Cultivation of Haemonchus contortus (Nematoda: Trichostrongylidae) from infective larvae to the adult male and the egg-laying female

The parasitic nematode Haemonchus contortus was cultured in vitro to the adult male and the egg-laying female. Gastric contents from 2 cannulated calves and 2 sheep as well as extracts of stomach mucosa from both hosts were added to a mixture of API-1 culture medium and Fildes' reagent (a pepsin digest of defibrinated bovine blood) adjusted to pH 6.4 for the first week and pH 6.8 thereafter. The gas phase was 85% N2:5% O2:10% CO2. Best results were obtained when API-1 culture medium plus Fildes' reagent was supplemented with ovine gastric contents. Adult males up to 10 mm were obtained, and they produced and stored sperm at about 28 days in culture. Spermatozoa were never seen in the uteri of females grown in vitro. After 36 days in culture, female worms up to 16 mm were obtained which produced and layed eggs. Some of the eggs underwent division up to the 8-cell stage but did not develop further. No live larvae were observed at a stage earlier than the fourth. Presumably, this was because no matings were observed, although large clumps of worms did occur in the culture medium and mating might have occurred. The growth of adults from young to mature phases of this stage included: for the female, oogenesis and laying of eggs; for the male, development of paired sclerotized spicules, and spermatogenesis with the production of spermatozoa.     

An extracellular serine protease of an isolate of Duddingtonia flagrans nematophagous fungus

This study aimed to present a protease produced by Duddingtonia flagrans fungus (AC001), and to evaluate its activity in the biological control of cyathostomin infective larvae (L₃). The crude extract from D. flagrans grown in liquid medium was applied first to a DEAE-Sepharose? and later to a CM-Sepharose? ion exchange column. Protease activity was determined under different pHs and temperatures. Subsequently, the effects of metal ions and phenylmethylsulfonyl fluoride (PMSF) inhibitor on activity were evaluated. Next, the protease activity in the biological control of nematodes was tested. A new 38 kDa serine protease (Df1) was purified. Optimum activity was obtained at pH 8.0 and 60°C; CuSO₄, ZnSO₄ and PMSF strongly inhibited the activity. Df1 (AC001) showed an L₃ reduction rate of 58%. In conclusion, a serine protease produced by D. flagrans (AC001) has been isolated, which is effective in the in vitro destruction of cyathostomin L₃.     

Culture medium characteristics on the predatory activity of the nematophagous fungus Duddingtonia flagrans

The influence of casein and pH on the activity of the nematophagous fungus Duddingtonia flagrans (AC001) on trichostrongylide larvae was evaluated. A ‘positive influence’ was observed contributing to the reduction of 63% in the average number of recovered L₃ in the media supplemented with casein and pH 7.0.     

Interaction of the nematophagous fungus Duddingtonia flagrans on Amblyomma cajannense engorged females and enzymatic characterisation of its chitinase

The present work aimed to evaluate the production and the characterisation of a chitinase from nematophagous fungus Duddingtonia flagrans (AC001) and observe the interaction of this fungus on engorged females of Amblyomma cajennense under laboratory conditions. In assay A, the engorged females of A. cajennense were separated and immersed for 5 seconds in a fungal suspension of 10⁶ conidia/ml of the fungus D. flagrans and placed in Petri dishes, in the dark. In assay B, wheat bran supplemented with 1% chitin and liquid minimal medium was used [K₂HPO₄ (5.0 g/l), MgSO₄ (0.10 g/l), ZnSO₄ (0.0050 g/l), FeSO₄ (0.001 g/l) e CuSO₄ (0.50 mg/l)], as a substrate for chitinase production. To demonstrate the presence of chitinase in the crude extract obtained after the enzymatic extraction, a purification process was developed using a specific adsorption technique. The results from assay A demonstrated the interaction of the D. flagrans conidia produced from chitin-agar on engorged females of A. cajennense. In the assay B, D. flagrans produced a chitinase successfully, with a high value for enzyme activity. The molecular mass of semi-purified enzyme was estimated at approximately 34 kDa. It was concluded that the fungus produced a chitinase and has some entomopathogenic activity, as demonstrated here for the first time; however, it is strongly suggested that further studies are needed to elucidate the molecular mechanism of infection of target organisms by this fungus.     

