Induction of nematode-trapping organs in the predacious fungus Arthrobotrys oligospora (Hyphomycetales) by infective larvae of Ostertagia ostertagi (Trichostrongylidae)

Laboratory experiments were designed to study the influence of temperature, concentrations of nematodes, oxygen tension, light, and nutrient levels, on the induction of nematode-trapping hyphal nets in the predacious fungus Arthrobotrys oligospora. When induced by infective Ostertagia ostertagi larvae, a maximum number of nets was produced at 20 degrees C, at which temperature nets in surplus were produced at larval concentrations up to 1,000 larvae per cm2. A. oligospora did not produce nets in an anaerobic atmosphere containing 21% CO2 (v/v), and net induction was suppressed to a certain degree by exposure to light. The composition of the medium had an important influence on the saprophytic growth and the net-forming capability of A. oligospora as a maximum number of nets was induced at a relatively low concentration of corn meal supporting the relatively sparse mycelium. It was shown that a proportion of trapping nets in A. oligospora maintained their trapping potential for more than 7 weeks when the temperature was below 25 degrees C. Induction of nematode-trapping organs in A. oligospora is discussed in relation to control of infective nematode parasite larvae in cow pats.     

不同氨基酸对少孢节丛孢菌(Arthrobotys oligospora)捕食器诱导效果的研究

试验研究了11种氨基酸在玉米粉琼脂培养基上对捕食线虫性真菌--少孢节丛孢茵(Arthrobotrysoligospora)产生捕食器及对线虫第3期幼虫捕食情况的影响.结果表明:氨基酸可以诱导少孢节丛孢茵产生捕食器,且不同种类氨基酸对其产生捕食器的影响表现不同,各个浓度之间差异明显,其中以浓度为0.05g/L的L-苯丙氨酸(L-Phe)、L-蛋氨酸(L-Met)、DL-缬氨酸(DL-Val)的诱导效果较好:L-苏氨酸(L-Thr)、L-异亮氨酸(L-Leu)、L-亮氨酸(L-IJeu)、L-色氨酸(L-Trp)和L-缬氨酸(L-Val)属于中等;L-赖氨酸(L-Lys)、L-谷氨酸(L-Glu)和L-组氨酸(L-His)表现较差.于茵丝生长初期、中期、后期分别加入氨基酸时,不同时期捕食器的产生差异也较为显著,生长初期和生长中期加入氨基酸的试验组捕食器产生良好;前者又明显好于后者.上述现象提示,少孢节丛孢茵在没有线虫诱导的情况下,加入一定浓度的某些种类氨基酸,同样可以刺激其产生大量捕食器.而且捕食情况表明这些捕食器与线虫诱导刺激产生的捕食器一样,完全可以捕食线虫幼虫.     

Efficacy of an Arthrobotrys oligospora N mutant in nematode-trapping larvae after passage through the digestive tract of sheep

For oral applications, biocontrol of animal parasitic nematodes in ruminants, ion beam implanted nematode-trapping fungi must have the capacity to survive the passage through the digestive tract and be efficient in reducing infective larvae of nematodes in the faeces.Ion beams induced mutation in the spores of nematode-trapping Arthrobotrys oligospora. Mutants with genetic stability were bred. The fungi were cultured in bottles with corn kernels as growth media, and spores of different doses were, respectively, administered orally to each group of sheep naturally infected with gastrointestinal nematodes. The control group did not receive fungi. The faeces of these experimental animals were collected and faecal cultivations carried out. The fungal germination, growth, reproduction and predation of livestock parasitic nematode larvae were tested in laboratory. The efficacy of an A. oligospora N mutant in nematode-trapping larvae after passage through the digestive tract of sheep was tested. The results indicated that the ion beam implantation induced mutation of the nematode-trapping fungi is a positive mutation. The mutant spores through the digestive tract of sheep can kill livestock parasite nematode larvae in vitro. These results indicate the potential of the A. oligospora N mutant as a biological control agent for sheep nematodes.This study showed that such biotechnology could be explored for improving the effectiveness of the use of fungal infections to control livestock parasitic nematodes. This work represents the first application of nematode-trapping fungi in eukaryotic microorganisms.     

Interaction between the nematode-destroying fungus Arthrobotrys robusta (Hyphomycetales) and Haemonchus contortus infective larvae in vitro.

