嗜线虫真菌Duddingtonia flagrans保存方法的研究

目的探讨嗜线虫真菌Duddingtonia flagrans长期保存以及复苏后生物学活性测定技术。方法分别将菌株置4℃、-25℃和液氮保存,定期进行菌落生长速度、厚垣孢子生成时间以及捕杀绵羊粪便中感染性幼虫活性测定。结果4℃保存15个月、-25℃保存30个月以及液氮保存5年对该菌生长和捕杀感染性幼虫能力无明显影响。结论嗜线虫真菌D.flagrans具有较高的生物学稳定性。     

嗜线虫真菌Duddingtonia flagrans培养基的筛选

2000年新疆畜牧科学院从丹麦皇家农业和兽医大学引进了Duddingtonia flagrans菌株,经过3年多的研究,对该真菌进行了实验室驯化、基础培养、生物学特性和体内外捕杀新疆牛羊胃肠道线虫的效果检测。结果表明该真菌具有与国外研究一致的良好的生物防治效果。嗜线虫真菌D.flagrans经培养后产生大量的厚垣孢子能通过动物的胃肠道不被消化而随粪便一起排出体外,在适宜条件下该厚     

嗜线虫真菌Duddingtonia flagrans防制绵羊胃肠道线虫病

将 30只 4～ 6月龄羊分为 2组 ,分别在各自的围栏草场中放牧。用毛圆线虫和血矛线虫为主的第 3期幼虫对所有羊只人工感染 2次后 ,一组羊按每千克体重 1× 10 6 个的剂量每天饲喂嗜线虫真菌的厚垣孢子制剂 ;另一组羊作对照 ,仅饲喂等量的上述制剂基质——小麦。第 2次人工感染后 8周 ,2组羊粪便 EPG基本相近 ,但与对照组相比较 ,真菌组粪便培养物中感染性幼虫减少了 6 9.7% ;草场草样中感染性幼虫数也较对照组减少 6 7.8%。再把以上 2组羊驱虫后分别放养于上述围栏草场 ,8周后真菌组羊粪便 EPG较对照组减少 87.3% ;草场感染性幼虫也较对照组减少82 .6 %。上述结果显示 ,嗜线虫真菌 Duddingtonia flagrans在绵羊胃肠道寄生性线虫病生物防制中具有良好的应用前景     

嗜线虫真菌Duddingtonia flagrans减少绵羊粪便中线虫感染性幼虫剂量测定

嗜线虫真菌Duddingtonia flagrans是目前发现的一种最具有动物胃肠道线虫病生物防治应用潜力的真菌。为了解该菌捕杀绵羊粪便中感染性幼虫效果与剂量的关系,为今后该制剂应用和质量检验标准的制定提供依据,用不同剂量的厚垣孢子,分别以不经消化道直接加入感染羊粪便,或作添加剂通过饲喂进入消化道后采集直肠粪便,经培养检测感染性幼虫数量的变化。结果表明,嗜线虫真菌Duddingtonia flagrans具有良好的捕杀胃肠道线虫感染性幼虫的生物学特性。以制剂厚垣孢子4×103/g剂量加入感染羊粪便,或以每天5×105/kg体质量的剂量饲喂绵羊,可使粪便培养物中线虫感染性幼虫数减少83.6%~87.5%。     

