Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
  • A01N25/26—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests in coated particulate form
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom

Definitions

• a method of controlling nematode infection in animals and a composition therefor A method of controlling nematode infection in animals and a composition therefor.
• the present invention relates to a method of reducing the number of infective, animal parasitic nematode larvae in an environment of ani ⁇ mals so as to reduce the transmission of nematode infection to the animals inhabiting said environment; a composition for controlling animal parasitic nematodes; and a process for preparing a fungal material of a nematode-destroying fungus.
• Nematode-destroying fungi are an extremely common group of more than 150 microfungi (Barron, 1977 (1)). Most species are found within the order Honiliales (Hypho ycetes) of the class Fungi imperfect! , while some belong to the order Zoopagales of the class Phycomycetes . A key by Cooke & Godfrey (1964) (2) lists more than 90 ' species. Today there are several reviews on aspects .of the biology of nematode-destroying fungi (Drechsler, 1941 (3); Soprunov, 1958 (4); Duddington, 1962 (5); Pramer, 1964 (6); Sayre, 1971 (7); Barron, 1977; Peloille, 1981 (8); and Mankau, 1981 (9)) .
• Nematode-destroying fungi may be divided into three groups according to their mode of action: a predatory, an ovoparasitic and an endo- parasitic group.
• Protozoans, rotifers and especially nematodes are organisms that may be trapped by the group of predatory fungi.
• the predatory fungi develop different kinds of cap ⁇ turing organs like constricting or non-constricting rings, sticky hyphae, knobs, branches or, for instance in the case of Ar throbo rys oligospora , three-dimensional sticky networks which are the most com ⁇ mon type of trapping organ.
• the group of nematode-des roying fungi is widely distributed and may be isolated fro leaf mould, rotting wood, partially decayed plant residues, moss and dung (Duddington, 1951 (16)).
• Arthrobotrys oligo ⁇ spora one of the most widely distributed predatory fungi, is the most common predatory fungus in agricultural soils in a temperate climate (Shepherd, 1961 (17)), and it may also be isolated from cattle dung.(Juniper, 1957 (18)).
• Zopf (1888) (19) described it as a dung-inhabiting fungus.
• predatory fungi are capable of sapro- phytic development but they are poor competitors in many soils which often contain factors fungistatic to conidia and perhaps other germi ⁇ nating units (Cooke, 1968 (20)). Conidia in such soils are lysed or capturing organs are formed directly from the conidia (Mankau, 1962 (21)). Traps created directly from conidia may represent an adaption of the predatory fungi to survive in highly antagonistic soils using only nematodes as feed.
• Predatory fungi occur in nature but it has proved very dif icult to alter the environment of the soil to favour predatory fungi by the admixture of organic matter (defined as a residue or waste of bio ⁇ logical processes or decomposing matter of biological origin, e.g. compost or manure) or fungal material.
• organic matter defined as a residue or waste of bio ⁇ logical processes or decomposing matter of biological origin, e.g. compost or manure
• predatory fungi are more or less inactive until a fresh energy source such as organic matter is added as this stimulates bacterial growth and hence nematode multiplication.
• a fresh energy source such as organic matter
• the activity of predatory fungi may decrease as a result of an increased intensity of competition from other soil fungi.
• predatory fungi are applied directly to soil, some species ap ⁇ pear to be able to compete successfully with the soil microflora at higher inoculum levels (Cooke, 1963 (23)).
• Cayrol et al. (1978) (28) developed a product based on a horse manure isolate of Arthrobotrys robusta cultivated on rye grains (Royal 300 ⁇ ) .
• the fungal product was reported to protect the commercial mushroom, Agaricus bisporus, from attack by the mycophagous nematode Ditylenchus myceliophagus .
• A. robusta had no adverse effect on the development of the mushroom mycelium and grew rapidly in mushroom compost.
• mushroom cultures seeded with 1% A. robusta product there was a 40% reduction of the D. myceliophagus population, and the mushroom harvest was increased by more than 20%.
• oligospora may destroy parasitic strongyle nematode larvae of the donkey (Soprunov, 1958 (4)), infective larvae of Trichostrongylus axei and Ostertagia ostertagi of cattle (Pandey, 1973 (33)), Haemon- chus contortus of sheep (Virat & Peloille,1977 (34)) and Cooperia spp. of cattle (Gr ⁇ nvold et al., 1985 (35)).
• Nansen et al. (1987) (36) showed that A.
• oligospora is capable of destroying a number of infective parasitic nematode larvae of cattle (Ostertagia ostertagi , Cooperia oncophora, Dictyocaulus viviparus) , sheep (Cooperia curticei , Haemonchus contortus) , pigs (OesophagostO- mum dentatum, Oe. quadrispinulatum) and horses (Cyatostoma spp.). Comparisons made among the first, the second and the third and infec ⁇ tive larval stages of Cooperia oncophora showed no difference in the ability of the different stages to induce networks in A. oligospora (Nansen et al. , 1986 (37)). All stages were trapped with the same efficiency.
• Roubaud & Deschiens (1941) (38) carried out an experiment in which two small plots of pasture (5 x 5 m) infected with Strongyloides papillosus and SunosComuz ⁇ spp. were each grazed by two ten-month-old lambs.
• One plot had been treated with spore powder of Arthrobotrys oligospora , Dactylella bembicodes and Dactylaria el- lipsospora .
• the lambs were examined. It was found that the animals which had grazed in the treated plot had a significantly lower parasite burden than the lambs from the control plot.
• Soprunov (1958) (4) tested the viability of Arth ⁇ robotrys oligospora after passage through the intestinal tract of a donkey A. oligospora was cultivated on chopped corn at temperatures between 25°C and 30°C and high humidiy for about one month. The final product contained 1.5-2 million conidia per gramme of spore powder. For five days, the donkey's odder was supplemented with 150 g of spore powder. From the 2nd to the 9th day, A. oligospora was detected in the faeces of the donkey which was infected with strongyle nema ⁇ tode parasites.
• One object of the present invention is therefore to provide a method of controlling animal parasitic nematodes wherein a fungal material of a nematode-destroying fungus is provided-in animal faeces by gas ⁇ trointestinal passage, the fungal material retaining an adequate de ⁇ gree of viability after the gastrointestinal passage.
• the present invention relates to a method of reducing the number of infective, animal parasitic nematodes in an environment of animals so as to reduce the transmission of nematode infection to animals inhabiting said environment, the method comprising admini- stering to an animal a composition comprising a fungal material of a nematode-destroying fungus in order to provide an adequate nematode- controlling amount of the fungal material in the animal f eces, the composition being formulated in such a way that an adequate nematode- controlling proportion of the fungal material remains viable after passage through the gastrointestinal tract of the animal to which the composition is administered.
• the nematodes which the method of the invention aims to control may be any of the nematode species known to infect animals, including human beings, and to cause more or less severe damage in the host which they infect.
• Examples of nematodes to be controlled by the me ⁇ thod of the invention are Ostertagia spp., Trichostrongylus spp., Haemonchus spp., Dictyocaulus spp., Oesophagostomum spp., Cooperia spp., Cyathostoma spp., Strongyloides spp., Strongylus spp., Bunosto- mum spp., Ancylostoma spp.