In an in vitro trial, the effect of the nematode-destroying fungus Arthrobotrys robusta on Haemonchus contortus infective larvae was evaluated in petri dishes containing corn meal agar. After seven days incubation at 25 degrees C, 92.33% (+/- 4.1) predation was recorded.     

少孢节丛孢菌的分离鉴定及捕食线虫活性研究

为研究新疆地区捕食线虫性真菌的生物活性,本实验采用玉米粉琼脂培养基(CMA)培养分离自新疆不同地区的捕食线虫性真菌,通过对分离株纯培养物的形态学观察和18S rDNA基因序列分析,鉴定4株少孢节丛孢菌,分别命名为XA3、XA6、XD1和XD4。18S rDNA基因遗传进化分析表明,XA3、XA6、XD1和XD4新疆分离株之间同源性较高,与GenBank登录的少孢节丛孢菌CBS289.82株同源性最高,分别为99.6%、99.5%、99.1%和98.9%,与形态学鉴定结果一致。捕食线虫活性测定试验表明,4个分离株在CMA中的捕食率为92.8%～97.6%;体外对绵羊粪便中线虫幼虫的捕食率为90.7%～95.4%,均具有很强的捕食活性。本研究为研究绵羊消化道线虫生物防控制剂奠定基础。     

The phototoxic effect of erythrosin B on third-stage larvae of gastrointestinal nematodes in sheep.

The phototoxic effect of erythrosin B on the infective third-stage larvae (L3) of naturally acquired mixed populations of ovine gastrointestinal nematodes was investigated. This xanthene dye was phototoxic when administered orally to parasitized lambs or applied directly to feces containing nematode ova. Phototoxicity was assessed by the lack of motility (non-swimming) exhibited by the L3 following their collection by Baermannization from cultured feces and exposure to fluorescent light for 360 min. When lambs were administered erythrosin B orally at dosages of O (control), 40, 60 and 80 mg dye kg-1 body weight daily for 10 consecutive days, the percentages of non-swimming L3 were 16%, 46%, 55% and 62%, respectively. However, erythrosin B phototoxicity did not persist after administration of the dye was discontinued and the percentage of non-swimming L3 declined to a level similar to that of the untreated controls within 2 days. The highest percentage of non-swimming L3 was observed when erythrosin B was added directly to feces containing nematode ova. A dose-response curve was evident from the successively higher percentages of non-swimming L3 with increasing concentrations of erythrosin B. Xanthene dyes have the potential to control parasites acquired by livestock on pasture by inducing a phototoxic reaction in the infective L3.     

少孢节丛孢菌捕食器表面聚合物的诱导与提取方法研究

为研究捕食线虫性真菌——少孢节丛孢菌(Arthrobotrys oligospora)捕食器表面聚合物的生化性质与作用,建立一种高效的表面聚合物诱导与提取方法尤为关键。试验首先用大豆蛋白胨液体培养基在氨基酸诱导作用下,产生大量含捕食器菌丝,然后分别用了5种不同化学性质溶液提取表面聚合物,经透析和浓缩后,测定了其中蛋白质的含量及其对线虫幼虫的毒性作用,同时还进行了提取液对真菌菌丝生长及其捕食作用的研究。通过综合分析得出,用5 mol/L LiCl溶液提取表面聚合物最为有效,其提取的表面聚合物溶液对线虫没有明显的杀线虫活性,但与捕食性真菌黏附捕获线虫过程密切相关;并通过比较源自含捕食器菌丝和普通营养菌丝的表面聚合物物质之间的差别,找到了参与黏附捕获线虫过程的物质,这为今后深入开展对捕食线虫性真菌表面聚合物的研究,进而阐明其作用机理提供了有价值的资料。     

Viability and nematophagous activity of the freeze-dried fungus Arthrobotrys robusta against Ancylostoma spp. infective larvae in dogs

