Field experiments on the ability of Arthrobotrys oligospora (Hyphomycetales) to reduce the number of larvae of Cooperia oncophora (Trichostrongylidae) in cow pats and surrounding grass

In field experiments, conducted on parasite-free grass plots in two consecutive summers, artificially prepared cow pats containing Cooperia oncophora eggs were inoculated with the nematode-trapping fungus Arthrobotrys oligospora. Numbers of infective C. oncophora larvae isolated from the pats as well as from the surrounding herbage were subject to an approximately ten-fold reduction as compared with numbers in fungus-free pats and herbage surrounding these. This reduction was undoubtedly a result of entrapment of the parasite larvae within the faecal pats.     

Predacious activity of Duddingtonia flagrans within the cattle faecal pat

Two studies were conducted to investigate the growth and activity of the fungus, Duddingtonia flagrans, within cattle faecal pats. Artificial faecal pats were constructed with the centre separated from the outer layer by a nylon mesh. Eight treatments were tested, by varying the presence/absence of Cooperia oncophora eggs and fungal spores within each layer. With parasite eggs in the centre layer, a statistically lower recovery of larvae was observed compared to both pats with parasite eggs in the periphery and pats with parasite eggs throughout both layers. Regardless of location within the pat, if co-located with the parasite egg, D. flagrans was found to be effective in trapping developing larvae. The reduction in recovery of larvae from pats with parasite eggs and fungal spores in the centre was found to be significantly higher than when parasite eggs were in the centre and fungal spores in the periphery. In the second study, pats were made up in two treatments: pats containing fungal spores and C. oncophora eggs (fungus) and pats containing C. oncophora eggs (control). The pats were incubated at low or high humidity. Ten pats were used in a cross over where five pats incubated at low humidity for 7 weeks were removed, water added and then incubated at a high humidity for 1 week. Another five pats were incubated at a high humidity for 7 weeks, aerated and incubated at a low humidity for 1 week. There was no apparent growth of fungus in faecal pats incubated at a high humidity and less than 20% of larvae were recovered. The growth of D. flagrans was observed in faecal pats incubated at a low humidity, but a corresponding reduction in the percentage recovery of larvae did not occur, except in week 4. No statistical difference between fungal and control pats was seen in the change over pats. Nematophagous activity was assessed throughout the study and observed in the first 4 weeks within the pats containing fungus.     

The capability of the predacious fungus Arthrobotrys oligospora (Hyphomycetales) to reduce numbers of infective larvae of Ostertagia ostertagi (Trichostrongylidae) in cow pats and herbage during the grazing season in Denmark

Artificially prepared cow pats containing Ostertagia ostertagi eggs were deposited on two pasture plots in May, June and July 1986. Half of the cow pats, placed on one plot, were inoculated with 2000 conidia per gram faeces of the predacious fungus Arthrobotrys oligospora. On the other plot fungus-free control cow pats were placed at the same time. In the faeces generally fewer infective O. ostertagi larvae developed in the inoculated than in the control cow pats. On the herbage around the control cow pats deposited in May, June and July a maximum concentration of infective larvae was found at the same time on the 6th of August 1986. At that time the herbage larval infectivity around inoculated cow pats deposited in May, June and July was subject to a reduction of 48%, 89% and 46%, respectively, compared with fungus-free control cow pats. This experiment indicates that a concentration of 2000 A. oligospora conidia per gram faeces results in a significant lowering of the herbage larval infectivity during the grazing season in Denmark.     

Reduced splash dispersal of bovine parasitic nematodes from cow pats by the dung beetle Diastellopalpus quinquedens.

From a thoroughly mixed portion of cattle feces with Cooperia sp. eggs, 1-kg artificial pats were placed in 6 buckets containing 6 kg of soil each. Ten dung beetles, Diastellopalpus quinquedens, were added to each of 3 buckets. The remaining 3 buckets served as controls without beetles. When infective parasite larvae (L3) had developed in the cow pats indoors, the following procedure was followed. During occasions of rainfall each bucket was placed outdoors in the center of a wider and higher container. When the rain stopped all buckets were brought indoors, and infective larvae spread by splash droplets during the rain were collected in the containers and counted. After 33 days, the remaining dung on the soil surface in buckets with dung beetles constituted only 38% of that in the controls. Moreover, the number of L3 in feces left on the soil surface in the buckets with beetles was reduced by 88%, presumably due to beetle activity. This may explain the 70-90% reduction in splash dispersal of L3 of Cooperia sp. from cow pats attacked by beetles. The dung-burying activity of the beetles did not result in increased numbers of L3 in the soil under the cow pats, suggesting that many of the parasites in buried feces were destroyed.     