• the composition to be administered may include, as the fungal material of the nematode-destroying fungus, a fungal material from a predatory fungus (which is capable of captur ⁇ ing nematode larvae) , an endoparasitic fungus (which is capable of establishing itself in nematode larvae) or an ovoparasitic fungus (which is capable of establishing itself in helminth eggs) .
• the nema ⁇ tode-destroying fungus is suitably a predatory fungus.
• the trapping devices of predatory fungi may take various forms as indicated above.
• a further type of trapping de- vices consists of constricting rings where a branch forms a closed ring of three curved cells which swell up when a larva enters the ring so that the larva is held tightly and cannot escape. The trophic hyphae then penetrate the cuticle and consume the body contents of the larva.
• the term "transmission” is intended to mean that the nematode infection is transferred from infected animals to non-infected animals present in the same environment.
• fungal material is understood to mean a material which comprises all parts of a fungus; including the mycelium (hyphae) and all types of spores, e.g. conidia and chlamydospores, or a mixture thereof. It has surprisingly been found that the mycelium may also be employed as the material from which the active, that is nematode- destroying, fungus may be developed at the site where such fungi are usually active (i.e. in faeces).
• the term "adequate nematode-controlling amount" is intended to indi ⁇ cate that the amount of fungal material present in the animal faeces is sufficient to bring about a significant reduction of the number of infective nematodes in the environment, for instance an enclosure, of an animal to which the composition containing the fungal material is administered, and a consequent reduction of the transmission of nema tode infections to animals present in the environment treated with the fungal material.
• Such an adequate amount of the fungal material may presumably be provided by administering large quantities thereof to the animals which are to be .protected from nematode infection, so as to ensure the survival of an adequate proportion of the material even in unprotected form in the alimentary tract of the animal in question, but this is a less desirable procedure for two reasons, namely the problems associated with the production of large quanti ⁇ ties of fungal material and the possible difficulties in making the animal ingest the requisite large amount of fungal material. It is therefore preferred to provide the fungal material in protected form so that an improved viability of the fungal material is ensured, requiring the administration of, presumably, far smaller quantities of the fungal material, thereby improving the reproducibility of the method of the invention.
• the term "reproducibility" is intended to indicate that it may be possible to make substantially all animals which, for instance, belong to the same stock ingest sub ⁇ stantially equal amounts of the fungal material per kg body weight.
• the protection of the fungal material may also serve to improve the storage stability of the fungal material in that it may further pro ⁇ tect it from, for instance, absorption of water and exposure to oxy ⁇ gen which may result in a deteriorated long-term viability of the fungal material. This would tend not to be the case with correspond ⁇ ing unprotected fungal material, and in order to ensure an adequate supply of this viable unprotected material, it would be necessary to produce it in the immediate vicinity of the site of use and use it virtually immediately upon reaching a sufficient density of the pro ⁇ duction culture.
• the protection of the fungal material to be employed in the method of the invention therefore contributes to lowering the production costs of the fungal material by permitting a more ratio ⁇ nalized production which, together with the decrease of the amount of fungal material necessary to administer to obtain a sufficient nema ⁇ tode control when the fungal material is in protected form, makes it less expensive to employ the method of the invention for nematode control than utilizing a corresponding method which does not make use of a protected fungal material.
• nematode-destroying fungus The choice of a particular species of nematode-destroying fungus to be incorporated in the composition employed in the method of the in ⁇ vention will of course in each case depend on the specific type of nematode to be combated, i.e. whether it is susceptible to an endo- parasitic, ovoparasitic or predatory fungus. Many nematodes which constitute a serious problem in the field of animal husbandry may, however, be controlled by means of predatory fungi, and the composi ⁇ tion to be administered to animals in accordance with the principles of the invention therefore advantageously contains a fungal material of a predatory fungus.
• the fungus to be used may therefore be selected accord ⁇ ing to other criteria such as its fermentation properties (determin ⁇ ing whether it is feasible to produce it on a large scale, and hence the production costs) , growth properties at the site of use (it should preferably exhibit fast growth so as to be able to compete successfully with other organisms in the animal faeces) and optimum growth temperature. It is assumed, however, that for most practical applications, a predatory fungus belonging to an Arthrobotrys spp. or a Dactylaria spp. may be employed.
• compositions may further include mixtures of any of these fungal species with, for instance, different temperature optima or other growth properties in order to provide a composition which may be employed under widely varying climatic conditions, e.g. the tem ⁇ perature, or the structure and composition of the soil.
• the animals to which the composition is administered according to the method of the invention are usually domestic animals, that is, ani ⁇ mals which are bred for commercial purposes, and are most often young animals since young animals are particularly susceptible to nematode infections. Furthermore, the animals are typically untethered animals as these are more vulnerable to nematode infection than those which are kept in separate enclosures.
• the animals to which the composition comprising the fungal material is to be administered according to the invention may be pigs, in which case the environment where nematode control is to take place may be a pigsty or a pasture.
• the composition containing the fungal material is suitably administered at least once every two days, preferably at least once a day, for at least one month, pre ⁇ ferably for at least two months, during a period where contamination of the enviroment by nematodes is critical.
• peripheral where contamination of the environment by nematodes is critical should, in the present context, be understood to mean a period where the potential for development of infective nematode larvae and consequently the risk of transmitting infective material from one animal to the other is particularly high.
• An estimated daily dosage of active fungal material is in the range of 1-10 mg (dry weight) of the fungal material per kg of faeces.
• a group of animals to which the method of the invention may advan ⁇ tageously be applied is domestic fowl.
• the frequency with which the composition containing the fungal material is administered is typi ⁇ cally similar to that applicable to pigs.
• composition containing the fun ⁇ gal material may be administered is carnivores, for instance fur ani ⁇ mals such as mink or fox, or pets such as dogs or cats.
• the frequency with which the composition containing the fungal material is admini ⁇ stered as well as the estimated daily dosage thereof is t pically similar to that applicable to pigs.
• the method of the present invention may advantageously be practised on herbivores.
• a herbivore which is of particular interest in connection with the method of the present invention is the horse, as the problem of nema ⁇ tode resistance to conventional chemical anthelmintics has become in ⁇ creasingly serious with respect to horses in recent years.
• the composition may be administered both to horses kept in a stable and kept in a pasture, but is, perhaps, of the greatest relevance to horses more or less permanently pastured or at the time of the year when the animals are turned to grass since horses are usually kept in separate enclosures in a stable where the risk of contamination is less grave.
• composition should advantageously be administered at least once every two days, preferably at least once a day, for at least two months, during a period where contamination of the environment by nematodes (in this case, typically when the animals are turned to grass) is critical.
• An estimated daily dosage of active fungal material is in the range of 1-10 mg (dry weight) of the fungal material per kg of faeces.
• rumi ⁇ nants e.g. cattle, sheep, deer or goats.
• ruminants it is of the greatest importance to administer the composition to animals kept in a pasture where the intensity of nematode infec ⁇ tion is particularly high.
• the composition should preferably be ad ⁇ ministered to the ruminants at least once (the frequency of admini ⁇ stration depending on how the composition to be administered is for ⁇ mulated) , at a time of the year when contamination of the environment by nematodes is critical for the build-up of high infectivity in the environment.
• composition is suitably administered at least once over a period of at least 1 month, although the frequency of administration will vary widely de ⁇ pending on the type of formulation administered.