The predatory capability of three nematophagous fungi in the control of Haemonchus contortus infective larvae in ovine faeces

The effect of oral administration of three different nematode-trapping fungi, in aqueous suspension containing either Dactylaria sp. or Arthrobotrys oligospora conidia or Duddingtonia flagrans chlamydospores, on the number of Haemonchus contortus infective larvae in sheep faeces, was evaluated. The three selected species of fungi produce three-dimensional adhesive nets in the presence of nematodes. Sixteen Creole sheep were divided into four groups of four animals each. Groups 1 and 2 were orally drenched with a suspension containing 2x10(7) conidia of either A. oligospora or Dactylaria sp. Group 3, received a similar treatment, with D. flagrans chlamydospores, instead of conidia, being administered, at the same dose. Group 4 acted as control, without any fungi. Faecal samples were collected directly from the rectum of each sheep and faecal cultures were prepared and incubated at 15 and 21 days. Larvae were recovered from faecal cultures and counted. The highest reduction of the nematode population occurred in the D. flagrans group, reaching reductions of 96.3% and 91.4% in individual samplings in plates incubated for 15 and 21 days, respectively. Arthrobotrys oligospora showed moderate reductions in the faecal larval population, ranging between 25-64% at 15 days incubation. In general, Dactylaria sp., was less efficient in its trapping ability. Despite the inconsistent results with Dactylaria sp., reduction percentages of 73.4% and 80.7% were recorded in individual samplings during the first and second days, in plates incubated for 15 days. Duddingtonia flagrans, was shown to be a potential biological control agent of H. contortus infective larvae.     

Dense molecular marking reveals genetic diversity in morphologically similar isolates of the nematophagous fungus Duddingtonia flagrans

Three morphologically similar isolates of Duddingtonia flagrans [(Duddington) R. C. Cooke] viz., Df-2550, Df-2507 and Df-BJ were subjected to RAPD (Randomly Amplified Polymorphic DNA) and SRFA (Selective Fragment Length Amplification) mode of DNA fingerprinting analysis to generate 233 different anonymous DNA markers. Mean number of alleles per primer/primer pair for RAPD and SRFA primers was 13.75 and 29.66 respectively. Phylogenetic analysis through bootstrapping of 1000 simulated replicates of the data set demonstrated that Df-2550 was ancestral in the group of three and did not align with Df-2507 and Df-BJ, which appeared to diversify recently and therefore remained at the end of the phylogenetic tree. Genomic islands were also identified by three SRFA primer pairs, where Df-2550 aligned with Df-BJ, which is reverse to the master consensus-grouping pattern. Scanning image of the amplicon profiles when represented graphically generated unique molecular signature for the isolates.     

Influence of the preservation period in silica-gel on the predatory activity of the isolates of Duddingtonia flagrans on infective larvae of cyathostomins (Nematoda: Cyathostominae)

The continued maintenance of nematophagous fungi predatory activity under laboratory conditions is one of the basic requirements for a successful biological control. The purpose of this study was to evaluate the influence of time on the preservation of the fungus Duddingtonia flagrans (AC001 and CG722) stored in silica-gel for 7 years and their subsequent predatory activity on cyathostomin L₃ larvae in 2% water-agar medium (2% WA). Samples of the isolates AC001 and CG722, originating from vials containing grains of silica-gel sterilized and stored for 7 years, were used. After obtaining fungal conidia, the predation test was conducted over 7 days on the surface of 9.0 cm Petri dishes filled with 2% WA. In the treated groups each Petri dish contained 500 cyathostomin L₃ and conidia of fungal isolates in 2% WA. In the control group (without fungi) the plates contained 500 L₃ in 2% WA. The experimental results showed that isolated AC001 and CG722 were efficient in preying on cyathostomin L₃ (p < 0.01) compared to control (without fungus). However, no difference was observed (p > 0.01) in the predatory activity of the fungal isolates tested. Comparing the groups, there was a significant reductions of cyathostomin L₃ (p < 0.01) of 88.6% and 78.4% on average recovered from the groups treated with the isolates AC001 and CG722, respectively, after 7 days. The results of this test showed that the fungus D. flagrans (AC001 and CG722) stored in silica-gel for at least 7 years maintained its predatory activity on cyathostomin L₃.     

