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A Bell & Howell Information Company 300 North Zeeb Road. Ann Arbor. Ml 48106-1346 USA 313/761-4700 800/521-0600 EFFECT OF NEMATOPHAGOUS FUNGI ON THE DEVELOPMENT OF INFECTIVE LARVAE OF COMMON ENDOPARASITES OF HORSES, SHEEP, AND CATTLE
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the degree Doctor of Philosophy in the Graduate School of The Ohio State University
by
Jacqueline Bird, B.S., M.A., D.V.M.
*****
The Ohio State University 1995
Dissertation Committee: Approved by
K. Hinchcliff Adviser Department of Veterinary Preventive Medicine M. Wellman
R. P. Herd (Adviser) UMI Number: 9612150
OMI Microform 9612150 Copyright 1996, by OMI Company. All rights reserved.
This microform edition is protected against unauthorized copying under Title 17, United States Code.
UMI 300 North Zeeb Road Ann Arbor, MI 48103 To Will. ACKNOWLEDGEMENTS
I am grateful to my adviser, Dr. R. P. Herd, for his encouragement and support during this research. I am indebted to Dr. G.L. Barron for allowing me to access his laboratory for technical training, supplying fungal specimens used in this research, and generously permitting the use of several illustrations from his book, The Nematode-destrovina Fungi. Special thanks are due to Dr. LiHua Xaio for helpful discussions. Dr. George Majewski, Mr. Ken McClure, Mr. Gary Lowe, Mr. Bill Nichol and Mr. Jeremiah Snyder were very generous with their time and patience during critical times for handling animals and making collections. Mr. William Hamilton showed unflagging willingness to work with difficult animals at all hours and supplied some of the superb illustrations in this volume. Ms. Dolores Fisher patiently typed the many tables in this thesis. Suggestions that much improved the writing of this thesis came from Drs. Maxey Wellman and Kenneth Hinchcliff. A grant from the United States Department of Agriculture Formula Fund is gratefully acknowledged.
iii VITA
1983 ...... D.V.M. New York State College of Veterinary Medicine Cornell University
1979 ...... M.A. Department of Ecology and Evolution State University of New York at Stony Brook 1 9 7 1 ...... B.S. Michigan State University
PUBLICATIONS
1995 Bird, J. and R. Herd. In vitro assessment of two species of nematophagous fungi (Arthrobotrys oligospora and A. flagrans) to control the development of infective cyathostome larvae from naturally-infected horses. Veterinary Parasitololoay 56:181-187. 1994 Bird, J. and R. Herd. Nematophagous fungi for the control of equine cyathostomes. Compendium on Continuing Education 16:658-665. 1993 Bird, J. and L. Hudson. General Information and Physical Examination. In Atlas of Feline Anatomy for Veterinarians. L. Hudson and W. Hamilton, eds. W.B. Saunders Co. pp. 1-7.
iv 1980 Sokal, R.R., J. Bird and B. Riska. Geographic variation in Pemphigus populicaulis (Insecta: Aphididae) in Eastern North America. Biological Journal of the Linnean Society 14(2): 163-200. 1979 Bird, J. Geographic patterns in the variability of Pemphigus populicaulis in eastern North America. Master's Thesis. State University of New York at Stony Brook, Department of Ecology and Evolution. 1979 Bird, J . , D.P. Faith, L. Rhomberg, B. Riska, and R.R. Sokal. The morphs of Pemphigus populitransversusz Allocation methods, morphometric and distribution patterns. Annals of the Entomological Society of America. 72: 767-774.
FIELD OF STUDY
Major field: Veterinary Preventive Medicine Studies in: Veterinary Parasitology Dr. Rupert Herd, Adviser
v TABLE OF CONTENTS
DEDICATION...... ii ACKNOWLEDGEMENTS ...... iii
VITA ...... iv TABLE OF CONTENTS ...... vi LIST OF TABLES ...... X LIST OF FIGURES ...... xiii LIST OF PLATES ...... XV
CHAPTER PAGE I. LITERATURE REVIEW ...... 1 Introduction ...... 1 Taxonomic classification ...... 2 Functional classification ...... 3 Predacious fungi ...... 4 Endoparasitic fungi ...... 7 Egg-attacking fungi ...... 9 Criteria for the selection of fungi ...... 9 Growth r a t e ...... 10 Predatory activity ...... 10 Survival through g u t ...... 11 Production of chlamydospores ...... 13 Fungi which meet selection criteria ...... 14 Drechmeria coniospora ...... 14 Harposporium anguillulae ...... 15 Arthrobotrys oliaospora ...... 16 A. flaarans...... 17
vi E c o l o g y ...... 17 Geographic distribution ...... 17 Local distribution...... 18 Factors influencing growth ...... 21 Development of free-living larvae of parasitic n e m a t o d e s ...... 22 Persistence of free-living larvae of parasitic nematodes ...... 23 Horse ...... 23 Sheep and c a t t l e ...... 25 Nematophagous fungi in the control of parasitic nematodes ...... 26 Early w o r k ...... 26 Recent w o r k ...... 30 Arthrobotrys oliaospora ...... 30 Other fungal species ...... 35 In vitro stress tests and in vivo survival tests ...... 36 Commercial application ...... 41 S u m m a r y ...... 42