• the fungal material employed in the method of the invention is provided in protected form so as to obtain a controlled release of the fungal material.
• the release of the fungal material should preferably be adapted to ensure the high- est possible survival rate of the fungal material which means that the release will usually be directed to occur in the distal part of the intestines or in the faeces so as to allow growth of the fungus in the faeces.
• the controlled release may be achieved by means of a coating or a matrix with specific solubility or disintegration cha- racteristics, and the composition to be administered according to the invention may therefore be in the form of a matrix tablet or pill in which the fungal material is embedded, a coated tablet or capsule containing the fungal material or coated granules of the fungal mate ⁇ rial which is released on disintegration of the coating.
• the composition may advantageously be in the form of a bolus in which the fungal material is embedded and from which it is slowly and con ⁇ tinuously released.
• a bolus preparation has the advantage that it need only be administered once or twice during the -critical period, whereas for any of the other dosage orms, the composition will have to be administered with the same frequency as applies to any of the non-ruminants mentioned above.
• biologcial nematode-controlling agent As the fungi do not act immediately in destroying nematodes, that is, they require a certain period of time to develop, it may be necessary or at least convenient to combine the biologcial nematode-controlling agent with a chemical agent (cf. also Sayre, 1971 (7)).
• a che ⁇ mical antiparasitic agen- such as an anthelmintic which acts immedi ⁇ ately to reduce the initial parasite "burden" (i.e. the number of parasites with an infective potential present in an animal) in each animal to which the composition is administered, thereby reducing the overall initial parasite population of the environment in which the animals are kept which may require a smaller amount of fungal mate ⁇ rial to be administered in order to provide a continuous reduction of the number of infective nematodes over a longer period of time.
• the antiparasitic agent may also be one which is effective against other parasites than those controlled by the fungus incorporated in the composition.
• the present invention further relates to a composition for control ⁇ ling animal parasitic nematodes, which composition comprises a fungal material of a nematode-destroying fungus and an excipient which sig ⁇ nificantly improves the viability of the fungal material in the gas ⁇ trointestinal tract of an animal to which the composition is admini- ' stered.
• the fungal material included in the composition may be de ⁇ rived from any of the sources (genera or species of fungus) indicated above, and may comprise mycelium or spores or a mixture thereof.
• the composition may be formulated in accordance with any of the known methods of formulating veterinary preparations.
• a form of the composition that ensures pro ⁇ tection of the fungal material in the alimentary tract of the animal to which the composition is administered, as explained in detail above.
• Such protection may be obtained by embedding the fungal mate ⁇ rial in a matrix which may be formulated as tablets or pills, the matrix material being insoluble in a gastrointestinal environment or soluble or erodible in the large intestine.
• the matrix formulation is optionally coated with a suitable coating such as one of those indi ⁇ cated below.
• the matrix material may suitably be selected from a natural or synthetic wax, a plastic, a polymer, a polysaccharide, such as an alginate, a dextrin, a starch, cellulose or a derivative thereof or agarose, a resin, a fatty alcohol, fatty acid and esters thereof, a mineral such as silica or a silicate, kaolin, bentonite, diatomaceous earth, vermiculite, pumice or mineral wool, and a vege ⁇ table material such as wheat bran or seeds, e.g. poppy or sesame seeds.
• a natural or synthetic wax such as an alginate, a dextrin, a starch, cellulose or a derivative thereof or agarose
• a resin such as silica or a silicate, kaolin, bentonite, diatomaceous earth, vermiculite, pumice or mineral wool
• a vege ⁇ table material such as wheat bran or seeds, e.
• the composition may also be formulated as a coated tablet or capsule comprising the fungal material, the coating being adapted to dissolve or disintegrate in the intestines to release the fungal material.
• the composition may, however, advantageously comprise coated granules containing the fungal material as these are usually easier to ad ⁇ minister since they can be admixed with the feed dispensed to the animals.
• the coating employed to protect the fungal material is preferably an enteric coating which is usually a coating soluble at the pH prevail ⁇ ing in the large intestine or in the faeces, i.e. typically a pH of 7 or more.
• enteric coating materials are selected from shellac, cellulose acetate esters such as cellulose acetate phtha- late, hydroxypropyl methyl cellulose esters such as hydroxypropyl methyl cellulose phthalate, polyvinyl acetate esters such as poly- vinyl acetate phthalate, and polymers of methacrylic acid and (meth)- acrylic acid esters.
• the coating may be one which is enzymatically degrad- able in the intestines, especially the large intestine, or in the faeces, or a water-permeable coating which may be selected from ethyl cellulose, cellulose acetate, cellulose propionate, cellulose buty- rate, cellulose valerate, polyvinyl acetate, polyvinyl formal, poly ⁇ vinyl butyral, polymethyl methacrylate, polycarbonate, polystyrene, polyester and polybutadiene.
• the water-permeable coating is, however, preferably a microporous membrane, for instance selected from poly- vinylchloride, and microporous polycarbonates, polyamides, acrylic copolymers and polyurethanes which permit a gradual diffusion of the fungal material, and if a water-permeable coating is used it is pre ⁇ ferred to combine it with an enteric coating so that diffusion of the fungal material is substantially prevented under gastric conditions.
• a microporous membrane for instance selected from poly- vinylchloride, and microporous polycarbonates, polyamides, acrylic copolymers and polyurethanes which permit a gradual diffusion of the fungal material, and if a water-permeable coating is used it is pre ⁇ ferred to combine it with an enteric coating so that diffusion of the fungal material is substantially prevented under gastric conditions.
• composition When the composition is to be administered to ruminants, it may be advantageous to provide a futher layer of coating as the coated gra ⁇ nules containing the fungal material.
• Such an outer coating layer may comprise a coating material which is soluble at an acid pH such as a pH of about 1.5- 2.5.
• a further coating layer on the composition of the invention, apart from the coating layer(s) provid ⁇ ing the desired release characteristics as described above.
• a coating layer serves to improve the handling properties, storage sta ⁇ bility, etc. of the composition.
• hydroxy ⁇ propyl methyl cellulose may be used.
• a preferred form of the composition is a bolus as .it per ⁇ mits administration of the fungal material at long intervals while ensuring a continuous release from the bolus of an amount of fungal material sufficient to obtain a satisfactory nematode control.
• the bolus may suitably be of the matrix type and may either be one which slowly dissolves in the rumen or which is gradually eroded in the rumen.
• Suitable bolus materials may be selected from a natural or synthetic wax, a plastic, a polymer (e.g. carboxymethyl cellulose or polyvinylpyrrolidone) , .
• the fungal material may conveniently be embedded in the bolus in the form of coated granules in order to protect the fungal material from ruminal fluids which are indicated (cf. Example 10 below) to be par ⁇ ticularly detrimental to the viability of the fungal material.
• the bolus may be prevented from leaving the rumen by its shape in ac ⁇ cordance with well-known practice or by including in the bolus matrix an agent which imparts an increased specific gravity to the composi ⁇ tion, e.g. barium sulphate, titanium oxide, a zinc oxide or an iron salt. .
• an agent which imparts an increased specific gravity to the composi ⁇ tion e.g. barium sulphate, titanium oxide, a zinc oxide or an iron salt.
• the amount of active fungal material incorporated in the bolus com ⁇ position may vary within wide limits, but is suitably adapted to the amount of fungal material which it is desired to provide in the faeces during the critical period as explained above.