Viability and in vitro and in vivo activities of freeze-dried conidia of the predatory fungus Arthrobotrys robusta (I-31) were evaluated against infective larvae (L(3)) of Ancylostoma spp. in dogs. A. robusta conidia were lyophilized and stored at 4°C for a month. Freeze-dried conidia were diluted to 1×10(3)conidia/ml and tested in vivo. The treated group consisted of a solution containing conidia (1ml) and 1000 Ancylostoma spp. (L(3)) placed on Petri dishes plated with 2% water-agar (2% WA), at 25°C, in the dark for 10 days. The control group consisted of 1000 Ancylostoma spp. L(3), plated on 2% WA. After 10 days, Ancylostoma spp. L(3) from both the treated and the control groups were recovered and counted. The in vivo test was performed on two dogs by administering a single oral dose of freeze-dried conidia (1.5×10(5)) in aqueous solution to one animal and only water to the other. Fecal samples were collected at 12, 24 and 48h after the treatments, plated 2% WA plates and incubated at 25°C for 15 days. A thousand Ancylostoma spp. L(3) larvae were spread on these plates. At day 15, infective L(3) recovered from the treated and control groups were counted. In the in vitro test, A. robusta was able to survive the freeze-drying process, grow in the plates, form traps and capture Ancylostoma spp. L(3). There was a 75.38% decrease in the number of infective larvae recovered from the treated group. The in vivo test showed that freeze-dried A. robusta conidia survived the passage through the gastrointestinal tract of the treated dog, was able to grow in the plates and capture Ancylostoma spp. L(3), reducing the number of recovered L(3) (p<0.01). Freeze-drying can be an alternative method for conservation of conidia of nematophagous fungi.     

少孢节丛孢菌新疆分离株几丁质酶AO-14基因的克隆表达

利用RT-PCR扩增获得少孢节丛孢菌新疆分离株XJ-A1几丁质酶AO-14目的基因片段,对该基因进行克隆和分子特征分析;构建重组表达质粒p ET32a-AO14,转化到大肠杆菌进行诱导表达,用SDS-PAGE和Western blot分析表达的重组蛋白。结果表明,AO-14基因c DNA全长为1 200 bp,编码399个氨基酸;编码蛋白几丁质酶AO-14属于糖苷水解酶18家族,具有典型的几丁质酶催化区保守序列SXGG和DGXDXDWE,氨基酸序列的第131~139为活性位点所在区域,无信号肽序列。SDS-PAGE分析结果显示,AO-14表达产物的分子质量约为60 ku;Western-blot分析结果表明,重组蛋白可以与抗少孢节丛孢菌多克隆抗体发生反应,证实在大肠杆菌中实现了少孢节丛孢菌XJ-A1几丁质酶AO-14基因的表达,为进一步研究少孢节丛孢菌几丁质酶AO-14生物学功能奠定了基础。     

Predatory capability of the nematophagous fungus Arthrobotrys robusta preserved in silica gel on infecting larvae of Haemonchus contortus

Biological control of gastrointestinal nematodiasis in ruminants is an alternative to reduce the number of infective larvae. The fungal isolates predatory activity preservation is a basic requirement for the success of this control type. The aim of this work is to evaluate the predatory capacity of the fungus Arthrobotrys robusta (isolate I-31), preserved on silica gel on infective larvae of Haemonchus contortus under laboratory conditions on 2 % water agar (2 % WA). In this essay, A. robusta storage on silica gel showed successful predatory activity on H. contortus L₃ larvae (p?<?0.01) compared to the control group. Nematophagous fungi were not observed in the control group during the experiment. There was a significant reduction (p?<?0.01) of 73.84 % in the means of H. contortus (L₃) recovered from treatment with isolate I-31 compared to the control without fungi. Results indicate that A. robusta (I-31) could survive stored on silica gel for at least 7 years and keep its predatory activity on H. contortus (L₃).     