II. GROWTH AND PREDACIOUS ACTIVITY OF FOUR SPECIES OF NEMATOPHAGOUS FUNGI IN VITRO ...... 45 Culture and predacious activity ...... 44 Introduction ...... 45 Materials and methods ...... 45 R e s u l t s ...... 56 D i s c u s s i o n ...... 60 S u m m a r y ...... 66 Quantitative studies of predacious activity: Preliminary observations ...... 67 Introduction ...... 67 Effect of three fungal concentrations (spores- to-egg ratios) on the development of infective cyathostome larvae in vitro ...... 67 Materials and methods ...... 67 Results ...... 70 Discussion...... 71 Effect of two fungal concentrations (spores-to- egg ratios) on the development of infective trichostrongylid larvae in vitro ...... 73 Materials and methods ...... 73 R e s u l t s ...... 75 Discussion...... 75
vii Effect of three fungal concentrations (spores per gram of feces) on the development of infective cyathostome larvae in vitro ...... 77 Materials and methods ...... 77 R e s u l t s ...... 78 Discussion...... 79 S u m m a r y ...... 81 Quantitative studies of predacious activity: Major observations ...... 82 Introduction ...... 82 Materials and methods ...... 82 R e s u l t s ...... 93 D i s c u s s i o n ...... 101 S u m m a r y ...... 107
Quantification of fungi in feces ...... 108 Introduction ...... 108 EIDso method ...... 109 Estimation of spore concentrations in water . . . 110 Materials and methods ...... 110 R e s u l t s ...... Ill Discussion...... 112 Estimation of spore concentrations in feces of horse, sheep, and c o w ...... 113 Materials and methods ...... 113 R e s u l t s ...... 114 Discussion...... 116 III. PREDACIOUS ACTIVITY OF ARTHROBOTRYS OLIGOSPORA AND A. FLAGRANS IN V I V O ...... 118 Dosing trials of fungi in horses, sheep and c a t t l e ...... 118 Introduction...... 118 Materials andmethods ...... 118 R e s u l t s ...... 123 Discussion...... 124 Effect of fungal dosing of livestock on migration of infective nematode larvae to pasture . . . 127 Introduction...... 127 Materials andmethods ...... 127 R e s u l t s ...... 138 Discussion...... 145 S u m m a r y ...... 150 APPENDICES ...... 152 A. T a b l e s ...... 153 B. F i g u r e s ...... 203 C. P l a t e s ...... 240
viii D. Procedures for making nutrient media used in culturing ...... 249 E. Method of estimating the number of spores per ml in suspension ...... 2 5 3 F. Calculation of estimated number of fungal propagules in feces from cultures of serial dilutions of feces ...... 2 5 5 G. Protocol for collection and recovery of infective larvae on pasture ...... 2 5 8
REFERENCES ...... 2 6 2
ix LIST OF TABLES
TABLE PAGE
1 . Mean number of infective cyathostome larvae
recovered from horse feces mixed with 0.1, 1 / or 10 spores of Arthrobotrvs oliaospora and A. flaarans per parasite eggs...... 153
2 . Mean number of infective larvae recovered from sheep feces mixed with 1 or 10 spores of Arthrobotrys oliaospora and A. flaarans per parasite egg ...... 154
3. Mean number of infective cyathostome larvae recovered from horse feces mixed with 50, 100, or 200 fungal spores per gram of feces. 155
4. Percent reductions in the number of infective larvae recovered from equine fecal cultures with different concentrations of Arthrobotrvs oliaospora and A* flaarans . 156
5. Sample size determination for in vitro studies ...... 157
6 . Percent loss of parasite eggs associated with sedimentation technique for harvesting eggs from large quantities of feces...... 158
7. Percent of total infective larvae recovered from control fecal cultures comprised by different genera ...... 159
8 . Mean number of total infective larvae recovered from low and high egg count horse feces mixed with 0, 400, or 800 spores of Arthrobotrys oliaospora and A* flaarans per gram of feces ...... 162
X Mean number of infective cyathostome larvae recovered from low and high egg count horse feces mixed with 0, 400, or 800 spores of Arthrobotrvs oliaospora and A* flaarans per gram of feces ...... 165
10. Mean number of total infective larvae recovered from low and high egg count sheep feces mixed with 0, 400, or 800 spores of Arthrobotrys oliaospora and A* flaarans per gram of feces ...... 168
11. Mean number of infective Haemonchus larvae recovered from low and high egg count sheep feces mixed with 0, 400, or 800 spores of Arthrobotrys QjLig.Qg.ppra and A- .Oagrans_ per gram of feces ...... 171
12. Mean number of infective Trichostronaylus larvae recovered from low and high egg count sheep feces mixed with 0, 400, or 800 spores of Arthrobotrvs oliaospora and A- flaarans per gram of feces ...... 174 13. Mean number of infective Teladorsaaia larvae recovered from low and high egg count sheep feces mixed with 0, 400, or 800 spores of Arthrobotrvs oliaospora and A- flggrans, per gram of feces ...... 177 14. Mean number of total infective larvae recovered from low and high egg count cattle feces mixed with 0, 400, or 800 spores of Arthrobotrys oliaospora and A« flaqrans per gram of feces ...... 180 15. Mean number of infective Cooperia larvae recovered from low and high egg count cattle feces mixed with 0, 400, or 800 spores of Arthrobotrvs oliaospora and A- fiagrans per gram of feces ...... 183 16. Mean number of infective Ostertaaia larvae recovered from low and high egg count cattle feces mixed with 0, 400, or 800 spores of Arthrobotrys oliaospora and A* flaarans per gram of feces ...... 186 17. Estimated number of spores suspended in water by the modified EID5Q method and by direct count using a hemacytometer ...... 169
