Target-based and whole-worm screening approaches to anthelmintic discovery

Experimental approaches for identifying new anthelmintics include target-based and whole-worm screening methods. The former involves basic research into characterising and validating new targets, mostly proteins, followed by identification of inhibitors or agonists through the use of target-based screening assays and/or in silico drug design. The latter experimental approach uses whole-worm assays to identify anthelmintic agents with unknown modes of action, or where the primary interest lies in whether analogues are able to kill (or disable) worms rather than in measuring their direct impact on their likely target. This paper focuses initially on the intestine and external layers of nematodes as potential drug targets. Specific anthelmintic agents targeting either tissue are discussed to illustrate the impact of disruption to these structures. In both cases, the activity of these agents against insects was known, and activity against nematodes was identified using whole worm screening assays. Recent literature identifying ecdysone signalling pathway receptors in nematodes is then used to provide an example of basic research into a specific target that may lead to the development of high-throughput target-based drug screening assays. Finally, the role of whole-worm screening approaches versus target-based screening is discussed briefly.     

Effects of mosapride, a 5-hydroxytryptamine 4 receptor agonist, on electrical activity of the small intestine and cecum in horses

OBJECTIVE: To examine the effects of various doses of mosapride, a 5-hydroxytryptamine 4 receptor agonist, on motility of the small intestine and cecum in horses by use of electrical activity and to determine the dose that provides the optimal response. ANIMAL: 6 healthy adult Thoroughbreds. PROCEDURE: Electrical activity of the small intestine and cecum was recorded before and after mosapride administration by use of an electrogastrograph. Mosapride (0.5, 1, 1.5, and 2 mg/kg) was dissolved in 200 mL of water and administered orally to horses through a nasogastric tube. Three hours after drug administration, mean amplitude of electrical activity calculated for a period of 30 minutes was expressed as the percentage of the mean amplitude of electrical activity for a period of 30 minutes before drug administration. RESULTS: Mosapride administered orally increased the percentage of the mean amplitude of electrical activity in the small intestine and cecum in a dose-dependent manner. Mean +/- SD values differed significantly for 1, 1.5, and 2 mg/kg (127.0 +/- 12.5%, 137.7 +/- 22.2%, and 151.1 +/- 24.0%, respectively) in the small intestine and for 1.5 and 2 mg/kg (130.1 +/- 34.5% and 151.6 +/- 45.2%, respectively) in the cecum. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of results of this study clearly documents that mosapride promotes motility in the small intestine and cecum of horses and that the optimal orally administered dosage is 1.5 to 2 mg/kg. Therefore, mosapride may be useful for treatment of horses with gastrointestinal tract dysfunction.     

33株临床分离菌的药敏试验分析

收集标准菌株2株和临床分离菌株共33株,采用NCCLS推荐的微量稀释法测定6种抗生素对33株病原菌的最低抑菌浓度.33株临床分离菌株对临床上常用的6种抗生素的平均敏感率为27.7%,平均耐药率为64.8%.对金黄色葡萄球菌抗茵作用最强的是氯霉素,敏感率为82.6%,MIC50为2 mg/L,MIC90为64 mg/L;耐药性最强的是青霉素,耐药率达100%.肺炎克雷伯菌的耐药性较为严重,平均耐药率高达80%,抗茵作用最强的为新霉素,但其敏感率仅为40%.33株临床分离菌株的耐药性较为严重,存在交叉耐药性和多重耐药性现象.     

温度对星斑川鲽消化酶活性的影响

通过改变酶反应过程中的温度,分析了星斑川鲽蛋白酶、淀粉酶和脂肪酶的活性。结果表明:胃、肠和肝胰腺蛋白酶活性的最适温度为40,45,45℃;其蛋白酶活性大小顺序为胃肝胰腺肠。胃、肠和肝胰腺淀粉酶活性的最适温度为35,40,35℃;其淀粉酶活性大小顺序为肠肝胰腺胃。胃、肠和肝胰腺脂肪酶活性的最适温度为35,30,30℃;其脂肪酶活性大小顺序为肝胰腺肠胃。     