• an ade- quate amount of fungal material in a bolus may be in the range of 1-15 g, in particular 2-10 g, per bolus, inter alia dependent on the rate at which the bolus disintegrates in the rumen which, in turn determines the interval at which a new bolus should be administered.
• the com ⁇ position of the invention may also be in the form of a feed block containing embedded coated granules comprising the fungal material.
• This form of the composition may, however, be less preferred since the amount of fungal material ingested by the animals by block feed ⁇ ing will not be uniform, depending on a variety of factors such as the availability of grazing or other feed or, most importantly, the variations in the individual uptake of the fungus from the feed block (some animals being less willing" to avail themselves of the feed block than others) .
• the block material may for instance comprise molasses as this may improve the palatability of the block and thereby contribute to more frequent use thereof by the animals.
• composition further comprises a chemical antiparasitic agent, such as a a thelmintic as discussed above, or another active agent such as a growth promoting agent, hormone, vitamin, micro- or macronutrient or amino acid. Any such further active agents may be incorporated in the composition in accordance with accepted practice in the field of veterinary pharmacology.
• a chemical antiparasitic agent such as a a thelmintic as discussed above
• another active agent such as a growth promoting agent, hormone, vitamin, micro- or macronutrient or amino acid.
• Any such further active agents may be incorporated in the composition in accordance with accepted practice in the field of veterinary pharmacology.
• the fungi from which the fungal material employed in the method and composition of the invention is derived may be grown under submerged conditions.
• the present invention further relates to a process for producing a fungal material of a nematode-destroying f ngus, which process comprises
• the process may be conducted under the ususal conditions for anaero ⁇ bic cultivation, such as in the presence of a suitable carbon source, e.g. selected from a sugar, e.g. sucrose, lactose, glucose, maltose, xylose, fructose, galactose, a starch, sodium lactate, malt extract, glucose syrup and lactose permeate, and a suitable nitrogen source, e.g. selected from asparagine, yeast extract, NaNO ⁇ , Bacto peptone, corn steep liquor and casamino acids.
• a suitable carbon source e.g. selected from a sugar, e.g. sucrose, lactose, glucose, maltose, xylose, fructose, galactose, a starch, sodium lactate, malt extract, glucose syrup and lactose permeate
• a suitable nitrogen source e.g. selected from asparagine, yeast extract, NaNO ⁇ , Bacto
• the fungal material is suitably harvested by filtration or centrifu ⁇ gation according to standard procedures, and the harvested mycelium may then be dried according to usual methods such as by means of dry air, e.g. in a fluid bed, in vacuum, by freeze-drying or by dessica- tion with a suitable dessicant such as anhydrous magnesium sulphate, silica gel, etc.
• a suitable dessicant such as anhydrous magnesium sulphate, silica gel, etc.
• the fungal material produced by the process of the invention may be derived from any of the fungal genera or species indicated above.
• the medium comprises a vegetable, organic or inorganic solid, support to which the fungus is able to adhere and on and/or in which it will grow.
• the solid support may suitably comprise a spongy or porous material such as seeds, e.g. poppy or sesame seeds, or an organic or inorganic polymer such as po ⁇ rous polyacrylic or glass beads.
• the fungal material by growing the fungus under aerobic conditions on a solid medium, the fungal material being either harvested from the medium or, if the medium is edible in itself and/or if the the concentration of fungal material is sufficiently high, the medium containing the fungal mate ⁇ rial may be used as such.
• the medium may suitably be inoculated with propargyles (the smallest segment capable of sporulation) of the fungal mycelium.
• the mycelium is subj cted to a gradual drying-out process in air after propagation of the fungus as this has been found to increase sporula ⁇ tion.
• the solid medium on which the fungus is grown may be any medium which has been found to favour the growth and/or sporulation of this type of organism.
• a suitable medium according to this criterion is one which contains a cereal component such as corn.
• the present invention further relates to a method of reducing the number of infectious nematodes in the environment of animals so as to reduce the transmission of nematode infections to animals inhabiting said environment, the method comprising administering to an animal a composition comprising a fungal material produced by submerged culti ⁇ vation as described above in order to provide an adequate nematode- controlling amount of the fungal material in the animal faeces.
• a composition comprising a fungal material produced by submerged culti ⁇ vation as described above in order to provide an adequate nematode- controlling amount of the fungal material in the animal faeces.
• the fungal material is preferably in protected form as indi ⁇ cated above, it may also be formulated into a composition where the fungal material is not protected.
• the fungal material may be formulated as granules to be admixed with the feed or spread over a pasture or an indoors enclo ⁇ sure for animals, or embedded as such in a bolus or feed block.
• Figs. la-Id show the number of loops per mm ⁇ and trapping efficiency of A . oligospora exposed to first-, second- and third-stage C. onco- 10 phora larvae and to R. choirgemuthi (juveniles and adults) . The ave ⁇ rage number of free individuals in fungus-containing dishes is ex ⁇ pressed as a percentage of that in fungus-free control dishes. Black. dots denote freely migrating nematodes in per cent of controls, and white dots denote the number of hyphal loops per mm . 5 Figs. 2a-2d show the number of loops per mm 7 and trapping efficiency of A.
• oligospora exposed to first- , second- and third-stage C . onco ⁇ phora larvae and to P . redivivus (juveniles and adults). The average number of free individuals in fungus-containing dishes is expressed as a percentage of that in fungus-free control dishes. It should be 0 noted that the figure on the scale for P. redivivus-induced loops are higher than those of the other scales. Black dots denote freely migrating nematodes in per cent of controls, and white dots denote the number of hyphal loops per mm 2 . 5 Fig. 3 shows the concentration of infective C. oncophora larvae in cowpats and surrounding grass at different times.. • denotes addition of A. oligospora to the faeces and A denotes no addition of A. oligo ⁇ spora to the faeces.
• FIGs. 4 and 5 show the concentration of infective C. oncophora larvae (L3) per gram of faeces either containing (+) or not containing (•) A. oligospora .
• Cornmeal agar plates containing 0.5 % of dextrose were inoculated with a 0.5 ml spore suspension of A. oligospora Fresenius (ATCC 24927) (used throughout the Examples). 22 days later, 1 x 1 cm seg ⁇ ments from the plates (containing mycelium and spores) were used to inoculate plates (9 cm in diameter) containing 2.7 % cornmeal agar (CMA; 77 % of cornmeal) media of different compositions:
• the plates were incubated at 26°C in the dark for 15 days and at 19- 23°C and 12 hours of light/12 hours of darkness a day for 14 days.
• the spores were harvested by 1) washing the plates with water, paraf- fin oil, 96% ethanol or water containing 0.1% of Tween ® 80, or 2) scraping off the spores by means of spatula without adding any li ⁇ quid.
• Procedure 1 was performed by adding 10 ml of washing liquid to the plates, loosening the spores by means of a spatula, filtering the spores on a B ⁇ chner funnel through a filter with a pore size of 8 um or centrifugating the spores at 6000 rpm. , drying the spores in a dessicator or by means of dry air at room temperature and storing the spores in a refrigerator or at room temperature.
• the spores obtained by procedure 2) were dried in a desiccator or by means of dry air at room temperature and stored in a refrigerator or at room temperature.