xi 18. Estimated number of spores/g equine feces by the modified EIDso method and by direct count using a hemacytometer...... 191
19. Estimated number of spores/g ovine feces by the modified EIDBO method and by direct count using a hemacytometer...... 193 20. Estimated number of spores/g bovine feces by the modified EIDso method and by direct count using a hemacytometer...... 195 21. Estimated number of viable fungal elements excreted per gram of feces from fungal-fed horses, sheep, and cattle sampled every 12 hours for 48 hours after dosing...... 197 22. Mean number of infective larvae per kg herbage surrounding treated and control equine dung pats...... 198 23. Estimated number of fungal spores excreted per gram of feces immediately prior to and 24 hours after dosing horses, sheep, and cattle...... 199 24. Mean number of infective larvae per kg herbage surrounding treated and control ovine dung p a t s ...... 200 25. Mean number of infective larvae per kg herbage surrounding treated and control bovine dung pats...... 201 26. Dates of dung pat placement on pasture and herbage collection...... 202
xii LIST OF FIGURES FIGURE Ut£
1 . Capture organs of predacious fungi ...... 204
2 . Endoparasitic fungi ...... 206 3. Egg-attackig fungus...... 208 4. Drechmeria coniospora ...... 210 5. Harposporium anauillulae ...... 212
6 . Baermann apparatus ...... 214 7. Pooled data on fecal cultures inoculated with Arthrobotrys oliaospora and A. flaarans from two preliminary equine studies...... 216
8. Percent reduction of total infective larvae and cyathostomes in Arthrobotrys oliaospora- treated equine feces cultured for 8, 16, and 24 d a y s ...... 218
9. Percent reduction of total infective larvae and cyathostomes in Arthrobotrys flaarans- treated equine feces cultured for 8, 16, and 24 d a y s ...... 220
10. Percent reduction of total infective larvae, Haemonchus. Trichostronaylus. and Teladorsagia larvae in Arthrobotrvs oliaospora-treated ovine feces cultured for 8, 16, and 24 days . 222
1 1 . Percent reduction of total infective larvae, Haemonchus. Trichostronaylus. and Teladorsagia larvae in Arthrobotrvs flagrans-treated ovine feces cultured for 8, 16, and 24 days . 224
12. Percent reduction of total infective larvae, Cooperia and Ostertaaia larvae in Arthrobotrys oligospora-treated bovine feces cultured for 8, 16, and 24 d a y s ...... 226
xiii 13. Percent reduction of total infective larvae, Cooperia and Ostertaaia larvae in Arthrobotrys flaarans-treated bovine feces cultured for 8, 16, and 24 days ...... 228
14. Percent reduction of total infective larvae in fungal-treated equine feces cultured for 8, 16, and 24 days...... 230
15. Percent reduction of total infective larvae in fungal-treated ovine feces culture for 8, 16, and 24 days...... 232 16. Percent reduction of total infective larvae in fungal-treated bovine feces culture for 8, 16, and 24 days...... 234
17. Visser filter apparatus ...... 236
18. Percent reduction of total infective larvae harvested from herbage surrounding fungus- treated feces at 4 and 8 weeks after place ment of treated and control dung pats from horses, sheep, and cattle ...... 238
xiv LIST OF PLATES PLATE EPGE I. Chlamydospores intercalated along the length of hyphae of Arthrobotrys flaarans grown in c u l t u r e ...... 241 II. Cyathostome infective larva with numerous Drechmeria coniospora conidia adhered to the cuticle...... 243 III. Several Arthrobotrys oliaospora spores with curving cells forming rings ...... 245 IV. Sheep fitted with size 44 jockey briefs for fecal collection during a 1-2hour period. . 247
xv CHAPTER I. LITERATURE REVIEW
INTRODUCTION In its current efforts to control parasites, the livestock industry faces serious problems of drug resistance, drug residues, ecotoxicity, and the limited ability of anthelmintics to kill encysted and hypobiotic nematodes (Herd, 1993; Waller, 1993). Heavy reliance on chemotherapy for parasite control serves to enhance these problems. Economic losses due to parasite infection of livestock in the USA were estimated at more than $3 billion annually by Drummond et al., (1981). Today, the costs may even be higher. Therefore, the need to investigate alternative methods of parasite control are of great importance. Microscopic fungi have been described that are natural enemies of both free-living and parasitic nematodes (Drechsler, 1933). These fungi are mostly found in the upper layers of the soil under widely varying conditions of moisture content, pH, and organic content (Gray, 1987). Their ability to infect and kill nematodes has led to
1 speculation about their use for the control of pre-parasitic larvae of parasitic nematodes of livestock of major economic importance. Ideally, control would be achieved by feeding fungi to livestock that then excrete viable fungal components in their dung. In this way, the fungi are present at the site of the development of the larvae, potentially reducing present and future pasture infectivity.