屠宰猪肠道沙门氏菌的分离鉴定、耐药性分析及致病性试验

为了解广西南宁市猪源沙门氏菌的污染状况、耐药状况及致病力情况,在南宁市某生猪屠宰场随机直接从131头屠宰猪的肠道采集样品,采用鉴别培养基分离,生化鉴定的方法对样品中的沙门氏菌进行分离鉴定,并采用标准K-B纸片法对分离菌株进行25种抗生素敏感试验,最后对分离株进行小白鼠致病性试验。结果从131份屠宰猪的肠道中共分离到沙门氏菌45株,检出率为34.35%;其中鼠伤寒沙门氏菌14株,甲型副伤寒杆菌2株,肠炎沙门氏菌3株。45株分离菌株全部耐药,耐药率高达100%,其中44株为多重耐药菌株,占97.78%。45株沙门氏菌中有40株对小白鼠具有致病性,致病率达88.89%。这表明南宁市的屠宰猪存在一定程度的沙门氏菌污染,并且分离菌株存在较严重的耐药现象以及具有较强的致病性。应采取有效措施控制沙门氏菌在猪群中的污染和限制抗生素在养猪过程中的使用并严格遵守休药期,以减少细菌耐药性的产生,保障猪肉及猪肉制品的食品安全。     

Some factors influencing colicin activity between pathogenic and commensal Escherichia coli from the pig.

Commensal strains of Escherichia coli derived from pigs had a broad spectrum of in vitro colicin acitivity against pathogenic serotypes. Of eight pathogenic serotypes tested, only three were colicinogenic and were active against relatively few commensal strains. Colicin activity was influenced by temperature, pH and oxygen tension as well as by the availability of certain nutrients and the presence of trypsin. Lack of colicin activity in intestinal supernatant fluid was ascribed to the concentration of trypsin present. It was concluded that colicins are unlikely to influence the dominance of pathogenic Escherichia coli serotypes in the pig's intestine, except possibly in the colon and rectum where the concentration of trypsin is low.     

Measurement in vitro of a larval migration inhibitory factor in gastrointestinal mucus of sheep made resistant to the roundworm Trichostrongylus colubriformis

Lambs were challenged by dosing with 2500 T. colubriformis larvae per day for 34 weeks, rested for 4 weeks and then re-challenged with the infective larvae for a further 10 weeks. A technique for the measurement of inhibition of larval migration from agar gels was used to investigate the antiparasitic activity of mucus, obtained from the small intestine and abomasum of the lambs, at the end of the re-challenge period. Measurements were also made on ileal digesta samples collected at different times during the development of the initial resistance, the rest period and the re-challenge period, and on faeces collected during the re-challenge infection. The mucus from the small intestine and abomasum paralysed and inhibited larval migration from agar gels significantly more (P less than 0.01 and P less than 0.05, respectively) than corresponding mucus from parasite-free control animals. This inhibitory activity was also detected (P less than 0.01) in the ileal digesta of infected animals from Week 6 of primary dosing. The magnitude of the inhibition in the ileal digesta increased with time during the infection period and was again detected during re-infection (P less than 0.01). It was not detectable in resistant sheep towards the end of the rest period. Slight inhibitory activity was detected in faeces after 2 weeks of re-infection. These observations suggest that substances secreted into the lumen of the small intestine of infected animals are responsible for resistance against ingested larvae.     

Hepatic and intestinal CYP3A expression and activity in broilers

Cytochrome P450 is involved in drug metabolism. Subfamily CYP3A shows a degree of similarity across different animal species. However, little information is available about its expression and activity in broiler chickens. A RT‐PCR method was developed for the quantification of CYP3A37 expression in the liver and small intestine of broilers. A higher expression in the jejunum was observed compared with that in the ileum. In the liver, a significantly lower expression compared with that in the jejunum was noticed. Thus, the role of the small bowel in drug metabolism cannot be neglected in broilers. CYP3A activity was studied in vitro using midazolam as a substrate. Two protocols for the preparation of intestinal microsomes were compared. Mincing of the tissues before ultracentrifugation seemed to be more appropriate than a protocol based on ethylenediaminetetra‐acetic acid separation. CYP3A activity revealed to be the highest in the duodenum with a decreasing trend towards the ileum. Activity in liver was comparable to duodenal activity.     