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°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°C. Induction of nematodetrapping organs in A. oligospora is discussed in relation to control of infective nematode parasite larvae in cow pats.     

Attempts to control infection with Ostertagia ostertagi (Trichostrongylidae) in grazing calves by adding mycelium of the nematode-trapping fungus Arthrobotrys oligospora (Hyphomycetales) to cow pats

Artificially prepared cow pats containing Ostertagia ostertagi eggs were deposited on two comparable grazing plots at weekly intervals during June and July 1987. Before deposition the cow pats, on the one plot, were inoculated with 0.250 g mycelial fragments per kg of faeces of the predacious fungus Arthrobotrys oligospora. On the other control plot comparable numbers of non-inoculated cow pats were placed at the same time. Subsequently, in July, two equal groups of calves were turned out one on each plot to graze for two months after which they were necropsied. The number of infective O. ostertagi larvae in inoculated cow pats was reduced by 42% and herbage larval infectivity around them by 50-71% as compared with the corresponding parameters in control pats and surrounding herbage. After a grazing period of two months the calves on the plot with inoculated cow pats had acquired worm burdens that were on average 37% lower than those of calves on the control plot. The lower degree of parasitism in these calves was also reflected in lower egg counts, lower serum pepsinogen levels and higher body weight.     

A Field Experiment on Rain Splash Dispersal of Infective Larvae of Ostertagia Ostertagi (Trichostrongylidae) from Cow Pats to Surrounding Grass

Ostertagia ostertagi is an important tricho-strongyle nematode parasite of cattle in several countries, including Denmark. It leaves its host as eggs with faeces. After hatching it develops in the cow pat up to the third and infective stage, which is then transmitted to the surrounding grass.     

Seasonal population dynamics of the nematode Cystidicoloides tenuissima (Zeder) from the River Swincombe, England

The seasonal population dynamics of adult and larval Cystidicoloides tenuissima were studied in its definitive hosts brown trout, Salmo trutta and juvenile Atlantic salmon, S. salar , and mayfly intermediate host, Leptophlebia marginata , from the River Swincombe, Dartmoor National Park, Devon, U.K. Infective larvae were present in each mayfly generation for almost its entire duration in the steam benthos. The infection parameters (prevalence and mean intensity) and maturation in the fish indicated C. tenuissima was an annual parasite exhibiting a seasonal periodicity and also systematic variation with the host age. Maturation was correlated to river water temperature. Infection parameters increased from September to May, then declined in June and July and remained relatively constant for the rest of the summer. Variation in the fish infection parameters over time, site, and host species appeared to be controlled by transmission related events; the availability of infective larvae, host feeding behaviour and water temperature. The availability of infective larvae and host diet controlled the rate at which parasites were added to the parasite population, but the pattern of gains and losses was determined by a temperature dependent rejection response.     

Interspecific competition between the nematode-trapping fungus, Duddingtonia flagrans, and selected microorganisms and the effect of spore concentration on the efficacy of nematode trapping

The fungus, Duddingtonia flagrans, is able to trap and kill free-living nematode larvae of the cattle parasite Cooperia oncophora when chlamydospores are mixed in cattle faeces. Isolates of Bacillus subtilis (two isolates), Pseudomonas spp. (three isolates) and single isolates of the fungal genera Alternaria, Cladosporium, Fusarium, Trichoderma and Verticillium were isolated from cattle faeces and shown to reduce D. flagrans growth on agar plates. When these isolates were added to cattle faeces containing D. flagrans and nematode larvae of C. oncophora, developing from eggs, none of the isolates reduced nematode mortality attributed to D. flagrans. Similarly, the coprophilic fungus Pilobolus kleinii, which cannot be cultivated on agar, also failed to suppress the ability of D. flagrans to trap and kill developing larvae of C. oncophora. Increasing chlamydospore doses of D. flagrans in faecal cultures resulted in higher nematode mortality. Thus, no evidence of interspecific or intraspecific competition was observed. The consequences of these findings are discussed.     