少孢节丛孢菌捕食器表面聚合物在捕食线虫过程中的作用研究

为了解捕食性真菌——少孢节丛孢菌捕食器表面聚合物的性质及在捕食线虫过程中的作用,作者首先用氨基酸诱导产生大量捕食器(菌环和菌网)菌丝,然后分别应用5种不同化学性质的溶液、2种酶和7种蛋白酶抑制剂处理捕食器表面聚合物,观察对捕食和致死线虫作用的影响,同时使用上述物质或溶液处理菌丝以确定对菌丝生长的影响。结果表明:5 mol.L-1LiCl溶液和链霉蛋白酶E可明显降低真菌的捕获线虫的能力;0.1 mol.L-1NaOH、SDS-巯基乙醇-尿素PBS溶液、1.5%(w/v)SDS除可抑制捕获虫体外,同时还抑制了真菌菌丝的生长;除菲啰啉外,其余6种酶抑制剂对真菌的黏附捕获作用无明显影响;但其中丝氨酸蛋白酶类抑制剂还可抑制真菌对线虫的致死作用;另外用酶抑制剂处理线虫后,对真菌的黏附捕获作用也无明显影响。上述结果表明少孢节丛孢菌捕食器表面聚合物在黏附捕获线虫过程中起关键作用,其为蛋白质或含蛋白质类物质;并间接证实黏附捕获阶段不需要蛋白酶参与,但其在致死线虫阶段会发挥重要作用。这为阐明捕食性真菌作用机理提供了非常有价值的资料。     

口服少孢节丛孢菌孢子对家畜粪便中线虫幼虫的杀灭研究

对捕食线虫性真菌——少孢节丛孢菌（Arthrobotrys oligospora）菌株进行了临床杀家畜寄生性线虫幼虫作用的研究。结果表明：培养的少孢节丛孢菌分生孢子经口饲喂动物后，对粪便中线虫幼虫的杀虫率分别可达96.3％（羊）、96．5％（牛）和97．4％（马）。说明少孢节丛孢菌菌株分生孢子可通过家畜消化道，并保持了其捕食寄生性线虫的能力。研究结果为捕食线虫性真菌的临床应用奠定了重要基础。     

不同碳氮源及其比例和培养基初始pH值对少孢节丛孢菌菌丝生长的影响

为研究不同碳源、氮源、碳氮比值及培养基初始pH值对少孢节丛孢菌菌丝生长的影响,采用10种碳源和8种氮源物质进行了试验,结果表明:在固体培养基上,少孢节丛孢菌菌丝生长的最适碳源和氮源分别是蔗糖和蛋白胨;在液体培养基内,最适碳源和氮源分别是红薯滤液和硫酸铵.当固体培养基中碳氮比为5:1、液体培养基中碳氮比为10:1时,茵丝生长最快.在初始pH值为3.7时,液体和固体培养基中的菌丝均不能生长;固体培养基中最适合的初始pH值范围是7.0～8.0,以pH值为7.5时菌丝的生长率最大;液体培养基中最适合的初始pH值范围是5.0～7.0,以pH值为7.0时菌丝生长最佳.说明不同的碳源、氮源、碳氮比及培养基初始pH值对少孢节丛孢菌菌丝生长率的影响是不同的.而且固体培养基和液体培养基中最适碳氮源、碳氮比、初始pH值也存在差异.     

Vertical migratory behavior of the infective third-stage larvae of Oesophagostomum dentatum

The vertical migratory behavior of third-stage infective larvae (L3i) of Oesophagostomum dentatum was investigated using upright truncated agarose cones and equivalent conical depressions in agarose. Geotactic response varied with the age of the infective larvae. Four-day-old L3i showed no preference for the sloping surfaces of either indented or upright cones, while the 8-day-old L3i showed a positive geotactic reaction, migrating down the sloping surface of the depressions.     

Protection of lambs with a purified metabolite of exsheathed third-stage Haemonchus contortus larvae

Helminth-free lambs (approx 4 months old) were inoculated SC with a purified metabolite of exsheathed third-stage Haemonchus contortus larvae. The metabolite was obtained from in vitro cultivation and was identified as XL3FA1. After 3 periodic XL3FA1 inoculations, lambs were challenge exposed with H contortus. Multiple vaccinations with XL3FA1 did not inhibit the development of worm populations in the lambs; however, worm egg production seemed to be inhibited.     