• the spores obtained by procedure 1) were subjected to a viability test by cutting the spore-containing filters into smaller pieces, placing the pieces in tubes and adding 2 ml of water. The tubes were vortexed, and a few drops were pipetted off and inoculated onto a CMA-9 plate. It was practically impossible to remove the spores from the filters resulting from the paraffin wash, so that no viability test could be carried out in this case. A sufficient amount of water was added to the tubes in which spores had been centrifugated to suspend the spores, and a few drops of the suspension were used to inoculate CMA-9 plates.
• the spores obtained by procedure 2) were tested for viability by sus ⁇ pending a loopful of the spores in water and inoculating them onto CMA-9 agar plates.
• the plates were examined for germinated and non-germinated spores and the viability of the spores was calcu ⁇ lated.
• procedure 1) when using procedure 1) , the best re ⁇ sults are obtained by using water as the washing liquid, drying the spores in a dessicator and storing the spores in a refrigerator. How ⁇ ever, procedure 2) is currently preferred as it comprises fewer steps and leads to a viability of nearly 100% after one week, which has only decreased by 2% after 3.5 months.
• Crushed corn was obtained by rolling, chopping or blending and sieved to a particle size fraction of about 1.25 mm.
• the crushed corn of a substantially uniform particle size was autoclaved for 30 minutes at 121°C and left in the autoclave overnight. 20 g of this medium was placed in petri dishes with a diameter, of 9 cm. To the medium was added 50-150% of water (calculated on the raw weight of the corn) .
• the dishes were inoculated with segments of A. oligospora mycelium, substantially as described in Example 1. The dishes were then incu ⁇ bated in the dark at 26°C.
• Growth of the fungus was determined as radial growth, i.e. the area of the petri dish covered by the fungus, and the growth intensity was graded as follows:
• the growth intensity of the fungus was determined by the degree to which the mycelium had grown through the substrate and was visible at the bottom of the petri dish. This was evaluated as fol- lows:
• CMA plates were inoculated with A. oligospora and incubated as de- scribed in Example 1. After 20 days, the plates were washed with 10 ml of sterile water, and the water containing the fungal material was used to inoculate shaking flasks (one plate per flask.) containing 100 ml of nutrient medium of the following composition:
• Czapek Dox medium 1 g of K 2 HP0 , 0.5 g of MgS0 » 7H 2 0, 0.5 g of KCl, 0.01 g of FeS0 4 , 1.5 g of CaCl 2 , 1-1 of distilled water, 1 ml of 1 g of ZnS0 4 /100ml of H 2 0, 1 ml of 0.5 g of CuS0 4 /100 ml of H 2 0;
• the flasks were incubated at 26°C on a shaking apparatus in the dark for one week.
• the contents of two flasks were used to inoculate a 10 1 fermenter (Braun) containing 7 liters of the nutrient medium defined above, corresponding to about 8-10 g of wet weight of A. oligospora .
• the fermenter was incubated for 3 days at 26 ⁇ 1°C, maintaining a cycle of 12 hours of light/12 hours of darkness/day.
• the initial pH in the fermenter was 5.8 which was maintained at this level by ti ⁇ trating with a 5N NH3 solution, using 100 ml in 3 days.
• the fermenter was aerated by means of an oxygen flow of 5.5 1/minute and rotated at 750 rp .
• 5% of an antifoaming agent was added, using 100 ml in 3 days.
• 6N HC1 was added to a pH of 7.0.
• the yield of mycelium (it should be noted that no sporulation was observed when the fungus was grown in submerged media) was about 700 g wet weight (i.e. about 10%).
• the fungus showed a filamentous growth which is particularly well suited for further processing; the filamentous growth is believed to be caused by the rotation at 750 rpm. All of the sugar in the malt extract had been consumed in 3 days, and it must be assumed that a more concentrated nutrient medium will lead to increased yields of mycelium.
• the plates were incubated at 12 hours of light/12 hours of darkness at 15, 20, 30, 37 and 40°C.
• the temperature optima of the different organisms were determined in terms of the maximum diameter of the growth zone.
• A. arthrobotryoides had a temperature optimum at 20°C
• a . tortor had a temperature opti ⁇ mum at 30°C
• all the other organisms tested had a temperature optimum at 25°C.
• the strain of A. oligospora Fres. was grown and main ⁇ tained on corn meal agar (CMA) adjusted to pH 7 as described by Lysek and Nordbring-Hertz (1981) (42) . This medium allowed good mycelial growth and formation of conidia but not trap development.
• Test Petri dishes 3.2 cm in diameter, were each filled with approximately 4 ml of CMA and inoculated with 3-5 weeks old fungal cultures. Using a metal cork borer (5 mm in diameter) , circular agar plugs were cut from the fungal lawn and placed with mycelium down in the centre of the CMA Petri dish.
• Soil nematodes Panagrellus redivivus was cultured in flasks con ⁇ taining a soy peptone-liver extract medium (Nordbring-Hertz, 1972 (43)). Rhabditis dockgemuthi was cultivated on serum agar plates (Monrad, pers. comm.). These nematodes were harvested from approxi ⁇ mately one week old cultures, using the Baer ann funnel technique, and washed several times by alternate centrifugations and resuspen- sions in sterile water. The resulting suspensions contained both adult and juvenile nematodes.
• Parasitic nematodes Eggs of Cooperia oncophora were harvested from the faeces of a calf carrying an experimental monospecific infection of the nematode. Larvae were allowed to develop in the faeces by a cultivation procedure of Henriksen and Korsholm (1983) (44) and were isolated by a modified Baermann technique. By starting cultures at different intervals, it was possible to have batches of L ⁇ , L and L3 larvae available simultaneously. The three external developmental stages of the larvae are L ] _ and L 2 in which the larvae are pre-infec ⁇ tive and feeding on bacteria, and L3 in which the larvae are encased in the cast cuticle of the second moult.
• the stage of development was checked by microscopic examination. Prior to use in the experiments, the larvae were washed by serial centrifugations and resuspensions in sterile water. ⁇ To compare the ability of the nematodes to induce capture organs (hyphal loops) and to become trapped in such organs, suspensions thereof were added to 4-day old cultures of A . oligospora . A drop of each nematode suspension adjusted to 100-150 nematodes per drop was added to each of three test and three control dishes. Counts of free- living nematodes included both adults and juveniles. To check whether any nematode-free substance of the inoculum would induce traps, fun ⁇ gus CMA dishes were exposed to one drop of the supernatant of each of the final nematode suspensions.
• test dishes were examined at 100 x using a binocu ⁇ lar microscope. Trap formation began as a stout branch erecting from a vegetative hypha. Subsequently it grew and curled back so its tip anastomosed with the parent hypha or with an adjacent trap already formed. Only traps which formed completely closed loops, either in isolated position or more commonly as part of complex, three-dimen ⁇ sional networks, were counted. Under the microscope, five randomly selected fields were counted in each dish giving a total of 15 fields. The average number of traps per mm was calculated.
• nematodes were also counted (x 20). Only the normal, freely moving individuals were enumerated. In order to ac ⁇ count for "natural" deaths among the nematodes, the average numbers of such free individuals recorded in the test dishes were expressed as a percentage of those in fungus-free control Petri dishes. In ad ⁇ dition, on several occasions the contact between individual worms and traps was studied in closer detail ' with higher magnification (100 x) .