In vitro efficacy of an ophthalmic drug combination against corneal pathogens of horses

OBJECTIVE: To evaluate the in vitro efficacy of an ophthalmic drug combination against common corneal pathogens of horses. SAMPLE POPULATION: Representative isolates of 3 bacterial and 2 fungal corneal pathogens of horses. PROCEDURES: Pathogens were subjected to minimum inhibitory concentration (MIC) testing of a drug combination that consisted of equal volumes of natamycin 3.33%, tobramycin 0.3%, cefazolin 5.5%, and equine serum. Proteinase inhibitory activity of the drug combination was assessed by use of a fluorescence microplate assay with gelatin and collagen I as substrates. The MICs of the drug combination were compared with those for each of the component medications and antiproteinase activity of the drug combination was compared with that of serum by use of paired t tests and a 2-way ANOVA, respectively. RESULTS: The drug combination was at least as effective as each medication separately for inhibiting microbial growth of all pathogens tested and was significantly more effective against B-hemolytic Streptococcus spp, Aspergillus spp, and Fusarium spp than the relevant medications separately. Serum and the drug combination both had significant antigelatinase activity, and serum had significant anticollagenase activity. Antiproteinase activity of serum was a concentration-dependent event, which enabled serum to achieve significantly greater activity than the drug combination after 3.5 and 4 hours of incubation [corrected] for the gelatin and collagen I assays, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Drug combinations have the attractive potential of minimizing the time, stress, and fatigue associated with topical treatment regimens consisting of multiple drugs used separately for horses with keratitis.     

Evaluation of praziquantel against induced Nanophyetus salmincola infections in coyotes and dogs

Efficacy of praziquantel against Nanophyetus salmincola, the trematode vector of salmon poisoning disease, was determined in coyotes (n = 29) and dogs (n = 25). The 10- to 14-week-old animals were fed fish or fish kidneys that contained metacercariae of N salmincola. To prevent salmon poisoning disease, all animals were treated with tetracycline on days 8 and 9 after they were fed fish. Ten days after ingestion of infected fish, 19 coyotes and 12 dogs were treated once with praziquantel, administered SC or IM, at dosages between 6.68 and 38.73 mg/kg of body weight. Within 8 days after treatment, the numbers of trematode eggs in feces and trematodes recovered from the small intestine were reduced by greater than 99% when compared with untreated controls. Signs of drug toxicosis were not observed. On the basis of these results, praziquantel at doses approved for use in dogs against cestodes was highly effective against N salmincola in coyotes and dogs.     

Chemotherapy of paramphistomosis in cattle

Controlled tests were used to assess the efficacy of anthelmintics against immature paramphistomes, predominantly Calicophoron calicophorum, in 127 calves which were exposed to contaminated pasture for 7 weeks, treated and slaughtered. When a combination product of oxyclozanide and levamisole was used, oxyclozanide at 18.7 mg/kg reduced parasite numbers in the small intestine, abomasum and rumen-reticulum by 61 to 96.1%, 50.0 to 92.6% and 56.5 to 98.1%, respectively. When 2 doses were given 3 days apart, oxyclozanide was 99.9%, 100% and 100% effective, respectively, in the above organs, and produced improvement in clinically affected calves. This treatment elicited transient diarrhoea. Hexachlorophene at 20 mg/kg as a single dose was 99.5%, 100% and 100% effective against the fluke in the small intestine, abomasum and rumen respectively but severe neurological signs were seen in some calves. Niclosamide at 160 mg/kg given as single or 2 doses 3 days apart was 91.1% and 92.6% effective, respectively, against the parasites in the small intestine. No toxicity was noted. Closantel, at 7.5 mg/kg was not effective. Oxyclozanide and niclosamide when given as a single treatment had varying activity. Two doses of oxyclozanide and a single dose of hexachlorophene gave consistent results. Further tests based on reduction of faecal egg counts, 10 to 14 days after treatment were conducted with oxyclozanide and hexachlorophene against mature paramphistomes in 207 cattle. Oxyclozanide as a single dose or 2 doses 3 days apart at 12.8 to 18.7 mg/kg was 93.6 to 97.5% effective in reducing egg counts. Hexachlorophene at 20 mg/kg was 83.0% effective.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional analysis and induction of four novel goose (Anser cygnoides) avian β-defensins in response to salmonella enteritidis infection

In the current study, four novel avian β-defensins (AvBDs) (AvBD2, 5, 9, and 10) were identified in tissues from the Chinese goose (Anser cygnoides). The antibacterial activity of the AvBDs showed that all of these AvBDs exhibited antibacterial activity against most of the bacteria investigated (P<0.01). In addition, antibacterial activity of all of the AvBDs against Staphylococcus aureus and Pasteurella multocida decreased significantly or was completely abolished at 150mM NaCl (P<0.01). None of the AvBDs showed hemolytic activity. AvBD2 and AvBD10 were expressed widely, whereas AvBD5 and AvBD9 mRNAs were expressed in a limited number of geese tissues. AvBD9 was significantly induced in some immune tissues from geese after Salmonella enteritidis infection. The others were significantly upregulated in small intestine and some immune tissues of the geese (P<0.01). The present results suggest that the AvBDs are part of the host defense mechanism of the goose.     

