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。     

Environmental Impact on Soil Nematodes Following the Use of the Ivermectin Sustained-release Bolus or the Nematophagous Fungus Duddingtonia flagrans to Control Nematode Parasites of Cattle in Sweden

Management of nematode parasites of grazing livestock is essential, but there is concern about the potential environmental risk from agents used in their control. In this experiment, parasites in young cattle were controlled, using ivermectin boluses or the predacious fungus Duddingtonia flagrans , over 3 years. Treatment differences were sought among the soil nematodes recovered from soil samples collected from the paddocks on which treated animals grazed, and compared with similar samples taken from an untreated control paddock. Analysis of the soil nematode fauna on 21 occasions failed to demonstrate any impact of parasite management on soil nematodes. In 0-22 mm soil total nematode abundance averaged 524 000 m -2 , with 18 nominal taxa and H' diversity 2.41. There were both underlying paddock and year-to-year, climate-related, differences. The results of this trial not only confirm the lack of any adverse environmental impact of D. flagrans , a promising biological control agent, on soil nematodes, but also fail to show any impact arising from the use of ivermectin boluses.     

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.     

食线虫性真菌Duddingtonia flagrans F088的生长和产孢特性

【目的】研究食线虫性真菌Duddingtonia flagrans生长特性和不同营养要素对产孢量的影响,为其规模化发酵工艺的确定提供参考。【方法】以D.flagrans F088分离株为研究对象,研究该菌培养过程中pH值、厚垣孢子产量、菌丝干质量等指标的变化;通过单因素试验及正交试验,研究几种常见碳源、氮源、无机盐、维生素和碳氮比对厚垣孢子产量和菌丝平均生长速率的影响。【结果】供试真菌在培养2 d后进入对数生长期,4 d时达到高峰(OD_(600)值为7.46,菌丝干质量为12.07 g/L),而pH快速下降至4.45;3 d开始产生厚垣孢子,4 d时呈爆发式增长。OD_(600)与菌丝干质量、厚垣孢子产量呈极显著正相关,与pH呈极显著负相关。单因素试验结果表明,产生厚垣孢子的最佳碳源为白糖,氮源为大豆蛋白胨,碳氮比为40∶1,维生素为V_(B_1)、无机盐为MgSO_4;菌丝生长较好的碳源为红糖,氮源为大豆蛋白胨,与对照相比,无机盐和维生素对菌丝生长无显著促进作用。4因素3水平正交试验结果显示,D.flagrans F088产生厚垣孢子和菌丝生长的最佳配方组合为:30 g/L白糖、4 g/L大豆蛋白胨、1 g/L MgSO_4和30 mg/L V_(B_1)。【结论】初步明确了D.flagrans F088培养过程中pH、厚垣孢子产量、菌丝干质量等的变化趋势,确定了该菌菌丝平均生长速率和厚垣孢子产量达到最佳时的营养要素和配方组成。     

Implications of Fungicidal Effects of Benzimidazole Compounds on Duddingtonia flagrans in Integrated Nematode Parasite Management in Livestock

An in vitro trial with carbendazim fungicide on the growth profile of the predatory fungus Duddingtonia flagrans was undertaken and in vivo trials in sheep and buffaloes, fed on chlamydospores of D. flagrans and administered albendazole anthelmintic, were conducted. Although no growth inhibition was detected at a carbendazim concentration of 0.05 ppm, growth inhibition was recorded of 50% and above at concentrations of 0.25 and 1.00 ppm (p < 0.001) and of around 90% at concentrations of 2.00 to 5.00 ppm (p <0.0001). Scanty recovery of the fungus was made from faecal culture 48 h following a single dose of albendazole both in sheep and buffaloes. However, profuse fungal recovery was made from 96 h post dosing onwards. When the drug was used as an intraruminal slow-release capsule, no faecal fungal recovery could be made from day 3 after administration of the capsule, when the albendazole sulphoxide concentration was around 1.0 microg/ml. However, profuse and scanty fungal recovery could be made on days 1 and 2, respectively, after administration of the capsule, when the plasma albendazole sulphoxide concentration was around 0.4 and 0.9 microg/ml, respectively. The implications for use of a combination of anthelmintics and biological control in sustainable parasite control programmes are discussed.     