• Series A included R . Commission - Calcitus and the three developmental stages of C. oncophora .
• Series B differed in that P . redivivus replaced R . congressgemuthi .
• the third stage (L3) C . oncophora larvae were added to the dishes where traps were already present and induced by the other nematodes .
• Fig. 1 (Series A) and Fig. 2 (Series B) show that the rates of trap development were virtually independent of the type of nematode added. After 3 hours, traps were induced in some of the nematode dishes, but at 6 hours traps were formed in all nematode-fungus combinations. Over subsequent hours, all dishes exhibited an almost parallel in ⁇ crease in number of traps. The dishes receiving nematode-free super- natants had no traps.
• Figs. 1 and 2 show that the decline in the numbers of the free migratory nematodes started 3-6 hours after they were added to the dishes. This coincided with the initiation of trap formation. After 9 hours, the majority of the nematodes were trapped and at 15 hours, there was an almost complete absence of freely migrating individuals. Checks using higher magnification (100 x) revealed that the majority of immobile nematodes were trapped. However, R. Carnegiegemuthi (Series A) seemed to present an exception in that a few migratory juveniles were observed at the end of the experiment.
• Faeces containing parasite eggs were obtained from a calf experimen ⁇ tally infected with a monoculture of Cooperia spp.
• Infective Cooperia spp. larvae were isolated from faeces by a modified Baermann tech ⁇ nique (J ⁇ rgensen & Madsen, 1982 (45)).
• Infective Cooperia spp. larvae were isolated from grass by the agar technique described by J ⁇ rgensen (1975) (46) and Mwegoha & J ⁇ rgensen (1977) (47).
• the inoculation material comprised both Arthro ⁇ botrys oligospora and its growth medium, in that the whole contents of Petri dishes were milled in a mincing machine.
• the milled material was subsequently divided into 10 portions of 150 g (dry weight 56 g) , which were added to each of 10 faecal portions of 1 kg.
• Another 10 faecal portions of 1 kg were kept as fungus-free controls. All the faecal portions, which contained a uniform distribution of 560 Coope ⁇ ria spp. eggs per gram, were placed as cow pats (diameter 18 cm) on a parasite-free pasture in June.
• the minimum distance between-the two groups was 2.5 m and the dis ⁇ tance between single cow pats in each group was 1.7 m.
• the inoculation in experiment 2 was rather large (150 g per 1000 g of faeces) and this itself could perhaps damage the composition of the habitat, e.g. its water content. Therefore, on day 21 and 36, 10 g of faeces from the two groups of cow pats were obtained and dried at 105°C for 48 hours, and the percentage water content was measured as; (Water loss (g)/weight of dry faeces (g)) x 100%.
• the nematode-trapping fungus could be demon ⁇ strated in the inoculated cow pats using the sprinkling technique on corn meal agar (Fowler, 1970 (48)).
• . lee ed faeces was immediately cooled to 5°C to prevent the eggs from .o developing.
• This material was subsequently divided into 24 portions of 1 kg of faeces which were moulded into cow pats with a diameter of 16 cm and a height at the centre of 3 cm.
• 12 of these experimental cow pats Prior to deposition in the pasture, 12 of these experimental cow pats were inoculated with 2 million A.
• oligospora conidia per 1 kg of faeces that is 2000 conidia/g
• 10 ml of fun ⁇ gus-free water had been added to the remaining cow pats which were used as controls.
• the fungus-inoculated and control cow pats were placed at intervals of 2.6 m in separate plots of pasture. The procedure described above was repeated three times so that the experimental cow pats were placed in the plots for four consecutive months at intervals of about four weeks.
• the cow pats were protected against dung-seeking birds by means of hemispherical nets with a diameter of about 50 cm and a mesh diameter of 6 cm.
• samples of a few grams of faeces were col ⁇ lected from the edge of the cow pats, and samples of grass were cut from the edge of the cow pats to a distance of 20 cm and in swaths of 7 cm. The grass was cut as close to the ground as possible.
• LPG infective 0. ostertagi larvae per gram of faeces
• parasite-free "tracer” calves were turned to grass for two weeks in the two plots, with and without fungus-inoculated cow pats, approximately two weeks after the last portion of cow pats had been placed in the pastures.
• the calves were then stalled for three weeks until they were slaughtered.
• the tracer calves were slaughtered and the number of parasitic nematodes in the abomasum were recorded. Recordings of the weekly maximum and minimum temperatures two metres above ground level and the total weekly precipitation were obtained at a weather station about two kilometers southeast of the experi ⁇ mental plots.
• calf No. 262 had an abnormal grazing behaviour in that it seemed to ingest the highly contaminated grass close to the cow pats. This grass Is normally avoided by calves as long as there is no general grass depletion in the pasture. If, therefore, the data from this calf presented in Tables 4 and 5 are deleted, a significant beneficial effect is ob ⁇ tained by adding viable A . Oligospora in cow pats both with respect to serum pepsinogen levels (0.6 in week 5) and worm counts (4630).
• the tracer calves had been turn ⁇ ed to grass at a time when the infective level of 0. ostertagi in fungus-treated and fungus-free plots approached comparable levels. This, too, might have contributed to the very small difference be ⁇ tween the two groups of tracer calves. If the tracer calves had been turned to grass two weeks earlier, the calves grazing in the fungus- treated plot would very likely have ingested a far smaller number of nematode larvae than the calves grazing in the fungus-free plot.
• A. oligospora The ability of A. oligospora to develop trapping devices and to trap a variety of animal parasitic nematodes in the third infective larval stage was examined, and comparisons were made with indigenous soil nematodes .
• Lar ⁇ vae were allowed to develop up to the third stage by standard culti ⁇ vation procedures and were subsequently isolated by modified Baermann techniques. Before use, the larvae were washed by centrifugations and resuspensions in sterile water. Stage of development and motility were checked by microscopial examination.
• the soil nematodes employed in this experiment were Fanagrellus redi ⁇ vivus which was cultured axenically in flasks containing soy peptone- liver extract (Nordbring-Hertz, 1972 (43)) and Rhabditis scrgemuthi which was cultured on bacteria-containing serum agar plates. Both species were subcultured once or twice a week. Before use in the ex ⁇ periment, they were harvested by baermannization and were subsequent ⁇ ly washed by alternate centrifugations and resuspensions in sterile water. The final suspensions contained both adults and juveniles.
• the fungus dishes were exposed to daylight and kept at room temperature (20-23°C) and 98- 100% of humidity.
• the mycelium usually reached the periphery of the dishes in 4-5 days.
• the dishes were supplied with a grid pattern on the bottom to facilitate nematode counting.
• the study comprised two series of experiments, each including a va ⁇ riety of the parasitic nematode species and in addition one or both of the soil nematodes (Tables 6 and 7) .
• the experiment started with adding one drop, i.e. approximately 500 nematode individuals of a given species, to the centre of a dish con ⁇ taining traps pre-induced by P. redivivus as described above.
• one drop was added to a fungus-free CMA dish.
• Nematodes were, observed under a stereoscope (x 20) starting 30 minutes after adding the nematodes and then with the same intervals as described above for trap recordings.
• the number of normal, freely moving individuals on the test dishes was expressed as a percentage of those on the fungus-free control dishes.