捕食线虫性真菌Duddingtonia flagrans冻干制剂临床模式

本试验选用耐高温高压的菌种袋作为玉米粒培养基的培养容器,对Duddingtonia flagrans厚壁孢子进行批量培养,然后经孢子洗脱液将孢子洗脱后,将其制备成冻干制剂,并在内蒙古地区的呼和浩特市和林格尔、包头市萨拉齐、呼伦贝尔西旗等地养殖场共165头绵羊中进行了临床应用研究。通过设立不同的试验组和对照组,使用常用驱虫药伊维菌素进行驱虫试验,同时配合口服捕食线虫性真菌Duddingtonia flagrans厚壁孢子,在不同时间进行动物直肠采粪,对粪便进行第三期幼虫培养,然后检测比较不同组别粪便中感染性幼虫的数量,评价捕食线虫性真菌临床应用模式效果。结果显示,将伊维菌素与D.flagrans冻干制剂联合应用于绵羊的寄生性线虫病防治,可使粪便中幼虫数量降低100%,效果优于单独用药组。结果表明,捕食线虫性真菌Duddingtonia flagrans冻干生物制剂与驱虫药物联合使用的临床应用模式,可以取得较好的家畜线虫病临床防控效果,值得进一步在生产实践中进行深入研究和推广。     

用嗜线虫真菌防治家畜胃肠道线虫病的研究进展

简要论述了嗜线虫真菌种类及其捕捉器官和国内外生物防治家畜胃肠道线虫病的研究进展。国内外防治该病主要是用各种驱虫药物定期驱虫，由此造成寄生虫抗药性、动物组织残留药物、环境污染、线虫反复感染等诸多负作用。用线虫天敌嗜线虫真菌生物防治家畜胃肠道线虫病成为该领域研究的重点课题之一。实验室和野外的大量研究结果证明，嗜线虫真菌在通过动物胃肠道时能不被消化，从而保持活性，在粪便中能有效捕食营自由生活阶段的线虫幼虫，使放牧草场中感染性线虫幼虫明显减少，呈现出良好的生物防治效果。文章重点介绍了嗜线虫真菌Duddingtonia flagrans，对用嗜线虫真菌生物防治家畜胃肠道线虫病的前景充满信心。     

嗜线虫真菌捕食绵羊粪便中捻转血矛线虫幼虫的效果观察

采集感染捻转血柔线虫等胃肠道线虫绵羊粪便培养、分离三期幼虫，加入嗜线虫真菌纯培养物中，72h后84％的幼虫被真菌丝绕和捕获；将该真菌培养物与感染捻转向矛线虫等胃肠道线虫的绵羊粪便便混合后共同培养10d，粪便培养物中的三期幼虫减少82％。     

嗜线虫真菌制剂研制及其防治绵羊胃肠道线虫病的效果

用WA或PA琼脂平板制备嗜线虫真菌一级种子,用以小麦为基质加土豆汁的固体培养基或以土豆汁、微量盐为基础的液体培养基制备二级种子。再以上述固体培养基进行扩大培养,26℃培养3-4周,制剂厚垣孢子量可达1×106/g。研究了该制剂不经胃肠道和经胃肠道捕杀幼虫的生物学活性。用1×106/kg体重的剂量饲喂绵羊,对围栏草场中人工感染胃肠道线虫的绵羊进行生物防治临床效果观察,试验组羊克粪便虫卵数较对照组减少87.5%,草场中感染性幼虫减少82.6%,该制剂对绵羊胃肠道线虫病有良好的生物防治效果。     

不同培养条件对捕食性真菌Duddingtonia flagrans生长的影响

为了研究不同生长条件对捕食线虫性真菌Duddingtoniaflagrans生长的影响,本研究将带有D.flagrans菌丝的琼脂块分别接种于不同碳氮比及不同pH值(4.0~8.5)的CMA固体培养基中,在不同温度下(10℃~45℃)进行培养,每天定时观察菌株的发育情况,并测量菌丝生长长度。结果表明,D.flagrans生长最适pH值范围为6.5~7.0,其中以pH 7.0中生长最快;最适温度为35℃;最适碳氮比范围为5∶1~10∶1,其中在碳氮比为5∶1时,生长率最高。菌丝呈辐射状生长,纵横交错,无色,分隔,生长后期有缠绕圈和厚壁孢子的形成。上述结果为进一步研究D.flagrans的生长特性及捕食作用条件奠定了基础。     