Cryopreservation of the infective larvae of the common nematodes of ruminants

Exsheathed infective larvae (L 3) of 19 species of nematodes were tested for infectivity in either sheep or cattle after they had been frozen in 0,9% NaCl solution, stored for a relatively short time in the gas phase of liquid nitrogen and subsequently thawed. In addition, 13 of these species were tested after similar storage for up to 18 months. In sheep, Haemonchus contortus, Ostertagia circumcincta, Trichostrongylus axei, Trichostrongylus colubriformis, Nematodirus spathiger and Oesophagostomum columbianum were viable after 2 years of cryopreservation, a mean of greater than 90% of the L 3 being alive when thawed after this period. Similar results were obtained with Chabertia ovina L 3 after 18 months and with Marshallagia marshalli, Trichostrongylus falculatus and Dictyocaulus filaria, after a short period of freezing. On the other hand, Gaigeria pachyscelis and Strongyloides papillosus survived freezing for up to 7 months but neither was viable at the end of this period, nor was exsheathed G. pachyscelis viable without freezing. Most of these infestations were established by inoculating the infective larvae into the abomasum and/or duodenum. M. marshalli, T. falculatus and C. ovina also proved infective after oral dosing. D. filaria, the only other species tested by this route, was not infective when dosed per os after thawing. The infective larvae of the bovine nematodes, Haemonchus placei, Ostertagia ostertagi, Nematodirus helvetianus, Oesophagostomum radiatum, Cooperia pectinata and Cooperia punctata survived freezing for a mean of 26 months, greater than 90% being alive on thawing, but infectivity was generally lower than with the same genera in sheep. Even when not frozen, exsheathed Bunostomum phlebotomum was non-infective. When Cooperia spp. after thawing were tested for infectivity by the oral route, more worms developed in one calf infested orally than in another infested by inoculation into the duodenum. Ova of H. contortus, M. marshalli, O. circumcincta, T. colubriformis, T. falculatus, N. spathiger, C. ovina, H. placei, O. ostertagi, Cooperia spp. and N. helvetianus were recovered from the faeces of animals infested with cryopreserved L 3. No ova of O. columbianum or O. radiatum were recovered from faeces, because differential larval counts were performed before they were patent. Nevertheless, gravid females were obtained post-mortem. Frozen L 3 of N. helvetianus were used to re-establish a pure strain in calves, 2,3 million ova being recovered from infestations with 10 670 L 3 frozen for 26 months. The infectivity of the progeny of frozen L 3 was tested with M. marshalli and C. ovina. In both instances infectivity was high and the worms which developed also produced ova, thus completing the cycle. This appears to be the first report of infective larvae of parasitic nematodes retaining their infectivity after being frozen in liquid nitrogen (gas phase) for longer than 2 years. This is also apparently the first time that M. marshalli T. colubriformis, T. falculatus, T. axei, N. spathiger, C...     

Comparison of two techniques used for the recovery of third-stage strongylid nematode larvae from herbage

A laboratory trial to determine the efficacy of two methods in recovering known numbers of third-stage (L3) strongylid nematode larvae from herbage was carried out. Herbage samples consisting almost entirely of star grass (Cynodon aethiopicus) that had no L3 nematode parasitic larvae were collected at Onderstepoort, South Africa. Two hundred grams samples were placed in fibreglass fly gauze bags and seeded with third-stage strongylid nematode larvae at 11 different levels of herbage infectivity ranging from 50 to 8000 L3/kg. Eight replicates were prepared for each of the 11 levels of herbage infectivity. Four of these were processed using a modified automatic Speed Queen heavy-duty washing machine at a regular normal cycle, followed by isolation of larvae through centrifugation-flotation in saturated sugar solution. Larvae in the other four samples were recovered after soaking the herbage in water overnight and the larvae isolated with the Baermann technique of the washing. There was a strong correlation between the number of larvae recovered using both methods and the number of larvae in the seeded samples, indicating that the two methods give a good indication of changes in the numbers of larvae on pasture if applied in epidemiological studies. The washing machine method recovered higher numbers of larvae than the soaking and Baermann method at all levels of pasture seeding, probably because the machine washed the samples more thoroughly and a sugar centrifugation-flotation step was used. Larval suspensions obtained using the washing machine method were therefore cleaner and thus easier to examine under the microscope. In contrast, the soaking and Baermann method may be more suitable in field-work, especially in places where resources and equipment are scarce, as it is less costly in equipment and less labour intensive. Neither method recovered all the larvae from the seeded samples. The recovery rates for the washing machine method ranged from 18 to 41% while those for the soaking and Baermann method ranged from 0 to 27%. Practical application of the two methods to estimate the number of nematode larvae on pastures without applying a correction factor would therefore result in a significant underestimation. This study provides a model, which can be applied in various laboratories to determine the larval recovery rates for techniques being used and the application of a correction factor when estimating the actual numbers of larvae on pasture.