NEMATOPHAGOUS FUNGI TAXONOMIC CLASSIFICATION Nematophagous fungi are a functional grouping of diverse fungi that independently evolved the ability to infect, kill and derive nutrition from nematodes. Fungal systematic classification is based on morphological characteristics and assumptions about the level of relatedness they represent. For example, fungi with nonseptate hyphae are considered more primitive than those with septate fungi. Because the nematode-destroying fungal grouping is a functional one, a great diversity of taxonomic classes are present (Barron, 1977). They include many of the lower (Chytridiomycetes. Oomycetes. Zygomycetes) and higher (Deuterorovcetes and Basidiomvcetes) groups. Morphology of both conidia and trapping mechanism are important for identification of nematophagous fungi. However, unrelated fungi can exhibit similar trapping mechanisms. For example, certain Zygomycetes produce adhesive conidia as do some Deuteromvcetes. There has been some confusion in the biological control literature concerning species of fungi belonging to the genera Arthrobotrvs and Duddinatonia. Schenck et al., (1977) revised the genus Arthrobotrys so that it included
nematode-trapping species of Dactylaria. Trichothecium. Didvroosophaga. Genicularia. Candelabrella. Dactylarjopsis, and Duddingtonia. This revision has been accepted and adopted by authorities such as Barron (1977), but has yet to gain universal acceptance. Workers in Denmark who have made major contributions to the study of nematophagous fungi for parasite control of livestock continue to recognize the genus Duddingtonia. in this thesis,- the classification schemes of Barron (1977) and Schenck efc al. (1977) have been followed with one exception. Owing to its recent reclassification (Gams and Jansson, 1985), the genus Meria has been renamed Drechmeria.
FUNCTIONAL CLASSIFICATION In addition to the taxonomic classification described above, it has been useful to classify nematophagous fungi into functional groups. Duddington (1962) classified all nematophagous fungi as predacious, but Barron (1977) later distinguished between those fungi that entrapped nematodes using capture organs that develop along the length of their hyphae (predacious fungi) and those which infected nematodes by adhesion, ingestion and subseguent penetration of the worm by conidial spores (endoparasitic fungi). Nordbring- Hertz (1988) proposed a third functional class, egg- attacking fungi, which invade eggs and females of cyst and root-knot parasitic nematodes. The main characteristics of the predacious fungi, endoparasitic fungi, and egg-attacking fungi are described below.
1. Predacious fungi Predacious fungi were the first nematode-destroying fungi described. Voronin (1870) described a fungus (Arthrobotrys oliaospora) with networks of rings produced along the hyphae. Zopf (1884) subsequently identified the function of networks as trapping organs. Drechsler (1933) later observed that the development of fungal traps was induced by the presence of nematodes. Couch (1937) experimentally demonstrated that trap induction was mediated by metabolites produced by nematodes. He demonstrated that a species of predacious fungus, Dactylella bembicoides. grew as a saprophyte producing few or no traps in pure culture, and trapping organs developed after the fungus was exposed to a suspension of nematodes. Commandon and deFonbrune (1938) showed that the sterile filtrate in which nematodes had been living induced ring formation. Predacious fungi develop an extensive mycelium with specialized capture organs being formed at intervals on each hypha. In pure culture, most nematophagous fungi rarely produce traps until induced by the presence of nematodes or
other live organisms, such as earthworms, fly larvae, or springtails (Nordbring-Hertz, 1968). The motility of the nematodes strongly influences this induction. More active species of nematodes induce a stronger response than sluggish ones (Jansson and Nordbring-Hertz, 1980; Nansen et al., 1988). Commandon and deFonbrune (1938) showed that water in which nematodes had been living induced trap formation. This ability was lost after heating the solution to 100°C, indicating that nematode secretions/excretions were involved. Numerous other substances of animal origin have been shown to induce trap formation, including blood serum and earthworm extract (Roubaud and Deschiens, 1939a). Different species of nematode-destroying fungi vary in their response to stimuli, and different isolates of the same species can show a wide range of trap induction activity to the same stimulus (Cooke, 1977). The different types of trapping organs have been reviewed by Barron (1977). The most primitive type, adhesive hyphae, has an adhesive material that covers the 6 entire surface of the hyphae. Prey can be caught at any site along the hyphae. This type has been found only in the lower, non-septate fungi. A second type of trapping organ produces erect adhesive branches along the length of prostrate, non-adhesive hyphae (Figure Id). This type is considered the most primitive observed in the higher nematophagous fungi and occurs in very few predacious fungi (Barron, 1977). Higher fungi commonly have capture organs consisting of stalked knobs coated with adhesive material (Figure la,b,c). Fungi bearing these adhesive knobs commonly produce non-adhesive solitary rings at intervals along the hyphae (Figure le). These solitary rings capture nematodes when they wedge themselves during an attempt to glide through. Another modification for capturing nematodes is the constricting
ring (Figure lh). Each ring is composed of three cells that instantly expand inward when touched on the inside surface by a nematode, tightly constricting its body. The most common trapping devices are adhesive loops
arranged in two or three dimensional networks (Figure lf,g). Adhesive material is elaborated over the entire looping network. The networks may be prostrate, but are commonly oriented perpendicular to the surface they grow on. Pandey (1973) studied 10 species of nematophagous fungi for their trapping behavior toward Trichostronaylus axei and 7 Ostertaaia ostertaai and documented that adhesive networks were more efficient at attacking nematodes than any other kind of trapping organ. There are two kinds of trap-forming fungi: spontaneous trappers which form traps in the absence of nematodes, and non-spontaneous trap formers which produce traps in response to the presence of nematodes. spontaneous trap-forming nematophagous fungi and endoparasites appear to have the greatest potential for parasite control because they are more widely distributed and nonselective in predation (Gray, 1987). Most studies on biological control have involved non-spontaneous trap-forming predacious fungi, particularly