Formulation of a strategy for the application of Duddingtonia flagrans to control caprine parasitic gastroenteritis

Experiments on the influence of egg density and varying quantities of chlamydospores on the nematode-trapping ability of Duddingtonia flagrans, influence of D. flagrans on the larval translation of gastrointestinal nematodes, doses of chlamydospores required for the effective control of gastrointestinal nematodosis and the epidemiology of nematode parasites were conducted in goats, which generated baseline data required for strategic application of the biocontrol agent. The nematode-trapping ability of D. flagrans, measured by numerical enumeration of infective third-stage larvae developed in the faecal culture, revealed that the efficacy is dependent on both nematode egg and chlamydospore density. Pasture plot studies revealed that D. flagrans, if deposited at the same time as nematode eggs, prevents translation of third-stage larvae of caprine nematodes from the faecal pats onto the grass blades. Feeding of 1 x 106 chlamydospores per kg body weight and above to goats virtually eliminated larvae from the pasture. Application of as few as 1 x 104 and 1 x 105 chlamydospores per kg body weight had a profound impact on larval recovery. The effect persisted as long as the chlamydospores were fed. Monthly faecal worm egg counts of adult goats maintained under a semi-intensive management system on the Chhattisgarh plain and pasture larval burden revealed that June to August were the months of high risk for nematodosis. Haemonchus was the dominant species recorded throughout the year. The present data can best be utilized by formulating a strategic control measure when the larval challenge to the animal is maximum (June to August), so as to prevent establishment of patent infection. The observations reinforced the strategy to be adopted for nematode parasite control in goats by applying the biocontrol option at the onset of the monsoon.     

Immune expulsion of the trichostrongylid Cooperia oncophora is associated with increased eosinophilia and mucosal IgA

Previous experiments have shown that a primary infection with 100000 infective larvae of the trichostrongylid Cooperia oncophora allows discrimination between different type of responder animals based on the speed by which the parasite is expelled from the host. In most of the animals (intermediate responders) the expulsion occurs 35-42 days after infection. This experiment was carried out to investigate which mechanisms contribute to the clearance of the parasite from the intestine. Sequential necropsy of the animals 14, 28 and 42 days after infection together with a segmental division of the small intestine, allowed us to characterise essential components associated with development of immunity and expulsion of the parasite from its niche. The results show that during the patent phase of the infection the parasite preferentially resides in the proximal gut. Forty-two days after infection ongoing expulsion is characterised by a migration of the worms to the more distal part of the intestine. Expulsion of the adult worm population appears to be mast-cell independent and is associated with a significant increase in parasite-specific mucous IgA and IgG1 as well as with an influx of eosinophils in the intestinal lamina propria. Although we did not observe a specific lymphocyte recruitment into the intestinal mucosa, the accumulation of eosinophils seems to be mediated by CD4+ cells. We measured significant negative correlations between the number of eosinophils and the expulsion rate of the parasite expressed by sex ratio and ratio eggs per gram faeces. Parasite-specific mucosal IgA levels were negatively correlated to the fecundity of the worms, expressed as number of eggs per female worm. Our results describe the involvement of both eosinophils and mucosal IgA in the regulation of C. oncophora expulsion and suggest the development of a Th2 effector immune response.     

Biological control of trichostrongylid infections in calves on pasture in Lithuania using Duddingtonia flagrans, a nematode-trapping fungus

The effect on the pasture contamination level with infective trichostrongylid larvae by feeding the nematode-trapping fungus, Duddingtonia flagrans at two dose levels to first time grazing calves was examined in Lithuania. Thirty heifer-calves, aged 3-6 months, were divided into three comparable groups, A, B and C. Each group was turned out on a 1.07 ha paddock (a, b and c). The paddocks were naturally contaminated with infective trichostrongylid larvae from infected cattle grazing the previous year. Fungal material was fed to the animals daily during a two month period starting 3 weeks after turnout. Groups A and B were given 10(6) and 2.5x10(5) chlamydospores per kg of live weight per day, respectively, while group C served as a non-dosed control group. Every two weeks the heifers were weighed and clinically inspected. On the same dates, faeces, blood and grass samples were collected. From mid-July onwards, the number of infective larvae in grass samples increased markedly (P<0.05)on paddock c, whereas low numbers of infective larvae were observed on paddocks a and b grazed by the fungus treated groups. However, the results indicate that administering fungal spores at a dose of 2.5x10(6)chlamydospores per kg live weight per day did not significantly prevent parasitism in calves, presumably due to insufficient suppression of developing infective larvae in the faeces. In contrast, a dose of 10(6) chlamydospores per kg lowered the parasite larval population on the pasture, reduced pepsinogen levels (P<0.05), and prevented calves from developing parasitosis.     

Observations on the seasonal prevalence, pathology and transmission of Dracunculus insignis (Nematoda: Dracunculoidea) in the raccoon (Procyon lotor (L.) in Ontario.