马蹄香镇痛作用的研究及对肠蠕动的影响

为研究马蹄香对小鼠的镇痛作用及对小鼠肠蠕动的影响,通过小鼠醋酸扭体法和小鼠热板法研究马蹄香的镇痛作用,通过小鼠碳末推进法研究马蹄香对肠蠕动的影响。在验证外周镇痛作用的扭体试验和验证中枢镇痛作用的热板试验中,马蹄香药物组和阿司匹林阳性药物组均与空白对照组存在显著差异;在碳末推进试验中,马蹄香药物组和多潘立酮阳性药物组均与空白对照组存在显著差异。结果表明,马蹄香具有外周和中枢镇痛药物活性,并能够有效促进肠蠕动。     

The role of absorbed drug in the efficacy of oxfendazole against gastrointestinal nematodes

Comparisons were made of the relative efficacy of ozfendazole (OFZ), administered to sheep at 5 mg/kg either as an oral drench, single intravenous injection or 12 and 24 divided intravenous injections over 24 and 48 hours, against benzimidazole-resistanthaemonchus contortus andTrichostrongylus colubriformis. A single intravenous injection was at least equally potent as the oral drench whilst the divided dose intravenous regimes significantly increased OFZ efficacy against both parasite species.These findings demonstrate that (i) absorbed drug is important for the efficacy of OFZ against nematodes in the abomasum and small intestine and may be more important than unabsorbed drug passing down the gastrointestinal tract, and (ii) the maintenance of plasma OFZ levels of approximately 2 g/ml by divided dose regime increased efficacy compared with that achieved with the same total dose given as a single administration.     

Pathophysiology of equine postoperative ileus: effect of adrenergic blockade, parasympathetic stimulation and metoclopramide in an experimental model

An experimental model of postoperative ileus was developed in ponies using trauma to, and exposure of, a length of small intestine which gave rise to a reproducible and reversible set of changes in gut activity. This was assessed by recordings of electrical and mechanical activity and by propulsion of spheres from stomach to anus. Activity was depressed, especially in the stomach and colon, and transit was slowed. All drugs given increased electromechanical activity but propranolol was the least effective and did not alter the delayed transit of spheres. Yohimbine was more effective and the addition of bethanechol produced a little extra propulsive action. Metoclopramide had the best effect, virtually returning transit to normal and was the only drug fully restoring coordination of gastric and small intestinal activity which was disrupted by the ileus procedure. Loss of gastroduodenal coordination is probably the central lesion in equine ileus and may be mediated by dopamine.     

Tissue distribution of 14C-diflubenzuron in Atlantic salmon (Salmo salar)

Diflubenzuron is a potent inhibitor of chitin synthesis, with potential use against salmon lice infestations. The absorption, distribution and elimination of the substance in Atlantic salmon was examined after a single, oral dose of 75 mg/kg bodyweight. The kinetic properties were studied by whole-body autoradiography, liquid scintillation counting and thin layer chromatography, using a 14C-labelled isotope of the substance. The drug was poorly absorbed from the intestine, but reached a concentration of more than 4 micrograms/g in the mucus layer of the skin 2 days after administration. If maintained for several days, this concentration is probably sufficient to control all moulting stages of sea lice in Atlantic salmon. The main route of excretion was via the bile.     

氢溴酸槟榔碱对小鼠小肠推进的时效及量效关系的影响

为将氢溴酸槟榔碱开发为驱畜禽绦虫新药,同时为确定临床用量等提供科学依据,进行了氢溴酸槟榔碱对小白鼠小肠推进率的时效及量效关系的研究。结果显示,小肠推进率随给药剂量增大而增大,表明氢溴酸槟榔碱对小鼠在体肠自发性蠕动有明显的促进作用,且呈剂量依赖性关系。     