• the motility of the various nematodes and the behaviour of the captured individuals were studied in closer detail at higher magnification (x 100) .
• Table 7 shows that, in both series, A. oligospora possessed a high and almost instantaneous trapping efficiency towards all nematodes, in that approximately 80% or more were caught after 30 minutes. Most species were completely trapped after six hours.
• One interesting ex ⁇ ception was R. committeegemuthi where a few free individuals were recorded as late as 27 hours after the experiment had started. On one occa ⁇ sion, a strangled and killed female was observed from which larvae were gradually released, and this may perhaps explain the persistent finding of few individuals of this species.
• nematodes of all categories were occasionally seen migrating freely with traps and hyphal branches attached to their cuticle, suggesting that they had succeeded in liberating themselves. However, they were apparently recaptured.
• the nematodes on the fungus dishes were widely dispersed over the surface and in close contact with the hyphae. On the fungus-free control dishes, they tended to accumulate at the edge of the agar.
• P. redivivus 100 1 2 0 0 0 0 0 0 0 0 0
• the concentration of fungal material in the starting material was -1 determined by preparing 10 " , 10 and 10 dilutions of spores by mixing 1 g of hyphal material with 9 ml of sterilized tap water * .
• Pe ⁇ tri dishes containing CMA medium (as described in Example 1) were inoculated with 0.5 ml of fungal material per Petri dish (5 Petri dishes per dilution) and incubated at 25°C overnight. On the follow ⁇ ing day, 0.5 ml per Petri dish of a Panagrellus suspension was added, and the recovery of the fungal material was determined by observing any growth of the fungus after 2 weeks of Incubation. During the first week, the Petri dish was " kept only partly covered.
• 1 ml of the diluted fungal material was added to 9 ml of water, 9 ml of pepsin-HCl and 9 ml of filtered ruminal fluid in order to simulate the gastrointestinal passage of the fungal material in calves.
• the pepsin-HCl was prepared by dissolving 2 g of 1:10,000 pepsin (Sigma P 7000) in 850 ml of sterilized tap water, adding 100 ml of 1M HC1 and making up to 1000 ml with sterilized tap water. Ruminal fluid was taken on the day when the experiment was started.
• the flasks containing the spores and hyphae were incubated at 39°C in a shaking bath for the first day and then in a thermostatic cupboard. One flask was used for each incubation time. 1 ml of fungal material added to 9 ml of water was incubated at 25°C as a control. After each incubation time, the fungal material was inoculated in Petri dishes containing Panagrellus as described above. Five dishes were used for each incubation time.
• Control 0, 1, 2 days, 1 week.
• 25°C in water show that the fungal material is viable for at least a week.
• 5 parasite-free donor calves were each orally infected with a mono ⁇ culture of between 30,000 and 100,000 infective larvae (L3) of a Danish strain of Ostertagia ostertagi Stiles (Trichostrongylidae) . Samples of fresh faeces containing 0. ostertagi eggs were collected daily for experimental purposes and stored for not more than 6 weeks at 5°C.
• Arthrobotrys oligospora was cultivated in a liquid medium containing 42 g of liquid malt extract; 1.68 g of asparagine; 1.5 g of CaCl 2 , 1 g of K 2 HP0 4 -7H 2 0; 0.5 g of KCl; 0.01 g of FeS0 4 /100 ml of H 2 0 and 1 litre of distilled water substantially as described in Example 4.
• the fungus did not develop conidia in this medium.
• 200 ml portions of in ⁇ oculated media were cultivated for between 1 and 2 weeks at 20°C in shaking bottles to ensure aeration of the medium.
• the liquid medium was removed and replaced by tap water containing 0.1% Tween ® 80 and the mycelia in the suspension were fragmented by placing them in an "Osterizer" blender for 2 minutes. The size of the mycelia fragments was between 30 and 300 urn. Finally the suspension was adjusted to different levels of gram mycelia per ml. For each mycelial suspension used, the number of mycelia frag ⁇ ments per ml was counted and the percentage of viable (germinating) mycelia fragments was measured on a corn meal agar medium (Lysek & Nordbring-Hertz, 1981 (42)). Only newly harvested and fragmented mycelia were used in the field experiments.
• a faecal portion from the donor calves was thoroughly mixed for 20 minutes in a cement mixer.
• the count of 0. ostertagi in the faeces was found to be 700 eggs/g faeces.
• ten 500 g por ⁇ tions were prepared.
• Two 500 g portions of faeces were separately in ⁇ oculated with each of the following amounts of A. oligospora mycelia/ kg faeces; 0.0 g (control), 0.03 g, 0.075 g, 0.19 g or 0.48 g.
• the proportion of germinating mycelia fragments in the inocula was found to be 88%.
• the faecal portions which were moulded into dome-shaped cow pats (diameter 16 cm) , were placed in small plastic buckets with a bottom layer of 1 litre of parasite-free sterile soil.
• the cow pats were kept in the laboratory at 20°C and 60-90% RH. Three times a week each cow pat was sprinkled with 10 ml of water.
• the experiment was carried out on a parasite-free pasture which had not been grazed for several years.
• the soil was a brown sandy clay with a pH of 6.5 and an organic matter content of 4% of soil dry weight.
• the pasture was divided into two comparable plots of equal size (20 x 100 metres; 0.2 ha).
• Infective larvae were isolated by a modified Baermann technique (J ⁇ r ⁇ gensen & Madsen, 1982 (45). When tracer calves were subsequently turned to grass in the two plots, they destroyed the cow pats so that no faecal samples could be taken after that time. 35 Determination of inf ctive Ostertagia ostertagi larvae in herbage around the cow pats
• the height of the herbage was kept between 8 and 15 cm.
• Herbage within a distance of 20 cm from the cow pats was cut to a height of between 20 and 30 cm in order to simulate the grass tufts around cow pats which normally occur on grazed pastures.
• the calves were weighed at monthly intervals and rectal, faecal and blood samples were taken at biweekly intervals.
• Faecal egg counts were made according to a modified McMaster tech ⁇ nique (Henriksen & Aagaard, 1976 (49)) and faecal larval counts were made by the method described by Henriksen & Korsholm, 1983 (44) .
• Blood samples were analysed for serum pepsinogen and serum albumin. Serum pepsinogen was determined according to Ross et al. , 1967 (50) and serum albumin was determined by an immunological method described by Mancini et al. , 1965 (51).
• the abomasum of each calf was analysed by sieving 1/10 of the mixed content of each abomasum through a sieve with a mesh size of 200 ⁇ m to retain adult parasites.
• the abomasal mucosa were removed by scrap ⁇ ing with a knife.
• the isolated mucosa was digested in a pepsin/hydro ⁇ chloric acid mixture (1000 ml of H 2 0; 150 ml of IN HCl, 8 g of pepsin (1:3000)) at 39 ⁇ C for half an hour.
• the digested suspension was siev ⁇ ed through a fine sieve with a mesh size of 36 ⁇ m to retain larval stages of the parasite.
• the retained materials from the abomasal con ⁇ tents and the mucosa were preserved in iodine until the parasites were identified and counted.
• the weekly mean, maximum and minimum temperatures (°C) 2 m above ground level and the total weekly rainfall (mm) were recorded at a meteorological station situated 1 km from the plots.
