 12 sows, respectively, were surveyed coprologically during 2006-2007. In one of the herds, while deworming a group of sows with levamisole, faecal samples were collected on Day -7, Day 0 (the day of treatment) and Day 10, in order to assess the therapeutic effect of the drug. Coprological investigation was performed also in 26 fatteners originating from other small farms and slaughtered in a local abattoir, with their intestines washed through for the presence of roundworms. In both herds examined, Ascaris suum and Oesophagostomum spp. were prevalent, whilst Trichuris suis appeared only very rarely. Mainly fatteners, replacement gilts and young sows were highly infected with A. suum. The roundworm occurrence in 2- 3-week-old piglets, with the intensity of 300 eggs per gram of faeces (EPG), indicated the possibility of parasite transmission to offspring very early in age. The highest level of Oesophagostomum spp. infection was observed in sows, but weaners were also much affected. For the group of dewormed sows, the mean faecal egg count reduction (FECR) was estimated to be 77.1- 80.4%, suggesting the presence of resistant nodular worms. A very high false-positive A. suum egg counts found in slaughtered animals (240 to 320 EPG) testified to a high contamination level of the environment of small piggeries, as well. Since the reciprocal transmission of parasites between pigs and poultry might occur, it implies that the flocks should be raised separately.

Highly water-soluble prodrugs of anthelmintic benzimidazole carbamates: synthesis, pharmacodynamics, and pharmacokinetics.

Highly water-soluble prodrugs 1a- g of anthelmintic benzimidazole carbamates 2a- g were synthesized. These prodrugs combine high aqueous solubility and stability with high lability in the presence of alkaline phosphatases. The veterinary utility of 1a was shown by a pharmacodynamic and pharmacokinetic study performed in swine. Comparable anthelmintic efficacy was observed with prodrug 1a or the parent fenbendazole 2a. The pharmacokinetic results showed that 2a is better absorbed when derived from 1a than when applied as such.