A. oligospora.
2. Endoparasitic fungi Endoparasitic nematode-destroying fungi have no extensive hyphal development outside the body of the host. In most species, only specialized structures producing spores occur externally: evacuation tubes for lower fungi and conidiophores bearing conidial spores for higher fungi (Barron, 1977) (Figure 2). Infection occurs either by adhesion to or ingestion by the nematode. In the lower fungi, zoospores occur. They are spores which move by use of one or more flagella. Under dry conditions, they move by amoeboid locomotion. Catenaria anauillulae. the most commonly occurring endoparasitic fungus, has zoospores capable of following a chemical gradient laid down by nematodes (Jansson and Thiman, 1992). They encyst on the cuticle of the nematode and subsequently
infect them. Other endoparasitic fungi utilize adhesive spores which
adhere to the cuticle of the nematode, penetrate and begin to develop assimilative hyphae called thalli inside the bodies. Nematodes are attracted to these adhesive conidia by chemical stimuli, often acquiring adhered conidia at
their anterior end at the sites of their chemosensory organs (Jansson, 1982a,b; Jansson and B. Nordbring-Hertz, 1983). Endoparasitic fungi that rely on the ingestion of their spores for infection have undergone remarkable modifications in spore morphology. These shape alterations aid in the ability of the ingested spores to become lodged in the buccal cavity or esophagus of the nematode (Barron, 1977). For example, the spores of Harposporium anauillulae resemble crescents with sharply pointed ends which become lodged in the esophagus immediately following ingestion. The mycelium of Harposporium is strongly attractive to nematodes. Nematodes are drawn into the region of the mycelium and ingest the non-attracting conidial spores as they feed (Jansson, 1982a,b). A lag time exists between infection by endoparasitic fungi and subseguent death of nematodes. Infected nematodes exhibiting considerable hyphal growth within their bodies have been observed to be quite active (Barron, 1977). The ability of nematodes to be active after infection enhances dispersal of the fungus.
3. Egg-attacking fungi Egg-attacking fungi infect the eggs or the females of cyst and root-knot parasitic nematodes by the ingrowth of vegetative hyphae; no specialized traps or spores have been observed (Figure 3). Some egg-attacking fungi appear to have the ability to detect the presence of eggs by as yet undefined factors and respond by growing hyphae to the eggs (Ellis and Hesseltine, 1962).
CRITERIA FOR THE SELECTION OF FUNGI Peloille (1991) listed the following criteria for consideration when selecting nematophagous fungi for biological control: (l) growth rate, (2) predatory activity, (3) ability to survive gut transit, (4) ability to produce chlamydospores. 10
GROWTH RATE Cooke (1963a) found that species of fungi producing adhesive branches and adhesive knobs grew faster than species producing adhesive networks. By contrast, Pandey
(1973) observed that adhesive branch and adhesive knob producers grew slower than adhesive network producers. These contradictory results may be due to the different species examined in each study. Olthof and Estey (1965) classified 10 species of nematophagous fungi as fast-, intermediate-, and slow-growing and found no correlation between species within the same genus, or between species with the same kind of nematode-trapping organ. Some variation in the temperature for optimum growth was seen, but all grew well between 20-25°C. Gronvold et al. (1985) reported that the predacious fungus, &. oliqospora. grew well at 7-20°C and a pH of 5 to 8. cow pats normally have a pH between 7 and 8, while temperatures vary widely. A. oliqospora and A. flaarans grew rapidly while Drechmeria coniospora and H. anauillulae grew slowly in culture at room temperature (Waller and Faedo, 1993).
PREDATORY ACTIVITY Investigation of nematophagous fungi and their predacious activity documented their ability to kill infective larvae of parasitic nematodes including 11
Ancvlostoma duodenale (Roubaud and Deschiens, 1939b; Soprunov, 1958), Bunostomum spp. (Roubaud and Deschiens, 1939b), Cooperia oncophora. £. curticei (Nansen et al., 1986, 1988; Gronvold et al ♦ . 1985, 1987), cyathostomes (Bird and Herd, 1995; Nansen et al., 1988), Dictvocaulus viviparus (Nansen at al., 1988), Haemonchus contortus (Gruner et al-, 1985; Hashmi and Connan, 1989; Mendoza-de Gives et al., 1992; Nansen et al., 1988; Parnell and Gordon, 1963), Necator americanus (Soprunov, 1958), Nematospiroides dubius (Nansen et al-, 1988), Oesophaqostomuin dentatum. and o. quadrispinulatum (Nansen et al- / 1988), Ostertaqia ostertaqi
(Gronvold et al- / 1988, 1989; Larsen et al-, 1991; Nansen et al-, 1988; Pandey, 1973), Stronqvloides fullerborni and S. papillosum (Roubaud and Deschiens, 1939b), Teladorsaqia circumcincta (Gruner et al-/ 1985; Hashmi and Connan, 1989; Peloille, 1991), Trichostrongvlus axei (Pandey, 1973) and T. colubriformis (Murray and Wharton, 1990). in general, fungi producing adhesive networks and adhesive branches were more efficient predators than species forming constricting rings and adhesive knobs (Pandey, 1973).