捕食性真菌Duddingtonia flagrans原生质体的快速制备与再生

为快速获得大量捕食性真菌Duddingtonia flagrans原生质体并使其再生,本试验研究了酶质量浓度、酶解温度、菌龄、酶解时间对D.flagrans原生质体产生的影响,以确定D.flagrans原生质体最佳快速制备条件。结果表明,在溶壁酶2mL/L、蜗牛酶8g/L、纤维素酶8g/L时,35℃恒温条件下,酶解菌龄为2d的菌丝7h,捕食性真菌D.flagrans产生原生质体数量最多且能够再生。这为下一步转化并标记和克隆捕食性相关基因奠定了重要基础。     

Capability of the nematode-trapping fungus Duddingtonia flagrans to reduce infective larvae of gastrointestinal nematodes in goat feces in the southeastern United States: dose titration and dose time interval studies

Infection with gastrointestinal nematodes, particularly Haemonchus contortus, is a major constraint to goat production in the southeastern United States. Non-anthelmintic control alternatives are needed due to increasing resistance of these nematodes to available anthelmintics. Two studies were completed in Central Georgia in August 1999, and April–May 2000, using Spanish does naturally infected with Haemonchus contortus, Trichostongylus colubriformis, and Cooperia spp. to evaluate effectiveness of nematode-trapping fungi as a biological control agent. In the first experiment, five levels of Duddingtonia flagrans spores were mixed with a complete diet and fed once daily to the does (three per treatment) in metabolism crates. The treatment concentrations were (1) 5×10⁵, (2) 2.5×10⁵, (3) 10⁵, and (4) 5×10⁴ spores per kilogram body weight (BW), and (5) no spores. Fungal spores were fed for the first 7 days of the 14-day trial, and fecal samples were collected daily from individual animals for analysis of fecal egg count and establishment of fecal cultures. Efficacy of the fungus at reducing development of infective larvae (L3) in the fecal cultures was evaluated. The mean reduction in L3 from day 2 of the treatment period until the day after treatment stopped (days 2–8) was 93.6, 80.2, 84.1, and 60.8% for animals given the highest to lowest spore doses, respectively. Within 3–6 days after termination of fungal spore feedings, reduction in L3 development was no longer apparent in any of the treated animals. In a second experiment, effectiveness of 2.5×10⁵ spores of D. flagrans per kilogram BW fed to does every day, every second day, and every third day was evaluated. Reduction in L3 development by daily feeding was less in the second experiment than in the first experiment. Daily fungal spore feeding provided more consistent larval reduction than intermittant feeding (every second or third day). When fed daily under controlled conditions, D. flagrans was effective in significantly reducing development of L3 and appears to be an effective tool for biocontrol of parasitic nematodes in goats.     

不同培养基及诱导物对捕食性真菌Duddingtonia flagrans发酵液中蛋白质的产生及杀虫作用的影响

为研究捕食性真菌Duddingtonia flagrans发酵液中蛋白质的产生及杀虫作用,使用不同营养成分组成的液体培养基及诱导物,对Duddingtonia flagrans进行培养和诱导,测定其发酵液中的蛋白质含量、蛋白酶活性、磷酸酶活性和杀虫作用,进而确定Duddingtonia flagrans产生胞外蛋白质和杀虫作用最适的培养基与诱导物。结果显示,不同培养基及诱导物对Duddingtonia flagrans的代谢过程会产生明显影响,导致其分泌的蛋白质种类、浓度以及蛋白酶和磷酸酶的活性显著不同;Duddingtonia flagrans发酵液不仅具有杀线虫活性,而且有杀蝇蛆作用。上述结果为进一步研究捕食线虫性真菌的杀虫机理提供了重要参考依据。     

Predacious activity of the nematode-trapping fungus Duddingtonia flagrans against cyathostome larvae in faeces after passage through the gastrointestinal tract of horses

This study was undertaken to examine the potential of the nematode-trapping microfungus Duddingtonia flagrans to survive passage through the gastrointestinal tract of horses and subsequently to destroy free-living stages of cyathostomes in faecal cultures. Three different oral dose levels were tested, two horses being used for each level. Faeces were collected twice daily and the number of parasite eggs per gram of faeces were determined. The numbers of infective third stage larvae which developed in faecal cultures were determined after the cultures had been incubated for 2 weeks at 24°C. Results showed a positive relationship between dose level and reduction in the number of infective larvae. Fungi were recovered in faeces at times which corresponded to high larval reduction.     