Trematodes enhance the development of the nematode-trapping fungus Arthrobotrys (Duddingtonia) flagrans

The capability of helminth (nematode and trematode) parasites in stimulating nematode trap and chlamydospore development of the nematophagous fungus Arthrobotrys (formerly Duddingtonia) flagrans was explored. Dead adult specimens of trematodes (the liver fluke Fasciola hepatica and the rumen fluke Calicophoron daubneyi) and nematodes (the ascarid Parascaris equorum and the strongylid Oesophagostomum spp.), as well as their secretory products, were placed onto corn meal agar plates concurrently inoculated with A. flagrans. Trapping organs were observed after 5 d and chlamydospores after 16 d, including in the control plates in the absence of parasitic stimulus. However, our data shows that both nematodes and trematodes increase trap and chlamydospore production compared with controls. We show for the first time that significantly higher numbers of traps and chlamydospores were observed in the cultures coinoculated with adult trematodes. We conclude that both the traps and chlamydospores formation are not only related to nematode-specific stimuli. The addition of secretory products of the trematode C. daubneyi to culture medium has potential for use in the large scale production of chlamydospores.     

Biochemical characterization of cuticle polypeptides from the infective larvae of Haemonchus contortus

1. Ecdysis of infective Haemonchus contortus larvae is effected by the enzymatic degradation of a specialized region of the second molt cuticle containing a biochemically unique polypeptide (mol. wt = 160,000). 2. The 160,000 mol. wt polypeptide and related polypeptides are synthesized at approximately 6 days of larval development. Antigenically similar polypeptides occur in other ruminant trichostrongyles. 3. Cuticle polypeptides digested during ecdysis differ from second molt cuticle collagens in amino acid composition and collagenase sensitivity. However, some antigenic homology between the 160,000 mol. wt polypeptide and cuticle collagens suggests structurally similar regions.     

Tyrosine-derived cross-linking amino acids in the sheath of Haemonchus contortus infective larvae

The sheath or second-molt cuticle (2M) was isolated from in vitro exsheathed Haemonchus contortus infective larvae (L3[2M]). Acid hydrolysates of 2-mercaptoethanol (2ME)-soluble and 2ME-insoluble cuticular proteins were analyzed by high performance liquid chromatography for tyrosine-derived cross-linking amino acids. Dityrosine and isotrityrosine were identified by their chromatographic behavior, absorbance spectra, and other chemical characteristics in both the 2ME-soluble and 2ME-insoluble fractions. Dityrosine and isotrityrosine were found in greater amounts in the 2ME-insoluble proteins. When intact 2M cuticles were labeled with 125I prior to acid hydrolysis, radiolabel was recovered in tyrosine but not dityrosine or isotrityrosine indicating that the tyrosine cross-links are not susceptible to iodination in the intact protein. The results are consistent with a hypothesis that tyrosine-derived cross-links are important components of H. contortus 2M cuticular proteins.     

Light and scanning electron microscopy of the ecdysis of Haemonchus contortus infective larvae

During the second ecdysis of ruminant trichostrongyles, a region of the second molt cuticle is digested by a 44-kDa Zn-metalloprotease. We have examined this digestion process by light and scanning electron microscopy (SEM). The substrate region of the cuticle appeared, during the ecdysis process, as an indented ring at the 20th cuticular annulus coincident with the anterior terminus of the lateral alae. Continued digestion of the cuticle resulted in holes in the ring region that expanded until they became continuous and separation occurred between the anterior and posterior portions of the cuticle. Mechanical movements of the L3 forced aside the cuticle cap that generally remained attached on one side to the posterior portion as the larva escaped from the sheath. The site of secretion of the 44-kDa ecdysing enzyme causing cuticle digestion was not clear from morphological observations; however, existing evidence strongly points to the release of enzyme from the esophageal (pharyngeal) glands through the mouth.     