SURVIVAL THROUGH GUT Deschiens (1939) first proposed feeding spores of nematode-destroying fungi to animals to disseminate spores in their dung. Early feeding trials indicated a measure of 12
safety to both pasture and livestock when high levels of fungi were ingested (Deschiens, 1941). Descazeaux and Capelle (1939) reported that conidial spores of the predacious fungi, Arthrobotrvs oliqospora and Dactvlella bembicoides. when fed to horses and guinea pigs, lost their viability after transit through the gut. Kornienko, Tendetnik and Charyev (1955) (cited in Soprunov, 1958) fed A* oliqospora spores to a donkey for 5 days and were able to isolate the fungus from excreted feces from day 2 to day 9. No adverse effects were associated with the feeding of spores during the study or at necropsy. Parnell and Gordon (1963) suggested that the
nematophagous fungus, Acrostalagmus (VerticiIlium) spp., survived gut passage in sheep. Decreased numbers of infective larvae were associated with its presence in fecal samples that had been collected in bags attached to the sheep. The authors assumed that the fungus had been ingested. However, contamination from the bag or other handling could not be ruled out.
Dactvlaria Candida. Arthrobotrvs mWBi^PrniB, A* tortor
(Gruner e£ al., 1985) and A. flaqrans (Peloille, 1991) were isolated from feces after dosing lambs and sheep. Hashmi and Connan (1989) reported that A* oliqospora was passed in the feces of cattle throughout the grazing season and peaked in spring and late summer. They also fed conidia of this species to calves and isolated the fungus in feces for five consecutive days after dosing. Larsen gt al. (1992) documented that two isolates of £. flacrrans grown on barley grains survived passage through the gut after dosing a single calf. They estimated the dose to be approximately one billion chlamydospores per calf per day for four days. One strain of A. oliqospora (ATCC 24927) did not survive passage through cattle, goats and pigs (Gronvold gt al. ,
1993a). Waller gt al- (1994) demonstrated survivability of
&. oliqospora. &. oviformis. and Geniculifera gudermata after dosing sheep with 1.2 - 2.4 million spores of each species.
PRODUCTION OF CHLAMYDOSPORES Chlamydospores are hardy, thick-walled structures formed from solitary or short chains of adjacent intercalary cells of a hypha (Talbot, 1971) (Plate I). They remain as viable units in the hypha when the hypha decays, functioning as resting spores. They play a significant role in survival during adverse environmental conditions. They are common among both predacious and endoparasitic fungi (Barron, 1977). However, the level at which they are produced varies highly. In A. oliqospora, chlamydospores are produced very rarely. However, another species, A. flaqrans. produces 14 them in great abundance. The endoparasite, Harposporium anauillulae. also produces numerous resting spores. Not all nematode-destroying fungi produce chlamydospores (Barron, 1977) and it is unknown how common the production of
chlamydospores is in the natural environment (Nordbring- Hertz, 1988). Peloille (1991) speculated that chlamydospores better survive transit through the gut of livestock.
FUNGI WHICH MEET SELECTION CRITERIA Four species of fungi which meet the selection criteria were selected for study. They belong to the class Deuteromycetes (Fungi Imperfecti), a diverse group of higher fungi for which sexual stages are undiscovered. Of the four species of fungi in this study, two were endoparasitic and two were predacious.
DRECHMERIA CONIOSPORA fi. coniospora Drechsler 1941 is an endoparasitic
fungus. It infects nematodes by adhesion of spores to the cuticle followed by hyphal penetration of the nematode. The hyphae are limited to the confines of the host nematode, except for short fertile hyphae that pass out of the body after death (Figure 4). Conidial spores are formed off these hyphae and released into the environment. These 15
conidia are nematode-attracting, which optimizes their ability to infect new hosts when numbers of nematodes are low (Gray and Bailey, 1985). Jansson (1982b) reported that this fungus possessed greater ability to reduce nematode numbers in soil compared with A. oliqospora. D. coniospora is commonly isolated from agricultural soils (Duddington, 1951b; Barron, 1977; Gray, 1983; Mahoney and Strongman, 1994). This characteristic may indicate an adaptation to the type of environment that animal pre- parasitic larvae exist in and an ability to exploit highly variable conditions. The potential for increasing the local
area over which this fungus acts is enhanced by the lag time between initial infection and death of the nematode (Barron, 1977).
HARPOSPORIUM ANGUILLULAE Infection by the endoparasite, H. anauillulae (Lohde 1874) Karling 1938, requires ingestion of arcuate conidial spores which are pointed at both ends. They lodge themselves in the esophagus and germinate into the body of the nematode (Figure 5). The hyphae are limited to the confines of the host, except for the short fertile hyphae that pass out of the body after death. These fertile hyphae form phialides which bear the spores that are released into the environment. These fungi also have been commonly isolated from agricultural soils (Gray, 1983). The mycelium that develops inside the host strongly attracts nematodes
(Jansson, 1982b). Jansson (1982b) suggested that nematodes are attracted to the vicinity of infected nematodes and incidentally ingest the non-attracting conidia while feeding. A lag period occurs between infection and death of the nematode, allowing infected nematodes to migrate, increasing the area over which the fungus is dispersed (Barron, 1977). Because it relies on ingestion for infection, only the feeding larval stages (first and second) would be susceptible to infection since the infective third stage larva (L3) does not feed. Thick-walled chlamydospores are commonly formed inside infected hosts.