Efficiency of feeding <Emphasis Type="Italic">Duddingtonia flagrans</Emphasis> chlamydospores to control nematode parasites of first-season grazing goats in France

A field trial, conducted over two consecutive years, was aimed at assessing the efficacy of the administration of spores of the nematophagous fungus Duddingtonia flagrans to young goats for the control of nematode parasite infections on a French commercial dairy goat flock. For both years, the first-year grazing kids were divided into two similarly managed groups (fungus and control groups): in 2003 a daily dose rate of 5 × 10⁵ spores/kg body weight was given to the fungus-group animals, while in 2004 a daily dose rate of 10⁶ spores/kg body weight was used; the other half of the kids, acting as control, did not receive the spores. Parameters measured every 3 weeks included nematode egg excretion, larval development in faecal cultures and pasture larval counts. Additionally, at the beginning, the middle and the end of each grazing season, the goats were weighed and blood samples for pepsinogen determination were collected. In 2003, similar results were recorded for all the measured parameters in the control and fungus groups. In contrast, in 2004, the kids receiving the spores showed lower faecal egg counts and pepsinogen levels at the end of the season and higher growth rate compared to kids of the control group.     

Potential environmental consequences of administration of anthelmintics to sheep

Anthelmintics, veterinary medicines for the control of endoparasites, enter into the environment largely through faeces of the treated animals. Sheep dung is a patchily distributed, ephemeral resource, with a functionally important decomposer community. The nature of this community and the pharmacokinetics of anthelmintics in sheep mean that the ecotoxic impacts of these drugs in sheep dung may differ markedly from those in cattle dung, where most research has been focussed. The period of maximum residue excretion is generally more transient in sheep than cattle dung, but low-level excretion may continue for longer, giving the potential for extended sub-lethal effects. Here, the environmental impacts of sheep anthelmintics, as well as alternative endoparasite control methods are reviewed. Impacts are discussed in terms of the potential for residues to enter into the environment, the toxicity and the impact on ecosystem functioning at an appropriate scale. Future research priorities are also discussed; these include the need for studies of the functional contributions of dung-colonising species, as well as the development of higher-tier ecotoxicological methods bridging the gap between laboratory and field experiments. Large-scale and long-term studies, including the development of appropriate models, are necessary to allow the consequences of anthelmintic administration to be assessed, particularly within the remit of sustainable animal production.     

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。     

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。     

Duddingtonia flagrans冻干制剂厚壁孢子优化培养及其杀线虫幼虫剂量研究

【目的】探究捕食线虫性真菌Duddingtonia flagrans冻干制剂厚壁孢子的批量培养方法及其对线虫幼虫的毒杀剂量。【方法】通过玉米粒或大麦粒培养基的单步培养法,以及先在含有0.05%琼脂粉的沙氏葡萄糖肉汤培养基中培养7d后继而转接到玉米粒或大麦粒培养基的双步培养法,以培养3~5周后洗脱的每克固体培养基中的厚壁孢子数为依据,对D.flagrans的最优培养方法进行筛选;然后使用最优培养法培养D.flagrans厚壁孢子并将其冻干后用于体外杀线虫幼虫试验,研究D.flagrans冻干制剂的杀线虫幼虫剂量。【结果】培养基质不同时,D.flagrans的菌落颜色有一定差异,玉米粒培养基中的菌落颜色呈微黄色,而大麦粒培养基中的菌落呈白色;D.flagrans的适宜培养时间为3周。采用单步培养法时,玉米粒培养基培养的厚壁孢子数量为2.4×10~5个/g,大麦粒培养基培养的厚壁孢子数量为3.2×10~5个/g;而采用双步培养法时,玉米粒培养基培养的厚壁孢子数量为3.0×10~5个/g,大麦粒培养基培养的厚壁孢子数量为3.5×10~5个/g。D.flagrans冻干制剂杀线虫幼虫剂量的阈值为每克粪便4×10~5个D.flagrans厚壁孢子,相应对线虫幼虫的杀虫率为95.2%。【结论】采用双步培养法培养厚壁孢子并将其制备成冻干制剂,在D.flagrans生物防治寄生性线虫病方面有较好的应用前景。