Lesions due to Dracunculus insignis in the legs of raccoons (Procyon lotor) in southern Ontario occur seasonally as most larvigerous females emerge in the spring and early summer (April-June). The pathology of dracunculiasis in the raccoon is described and the transmission of the parasite in the wild is discussed with respect to seasonality and local agricultural practices. Crayfish, fishes and frogs (including tadpoles) were given infective third-stage larvae of D. insignis to test their suitability as paratenic hosts. Most of the larvae fed to adult Rana pipiens and R. clamitans were recovered from the somatic musculature. Larvae had increased in size and were highly infective to raccoons.     

The influence of water and humidity on the hatching of Nematodirus battus eggs

This paper examines the influence of water on the ecology of the eggs of Nematodirus battus, with a view to estimating the importance of including rainfall in mathematical models of parasite abundance. The literature suggests that, under pasture conditions, the availability of moisture is unlikely to be limiting for egg development, while eggs and infective larvae are highly resistant to desiccation. In the presented experiment, eggs that had been kept in salt sludges at 95% and 70% RH and were subsequently put at 15°C produced only a mildly accelerated, but not a mass, hatch, in the first few days after return to water. Eggs kept at higher osmotic pressures died. Mass hatching of infective larvae, described at pasture when spells of rain follow periods of drought, is unlikely to occur as the result of a sudden water influx into eggs. Since water is not necessary for migration of infective larvae from the soil on to grass, such peaks in larval abundance are more likely to arise from the effects of temperature on hatching of eggs.     

Bionomics of larvae of Parelaphostrongylus odocoilei (Nematoda: Protostrongylidae) in experimentally infected gastropod intermediate hosts

Parelaphostrongylus odocoilei is a protostrongylid parasite that has recently been recognized at several locations in sub-Arctic, but not Arctic, North America. We investigated factors that may determine the distribution of P. odocoilei, including suitable gastropod intermediate hosts, temperature requirements for larval development in gastropods, and larval emergence facilitating overwinter transmission. We collected and experimentally infected gastropods from a site in the sub-Arctic where P. odocoilei is at the northern limit of its distribution. Deroceras laeve, Catinella sp., and Euconulus cf fulvus, but not members of the Pupillidae, were suitable intermediate hosts. We describe bionomics of larvae of P. odocoilei in D. laeve and Catinella sp. Infective larvae emerged from all slugs (D. laeve) and 60% of Catinella sp. snails, and emergence from D. laeve was intensity dependent. Emerged infective larvae survived up to 6 mo under conditions approximating that of the subnivean environment. In D. laeve, there was a direct relationship between temperature and development rate of larvae of P. odocoilei. Larvae of P. odocoilei did not develop to infective stage below the theoretical threshold (8.5 C), and required a minimum of 163 degree days to complete development. These developmental parameters can be incorporated into a model to predict larval development in the field. Knowledge of the factors influencing larval bionomics provides the foundation for predicting temporal and spatial patterns of parasite distribution, abundance, and transmission.     

The influence of weather and egg contamination on the development of third-stage larvae of Cooperia oncophora on pasture.

The influence of weather and egg contamination on the dynamics of herbage contamination with infective larave of Cooperia oncophora was investigated on artificially contaminated grass plots and in a grazing experiment with 24 first-year grazing calves from May to October 1987 in Lower Saxony, Germany. On the experimental plots the larval translation was highest at the beginning of July and in the second part of September, following high mean weekly temperatures. Between July and September peak recovery of larvae from herbage occurred 4 weeks after contamination. A seasonal pattern of larval translation similar to that on the experimental plots could be demonstrated on the grazed pastures when the number of larvae per m2 of pasture had been adjusted to the previous egg output by means of a contamination index. The resulting 'relative larval density' is regarded as a good indicator for larval development on pasture. From July to September the larval population on pasture resulted mainly from the egg contamination 2-3 weeks earlier. The short persistence of the infective larvae on herbage was probably due to the frequent and heavy rainfall throughout the season, causing a passive washout of larvae into the soil. On single pastures the larval density started to increase within 1 week after the calves had first contact with these fields. The impact of the calves on the distribution of larvae is discussed.     

Resistance to ivermectin by Haemonchus contortus in goats and calves.