Effect of biological control through the daily application of spores of Duddingtonia flagrans in lambs kept under an evasive grazing system in the Netherlands

In 2004, an experiment was carried out to evaluate the effect of biological control through feeding spores of Duddingtonia flagrans on parasitic gastroenteritis in lambs, kept under an evasive grazing system. In total 66 lambs were used. Forty naturally infected 3-month old ram lambs were weaned in mid June, and divided into four groups of 10 lambs. On 21 June, G1–G4 were moved to four separate virtually clean plots, they were moved after 4 and 8 weeks to similar plots, and housed after 12 weeks to be necropsied 16 days later. The other 26 lambs had been raised helminth-free, and were used as pairs of tracer lambs. All but one of these pairs, were grazed during the last 2 weeks on each plot. The remaining pair (TA) was grazed during the last 2 weeks on pasture (30 August to 13 September) on the plot that had been grazed by G3 between 19 July and 16 August, to study inhibited development in Haemonchus contortus. All lambs were fed 200 g of concentrates daily throughout the whole period, and those of G1 and G2 were also fed 500,000 spores of D. flagrans/kg bodyweight daily.

The faecal cultures demonstrated a high reduction in yield as a result of fungal application. However, no differences between groups were seen in weight gain, faecal egg counts, pasture larval counts, worm counts and tracer worm counts. H. contortus was the dominant species, and it is obvious that the moves at 4-week intervals prevented the development of severe haemonchosis. This is in particular demonstrated by the much higher worm counts in the two TA tracer lambs grazed. Nevertheless, increases to high faecal egg counts 3 weeks after the first and second moves, indicated acquisition of infection before these moves and at least subclinical haemonchosis. This was supported with the worm counts of lambs and tracer lambs. A higher proportion of inhibited early L4 than in other tracers and than in the permanent lambs were found in the pair of TA tracer lambs. This indicates that moves to new pastures in late summer and autumn delays the onset of inhibition.     

Soil Nematode Populations Beneath Faecal Pats from Grazing Cattle Treated with the Ivermectin Sustained-release Bolus or Fed the Nematophagous Fungus Duddingtonia flagrans to Control Nematode Parasites

The size and composition of the nematode assemblage in soil under faecal pats derived from young cattle treated or untreated with either ivermectin sustained-release boluses, or the nematophagous fungus Duddingtonia flagrans, were studied in each of three years. Soil samples taken 4, 6, 8 and 10 weeks after four deposition dates in 1998 showed significant temporal effects in many taxa and treatment effects in a few genera. In 2000, soil samples taken 10 weeks after deposition in July, August and September showed treatment effects in the plant-associated Tylenchus and Cephalenchus, and the bacterial-feeding Cephalobus 1 and Cephalobus 2 taxa. However, overall it was found that the nematode assemblages were similar below all three types of pat, and the assemblages varied with the season of deposition. D. flagrans, the novel biological control agent being tested against the free-living stages of nematode parasites of cattle, had no detectable impact on the size or the structure of the soil nematode communities under the faecal pats.     

捕食线虫性真菌菌种保存技术的研究

本试验对捕食线虫性真菌菌种保存技术进行了研究,测定了在不同介质保存条件下,捕食线虫真菌分生孢子和菌丝的存活情况及其不是寄生性线虫的能力.结果表明:在设定的保存条件下,分别经过1周冷冻融化或2周反复冷冻融化,被保存的分生孢子接种于玉米粉琼脂培养基上能生长发育:在加入线虫第3期幼虫后,能生产捕食器,对虫体发挥捕食作用.