• the seven different mycelial suspensions used contained 15-136 mil ⁇ lion mycelia fragments per 1 kg of faeces. The percentage of viable germinating fragments was found to be between 76 and 82%.
• the number of 0. ostertagi eggs per gram of faeces (EPG) in the seven batches of faeces from donor calves is shown in Table 13.
• the eggs per gram faeces (EPG) value in the first faecal batch was high (415) compared with the EPG counts from the remaining 6 batches (60-200) .
• the results presented in Table 13 also show that on average, admix ⁇ ture of A. oligospora mycelia fragments to half of the seven batches of faeces resulted in a 51% reduction in 0. ostertagi recoveries from faecal cultures.
• cow pat experiment was conducted (in the summer of 1987) , the weather was rainy with relatively low temperatures. Under these conditions, it took 0. ostertagi 1 to 3 weeks to develop from eggs to infective larvae. Therefore, no infective larvae were found in the rows of cow pats deposited 1 and 0 weeks before the tracer calves were turned to grass.
• infective 0. ostertagi larvae developed in inocu ⁇ lated cow pats than in control cow pats.
• the total number of infect ⁇ ive 0. ostertagi larvae measured in all rows of the inoculated cow pats in the period until the tracer calves were turned to grass was subject to a 42% reduction compared with the total number of infect ⁇ ive larvae measured in all rows of the uninoculated control cow pats during the same period.
• cow pats placed at the beginning of the experiment lead to increasing herbage infectivity about four weeks later. Most like ⁇ ly, the transmission of infective 0. ostertagi larvae from cow pats to herbage was facilitated by frequent rain about four weeks after the cow pats had been placed.
• infective 0. ostertagi larvae spread to the sur- rounding herbage in the fungus-inoculated plot than in the control plot.
• the total number of infective 0. ostertagi larvae measured in the herbage around cow pats in all rows in the inoculated plots was reduced by 50% compared with the total number of infective larvae measured around cow pats in all rows in the control plo.t over the first 8 weeks of the experiment.
• the predatory fungus A. oligospora was responsible for a 71% reduction in total herbage infectivity.
• L3PG infective 0. ostertagi larvae that developed in faecal cultures
• calves grazing in the inoculated plot were subject to a 41 and 33% reduction, respectively, compared with the group of calves in the control plot. These differences were statistically significant.
• the serum pepsinogen concentrations in both groups of calves rose to se- vere levels in the last part of the grazing period, which indicated that both plots were heavily infested with 0. ostertagi .
• CMA plates were inoculated with A . oligospora and incubated as de ⁇ scribed in Example 1. After 20 days, the plates were washed with 10 ml of sterile water and the water containing the fungal material was used to inoculate shake flasks containing 100 ml of a nutrient medium (YPG) containing 0.4% yeast extract, 0.1% KH 2 P0 4 , 0.05% MgS0 4 *7H 2 0 and 1.5% glucose. The flasks were incubated in the dark at 25°C on a shaking apparatus for 4 days. The fungal culture was then blended for 30 seconds at the highest speed In a Waring blender.
• YPG nutrient medium
• the culture medium was filtered off in vacua through a Whatman fil ⁇ ter.
• the harvested mycelium was suspended in isotonic saline (2.30 g of mycelium in 40 ml) .
• the suspension was adjusted to the following con ⁇ centrations per 5 ml corresponding to the dosage per 500 g of horse faeces:
• oligospora conidia were scraped from sporulating colonies, grown on YPG medium supplemented with 2% Difco Bacto agar and incubated at 25"C for 14 days in 12 hours of light/12 hours of darkness.
• the conidia were suspended in isotonic saline, and the following; dilutions were prepared:
• the germinating property of the conidia was 100% within 6 hours at 25°C which was tested on cellophane strips placed on 2% aqueous agar. Strips containing conidia were embedded in lactophenol cotton blue on slides and the percentage of germination was determined under a light microscope.
• A. oligospora 200 g portions of poppy seeds and sesame seeds were auto ⁇ claved in beakers containing 75 ml of deionized water. The beakers were then inoculated with 10 ml of a 3-day-old submerged culture of A. oligospora (cf. Example 4) grown on YPG medium containing 0.4% yeast extract, 0.1% H 2 P0 4 , 0.05% MgS0 » 7H 2 0 and 1.5% glucose at 180 rpm and 23-25°C.
• the inoculated seeds were covered with metal foil and incubated at 25 ⁇ 0.5°C for 21 days and were then air-dried at 20-25°C for 18 hours before coating and testing.
• the seeds were then coated with a coating material containing 44% Eu- dragit* RS 30 D (from Rohm Pharma GmbH, Federal Republic of Germany; a copolymer of acrylic and methacrylic acid esters with a low content of quaternary ammonium groups), 3% talc, 3% triethylcitrate, 50% wa ⁇ ter (w/w) and then with a coating material composed of 52.2% Eudra- git* L 30 D (from Rohm Phar a GmbH, Federal Republic of Germany; an anionic polymer based on polymethacrylic acid and acrylic acid es- ters), 1.7% talc, 1.7% triethylcitrate and 44.4% water.
• a coating material containing 44% Eu- dragit* RS 30 D from Rohm Pharma GmbH, Federal Republic of Germany; a copolymer of acrylic and methacrylic acid esters with a low content of quaternary ammonium groups
• 3% talc 3% triethylcitrate, 50%
• the respective coating materials were sprayed onto the seeds in a Uniglatt fluidized bed provided with a two-fluid nozzle.
• the coating materials which were stirred during the entire spraying process were fed to the fluidized bed through a peristaltic pump at 10-12 ml/mi ⁇ nu e.
• the pressure was 1 bar and the outlet air temperature 30°C.
• the amount of dry coating applied on the seeds was 3 mg/cm of Eudra- git ⁇ RS 30 D and 2 mg/cm 2 of Eudragit* L 30 D.
• oligospora colonized seeds to simulated gastric fluid (0.1 M HCl, 0.2% pepsin (Sigma) in water) was tested in order to determine whether the viability of the fungal ma ⁇ terial when passing through the gastrointestinal tract of an animal could be improved by providing the colonized seeds with a coating.
• the test was conducted in a Pharma Test (a laboratory stomach simula ⁇ tor device), type PTW, at 50 rpm and 38-40°C for 2 hours at pH 1.04 and 1 hour at pH 6.5 (deionized water adjusted with NaOH) . After the test, 50-65 poppy and sesame seeds, respectively, were incubated on YPG agar at 23 ⁇ 0.5°C for a maximum of 72 hours.
• Type of seed No. of No. of % seeds Cor- colonized by incubated seeds contain- rected A. oligospora seeds contain- ing viab- % x '
• Soprunov, F.F. "Predacious hyphomycetes and their application in the control of pathogenic nematodes", Academy of Sciences of the Turkmen SSR, Ashkhabad, 1958, pp. 1-292.
• Nansen et al. Veterinary Parasi tology, 1987, accepted, in press.
• Nansen et al. "Predacious activity of the nematode-destroying fungus, Arthrobotrys oligospora , on preparasitic larvae of Cooperia oncophora and on soil nematodes", Proc. Helminthol . Soc . Wash . 53 (2) , 1986, pp. 237-243-. 38. Roubaud, E. & Deschiens, R. , Comptes Rendus des Seances de la Societe de Biologie 135 , 1941, pp. 687-690.

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