Efficacy of ivermectin on susceptible or resistant populations of the parasitic nematode Haemonchus contortus was determined in cattle and goats held in a barn. Goats were each infected with 3000 infective, ivermectin-susceptible or -resistant H. contortus larvae on day 0 and reinfected with 2000 infective larvae on day 24. Goats were treated orally with 600 micrograms kg-1 ivermectin on day 31. No significant differences were detected in blood packed cell volume (PCV) or total protein (TP), prepatent period, or epg among the four groups of goats that were each infected with one of four parasite strains (one susceptible, three resistant). There were no differences among the four parasite strains in the numbers of infective larvae that developed to the third larval stage from fecal cultures or in the viability of cultured infective larvae when held in the laboratory at 27 +/- 1 degrees C for 14 weeks. After treatment with ivermectin, there were significant differences among the parasite strains in PCV, TP, and epg. Total worm counts were reduced by 94 to 97% with three times the recommended dose. Immature and adult Skrjabinema ovis were also present in two treated goats. In a second test, one goat infected once with 10,000 infective larvae of a resistant strain of H. contortus and then treated with nine doses of ivermectin, increasing from 500 to 2000 micrograms kg-1 over a period of 133 days, had 35 adult worms at necropsy. In a third test, three calves were readily infected with an ivermectin-resistant strain of H. contortus from goats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cow-dung colonization and decomposition following insect exclusion

The rate of dung-pat degradation in cattle pastures in south west England was examined between May and September 2002, using batches of standardized, 1.5 kg, artificially-formed cow pats. In pats in which insects were allowed free access, the rate of disappearance, measured as ash-free dry weight, was faster in spring than summer and the loss of organic matter ranged between 0.69 and 1.99% per day, equating to an estimated time for complete disappearance of 57-78 and 88-111 days in spring and summer, respectively. To assess the role of colonizing insects on decomposition, six batches of pats were constructed and either left uncovered or, using fine mesh cages, were covered for 2, 7 or 14 days following deposition to exclude colonizing insects, after which they were uncovered. After 35 days all pats were then retrieved from the field, the composition of the invertebrate community within each pat was determined and the degree of degradation, measured as ash-free dry weight, was assessed. Covered pats had significantly higher rates of ash-free dry weight loss than uncovered pats. From the 180 pats that were recovered and searched, larvae of Aphodius (Coleoptera: Scarabaeidae) were the most numerous taxon collected (1435 individuals). Aphodius larvae were significantly more abundant in pats that remained uncovered or were covered for 2 days only, compared to pats that had been covered for 7 or 14 days. The results show that the exclusion of insects for as little as two days following deposition causes a significant reduction in both the insect population and the subsequent rate of pat degradation.     

Habitat and weather affect reproduction and size of the dung beetle Aphodius fossor

1. The influence of sun exposure and moisture on reproduction and size of a north temperate dung beetle Aphodius fossor was investigated. Adult beetles were added to cow pats that were either shaded or exposed and treated with different watering regimes. 2. After 26 days, more eggs and larvae were recovered from shaded and wetter pats than from exposed and drier pats. 3. At the end of the experiment, more beetles had developed in shaded and wetter pats, and these beetles were larger than those from exposed and drier pats. 4. There was no between‐treatment difference in the ratio between adults in final sampling and larvae in first sampling. 5. It was concluded that exposure and moisture can influence both reproduction and size of A. fossor. It is suggested that weather may be a factor regulating reproduction in Aphodius not only through heat‐ or drought‐induced mortality, as suggested earlier, but also by limiting the time available for egg laying. Further studies are needed to clarify this suggestion.     

Expression and secretion of a larval-specific chitinase (family 18 glycosyl hydrolase) by the infective stages of the parasitic nematode, Onchocerca volvulus

A recently reported chitinase gene, expressed in the infective, third-stage (L3) larvae of the human filarial parasite Onchocerca volvulus, belongs to the family 18 glycosyl hydrolases and has been designated Ov-chi-1. The gene product of Ov-chi-1 is chitinolytic. Allosamidin ablates activity of the native enzyme in a dose-dependent manner but did not significantly inhibit the moulting of L3 larvae. Mono-specific antibodies were used to characterize Ov-CHI-1 as a 60-kDa protein expressed almost exclusively in L3 stages. Immunoelectron microscopy showed that Ov-CHI-1 expression is initiated in late L2 larvae and increases markedly in infective, L3 larvae. It is synthesized exclusively in the glandular esophagus and stored within discrete secretory granules. Secretion occurs through de-granulation during post-infective development, and the primary route of transport appears to be via the pseudo-coelom. An orthologue of Ov-chi-1 was detected in Caenorhabditis elegans by BLAST analysis. It is constitutively expressed at a low level and is overexpressed in dauer larvae and embryonated eggs. It is chitinolytic. We conclude that Ov-CHI-1 is a highly stage-specific enzyme that may have a role in infectivity of the parasite, aiding escape from the vector or participating in early post-infective migration and/or development. The identification of an orthologue in C. elegans opens the way for further studies into the biological function(s) of this intriguing parasite product.