ARTHROBOTRYS OLIGOSPORA A. oliqospora (Fresnius 1852) Drechsler 1937 is a predacious fungus which forms hyphal branches that anastomose to form complex three-dimensional adhesive networks used to trap nematodes. This species of fungus is readily cultured in the laboratory. Trapped nematodes are penetrated by an infective thallus. Assimilative hyphae develop inside the host body, absorbing the nutrients of the worm. Abundant conidial spores are produced on tall unbranched conidiophores. Larger, thick-walled chlamydospores, a resting stage, are occasionally produced. 17 In the presence of nematodes, many trapping organs are formed. This fungus is commonly found in agricultural soils (Barron, 1977; Duddington, 1951a; Gray, 1983). Most studies on biological control of animal parasites have used this
species.
ARTHROBOTRYS FLAGRANS A- flaarans Duddington 1950 is another predacious fungus which forms adhesive networks similar to A* oliqospora. This species is readily cultured in the laboratory. Conidial spores are rarely produced, but larger, hardier chlamydospores occur in great abundance, intercalating along the lengths of hyphae. This species can be isolated from agricultural soils (Barron, 1977).
ECOLOGY GEOGRAPHIC DISTRIBUTION Surveys have shown that nematophagous fungi occur throughout the world and in all types of climate. Surveys have been conducted in the U.S.A.: Maryland (Drechsler, 1937, 1950), Oregon, (Tolmsoff, 1959) , Florida and North Carolina (Feder, 1962), California (Mankau and Clark, 1959), and Illinois (Monoson and Williams, 1973; Monoson et al., 1975); in Canada: Quebec (Estey and Olthof, 1965) and Ontario (Barron, 1978); in Europe: England (Duddington, 18 1940, 1946, 1950, 1951a, b, 1954; Juniper, 1957); Scotland (Mackenzie, 1960); Ireland (Gray, 1983, 1984); Italy (Verona and Lepidi, 1970); Poland (Jarowaja, 1963); Germany (Fritsch and Lysek, 1983); France (Commandon and deFonbrune, 1938; Virat, 1977; Peloille, 1979, 1981; Peliolle and Cayrol, 1979); the former U.S.S.R. (Soprunov and Galuilina, 1951; Soprunov, 1958; Mekhtieva, 1972); Denmark (Shepherd, 1956) and Finland (Ruokala and Salonen, 1967; Salonen and Ruokola, 1968); in Asia: India (Das-Gupta, Shome and Shome, 1964; Sachchidanandia and Swarup, 1966, 1967; Dayal and Nand, 1973; Dayal and Gupta, 1975; Dayal and Singh, 1975; Dayal and Srivastava, 1978); Malaysia (Kuthubutheen, Muid and Webster, 1985); Japan (Muira, 1973; Mitsui, 1983;, 1985; Kobayashi and Mitsui, 1975); in Australia: Queensland (McCulloch, 1977); Western Australia (Tan-Han-Kwang, 1966);
in New Zealand (Fowler, 1970; Wood, 1973); and in the maritime Antarctic (Gray, Wyborn and Smith, 1982).
LOCAL DISTRIBUTION Nematophagous fungi have been found commonly in any kind of rotting vegetable matter and in dung (Duddington, 1957, p 170). Well established sites which have had time to develop an increased diversity and number of nematodes harbor the widest range of nematophagous fungi (Barron, 1977, p 117). Gray (1983) studied 10 types of habitats and 19 found nematode-destroying fungi were abundant in all the habitats, with over 90% of the samples originating from temporary agricultural pasture, coniferous leaf litter or coastal vegetation containing nematophagous fungi. The greatest species diversity of fungi occurred in old dung and
peatland. Gray and Bailey (1985) showed that the distribution of certain nematophagous fungi in soil varied with the depth, but found that network-forming fungi were able to succeed at all depths in which nematophagous fungi occur (0-35 cm). They were able to grow saprophytically in the upper soil
levels with higher levels of organic matter, but they used nematodes as an alternative source of nutrition in the lower levels where nutrients were limited. Some species produced nematode-attracting conidia (e.g., D. coniospora^ allowing them to survive when nematode numbers were low. Studies by Jansson and Nordbring-Hertz (1979) demonstrated that most nematophagous fungi attracted nematodes. The attraction intensity was lowest in fungi with moderate to high saprophytic ability, and increased with increasing predacity or parasitism. In general, predacious fungi possessed a comparatively good saprophytic ability, while endoparasitic fungi were often obligate parasites (Dijksterhuis gt al., 1994). Predacious fungi exhibited differences in their ability to utilize carbon and nitrogen sources 20 (Satchuthananthavale and Cooke, 1967a,b,c). Thorn and Barron (1984) suggested that nematode-trapping was an advantage for living in nitrogen-limiting habitats. Nematophagous fungi may use nematodes as an important source of nitrogen during growth on a carbohydrate diet. The presence of predacious fungi is more influenced by
pH and moisture than by organic matter and nematode density, while the distribution of conidia-forming endoparasites is more influenced by the amount of organic matter (Cooke, 1963b; Gray, 1987). Being obligate parasites, endoparasitic
fungi required high densities of nematodes to make the probability of finding a host great enough to support fungal populations. Species of endoparasitic fungi with conidial spores that do not attract nematodes were found in soils with higher nematode densities than those species producing nematode-attracting conidia (Cooke, 1963b; Gray, 1987). Non-spontaneous trap-forming predacious fungi were isolated from soils with low organic matter and low moisture (Gray, 1987). They grew saprophytically until nutrients or moisture improved, then they expanded to utilize the increased nematode populations for food. Spontaneous trap- forming predators were found in high organic matter/moisture soils where rich microbial flora and fauna can be exploited by traps that have already been made (Gray, 1987).