Efficacy of the nematode-trapping fungus Duddingtonia flagrans against infections by Haemonchus and Trichostrongylus species in lambs at pasture

The efficacy of the nematode-trapping fungus Duddingtonia flagrans against infections by trichostrongyle nematodes in sheep was assessed throughout 6 months. Twenty Ile de France lambs were divided into two groups (control and treated groups), which were kept in separate pastures. Animals of the treated group were fed with D. flagrans twice a week (Tuesdays and Fridays). Pellets were prepared with the fungus mycelia in liquid culture medium and contained approximately 20% fungus. They were mixed with the animals' diet at a concentration of 1 g pellet per 10 kg live weight. Faecal egg counts (FEC), packed cell volume (PCV), total serum protein and the animals' body weight were determined fortnightly from 7 October 2005 to 24 March 2006. Comparison of such parameters between groups showed no significant differences (P > 0.05), except on 10 February 2006, when the control group presented a higher mean FEC than the treated group (P < 0.05). Feeding sheep with pellets containing D. flagrans had no benefit to the prophylaxis of nematode infections under the experimental conditions used in the present study.     

Some analyses of exsheathing fluid from infective Haemonchus contortus larvae from Ontario

Infective Haemonchus contortus larvae from Ontario were exsheathed, and the exsheathing fluid was prepared, using several procedures some of which duplicated those of other researchers. Infective larvae were exsheathed successfully using the rapid (20-min) tetraborate system. Second-stage sheaths were dissected from infective larvae and were incubated with various preparations of exsheathing fluid. Up to 30% of the sheaths incubated with dilute exsheathing fluid for 1 h had refractile rings. When the fluid was concentrated by dialysis or lyophilization the exsheathing activity was not lost. Heat destroyed the ability of concentrated exsheathing fluid to cause refractile rings in dissected sheaths, but Cu²⁺, Hg²⁺ or diaminoethanetetra-acelic acid did not. The enzyme leucine aminopeptidase was not found in concentrated exsheathing fluid.     

Reversal of order of ecdysis in Haemonchus contortus (Nematoda)

Exposure of Haemonchus contortus infective larvae to carbon dioxide under specific conditions is believed to activate a succession of events that ultimately control both exsheathment and subsequent development to the fourth stage. Rarely at pH 6, but always at pH 2, worms fail to exsheath yet develop to the fourth stage surrounded by cuticles two and three. Failure to exsheath is probably a consequence of inactive enzymes in exsheathing fluid, because exposure to EDTA, which inactivates exsheathing fluid, reduced exsheathment and yielded sheathed fourth-stage worms. Although exsheathment is blocked at pH 2, CO2 can activate the receptor for exsheathment as readily at pH 2 as at pH 6. Therefore the hypothesis that an early common pathway controls both exsheathment and development is not invalidated.     

Redescriptions of Haemonchus mitchelli and Haemonchus okapiae (Nematoda: Trichostrongyloidea) and description of a unique synlophe for the haemonchinae

In the course of a revision of Haemonchus Cobb, 1898 (Nematoda), commonly referred to as large stomach worms, significant new morphological information was discovered that allows the recognition of 2 species believed for more than 50 yr to be synonymous. Both species, Haemonchus mitchelli Le Roux, 1929, from the eland Taurotragus oryx and other African ruminants and H. okapiae van den Berghe, 1937, from the okapi Okapia johnstoni, have a synlophe of 42 ridges, but the synlophe of H. mitchelli is longer than that of H. okapiae. The distal tip of the left spicule of H. mitchelli bears a barb that is about twice as long as the short barb and half as long as the long barb on the right spicule. In contrast, the barb on the left spicule of H. okapiae is similar in size to the short barb and about 25% as long as the long barb of the right spicule. The dorsal ray of H. mitchelli is bifurcated distally for 25-39% (32%) of its length and its stem is expanded proximally, but the dorsal ray of H. okapiae is bifurcated 37-50% (42%) and its stem is of uniform thickness.