Synthesis of citrulline from ornithine by the small intestinal mucosa of cattle

Three segments of cattle small intestine (duodenum, upper jejuno‐ileum and lower jejuno‐ileum) were examined in an in vitro system for activity of ornithine carbamoyltransferase (OCT; EC 2.1.3.3) which is involved in the synthesis of citrulline (Cit) from ornithine (Orn). The mucosa of the three segments of small intestine was collected from Japanese black cattle, homogenized and then centrifuged. The supernatant fraction was used as the crude OCT enzyme solution. The OCT activity was assayed by the production of Cit from Orn determined directly by HPLC. The optimal pH and temperature for OCT activities in the duodenum, upper jejuno‐ileum and lower jejuno‐ileum of cattle small intestine were 7.47 and 39°C. Little difference was observed between the three segments. The OCT activity in cattle kidney was also examined for comparison, and almost no activity was found. The OCT activities in crude enzyme solutions of the three segments of small intestine were stable for up to one month of storage at ?20°C in Tris HCl buffer solution. Finally, the role of the small intestine in supplying Cit as a precursor for arginine synthesis in cattle kidney was discussed.     

1~35日龄羔羊小肠消化酶活性的研究

选取初生体重(3.38±0.33)kg的小尾寒羊公羊56只,分为2组,从4日龄起分别饲喂牛奶粉代乳料或鱼粉代乳料,并在21日龄将原饲喂牛奶粉代乳料和鱼粉代乳料的羔羊各8只转喂开食料,在7、14、21、28和35日龄各屠宰4只,取小肠各段食糜,测定小肠食糜上清液的α-淀粉酶、乳糖酶、胰蛋白酶和糜蛋白酶的酶活性及体增重,以研究不同日龄和不同代乳条件下羔羊小肠消化酶的发育。结果表明,饲喂牛奶粉代乳料时羔羊小肠α-淀粉酶、乳糖酶、胰蛋白酶和糜蛋白酶活性随日龄变化,饲喂牛奶粉代乳料时羔羊日增重平均为157.2 g/(只·d),随日龄变化不显著;饲喂鱼粉代乳料时羔羊小肠的α-淀粉酶、乳糖酶、胰蛋白酶和糜蛋白酶活性在不同日龄均低于饲喂牛奶粉代乳料羔羊,平均分别低43.7%、46.0%、35.2%和10.2%,饲喂鱼粉代乳料时羔羊日增重降低,特别是在21日龄后,提示可能受鱼粉蛋白免疫反应的影响。在21日龄给羔羊转喂开食料,羔羊小肠的α-淀粉酶、乳糖酶、胰蛋白酶和糜蛋白酶活性降低,但之后逐渐恢复,在21日龄前饲喂牛奶粉代乳料的羔羊转喂开食料时α-淀粉酶、乳糖酶和胰蛋白酶活性降低的程度较饲喂鱼粉代乳料的大,但活性仍较高。由本研究得出结论,1~35日龄羔羊小肠α-淀粉酶、胰蛋白酶和糜蛋白酶活性随日龄增加,乳糖酶活性随日龄降低;羔羊饲喂鱼粉代乳料时小肠消化酶活性显著降低;在21日龄转喂开食料后,羔羊出现消化障碍,但较短期内可恢复。     

Myoelectric activity in the intestine of cows with strangulating obstruction of the distal small intestine

Myoelectric activity in 2 cows instrumented with permanent electrodes in the ileum, cecum, proximal loop of the ascending colon (PLAC), and spiral colon was analyzed after an obstruction developed in the distal small intestine. Results were compared with patterns from a group of 7 normal cows. Myoelectric activity in the ileum immediately orad to the occlusion was characterized by abolition of the migrating myoelectric complex (MMC) and a constant pattern of strong spike bursts of long duration. Cyclic activity was present in all parts of the large intestine, and propagation of phase III activity was evident from proximal to distal. A slight degree of disorganization in phase III propagation was restricted to the spiral colon. Activity cycles tended to be shorter in the cecum and PLAC of both cows with colic than in normal cows, and the intensity of spiking activity was generally lower. Changes in duration of the MMC in the spiral colon (bovine colonic MMC, bcMMC) were inconsistent, but the intensity of spiking activity tended to be lower in phases I and II of both cows compared to controls. The organization of phase III in several spindles typical of the bovine spiral colon was not disrupted, but phase IV of the bcMMC occurred only infrequently. Organized cyclic activity occurred in the large intestine of both cows despite complete disruption of the small intestinal MMC, indicating the presence of mechanisms able to initiate and regulate coordinated myoelectric patterns in the large intestine independent of the small intestine.