However, Cooke (1963b) observed non-spontaneous trap-forming 21 predators in soils with both low and high levels of organic matter and moisture, while spontaneous trap-formers were
limited to soils of relatively high organic matter and moisture. The occurrence of nematophagous fungi in dung (rich in both organic matter and moisture) has been reported numerous times (Bubak, 1906; Juniper, 1953, 1954a,b; Duddington, 1957; Levine, 1963; Pramer, 1964; Barron, 1977; Hashmi and Connan, 1989).
FACTORS INFLUENCING GROWTH Linford (1937) and Linford and Yapp (1939) reported that organic supplementation of soil increased the number of free-living nematodes and this spurred the growth of nematophagous fungi. The increased populations of fungi subsequently preyed on both free-living nematodes and pre- parasitic stages of parasitic nematodes. Cooke (1962)
demonstrated that sources of energy other than the presence of nematodes were necessary for increased fungal activity. However, the presence of nematodes in the soil was necessary for prolonged stimulation of predacious activity. Cooke (1963a,b) documented different growth rates in pure culture among the predacious fungi isolated from soil. He concluded that the development of predacious efficiency was associated with loss of the ability to be an efficient saprophyte as measured by rapid growth. Olthof and Estey (1965) also recorded different growth rates among different
species of nematode-destroying fungi. No consistent similarity was found among species of fungi belonging to the same genus or between those possessing the same kind of nematode-trapping organ. Pandey (1973) reported that, in general, adhesive network-forming fungi grew more rapidly than those using adhesive branches, adhesive knobs, or constricting rings. The growth spurts associated with the addition of nematode-destroying fungi to a locale is relatively short-lived, ranging from 4-8 weeks (Cooke, 1963b; Cooke 1968).
DEVELOPMENT OF FREE-LIVING LARVAE OF PARASITIC NEMATODES The development of infective larvae from worm eggs excreted in feces is dependent on many factors, the most important being temperature and moisture (Ogbourne, 1972; Armour, 1980; Michel, 1985). All 'strongyle-type' eggs of these parasites follow the same general scheme of development on pasture (Ogbourne, 1972; Michel, 1985). After passing out in the feces, eggs hatch in a day or more. The first stage larva (L3) feeds on bacteria in the feces and molts by shedding its old cuticle to the second stage (L2). This stage continues to feed on bacteria, and then molts to the infective third stage larva (L3), but retains the old cuticle as a protective sheath. 23
The infective larvae migrate from feces to herbage. This stage cannot feed and depends on energy reserves stored in intestinal cells until ingested or it dies.
PERSISTENCE OF FREE-LIVING LARVAE OF PARASITIC NEMATODES Horse: Viable cyathostome eggs from naturally-infected horses
hatched in feces on pasture within 7 days during spring, summer and early fall, but the hatching period varied from 2 to 10 weeks in late fall and winter (Ogbourne, 1972; English, 1979). The lowest temperature for egg hatching determined in the laboratory was 7.5°C. The development of cyathostome infective larvae from unembryonated eggs ranged from 3 to 15 days, depending on the temperature (Mikacic, 1953). Rates of development were influenced by both temperature and moisture. Fastest rates of development were associated with high temperatures accompanied by enough precipitation to keep feces moist. First stage larvae were rapidly killed by desiccation while second stage larvae were more resistant (Bazanova, 1948; Shumakovich, 1968). Warm temperatures occurring with a lack of rain allowed feces to dry out, delaying the development of infective larvae (Ogbourne, 1972). During dry conditions, second stage larvae may survive within the dung pat and resume development to the infective stage when 24
rains moisten the feces (Noller and Schmid, 1930). Waves of infective larvae migrate from feces to the surrounding herbage when feces become thoroughly moistened by rain. Even after adequate rainfall, not all of the infective migrate out of feces immediately. Horse feces act as reservoirs of infection from which infective larvae intermittently migrate onto the surrounding herbage when conditions are favorable (Ogbourne, 1972). The distance of active larval migration is generally assumed to be limited to less than 30 cm horizontally and 10 cm vertically from
the soil surface. Infective larvae may be spread over a wider area by temporary flooding, hooves, farm equipment and other agents. Many eggs are killed by prolonged exposure to low temperatures (Lucker, 1941; Parnell, 1936; Baker et al., 1939; Pukhov, 1941; Kazlauskas, 1959; Barus, 1963). Pre- inf ective larvae are also susceptible to freezing (Lucker,
1941), whereas infective larvae may survive over winter and infect horses in the following spring. Alternate freezing and thawing may kill both pre-infective and infective larvae (Parnell, 1934; Parnell, 1936; Britton, 1938). Few strongyle eggs develop into infective larvae during winter months in temperate climates (Ogbourne, 1972; Ogbourne, 1973; Polley, 1986). 25
Sheep and cattle: Estimates of critical low temperatures for development of most trichostrongylid infective larvae ranged between 5- 10°C (Michel, 1985); but variations occur between genera (Michel, 1969) and populations in warmer regions may have higher critical temperatures than populations in cooler regions (Crofton, 1963; Crofton et al., 1965). Climatic factors such as desiccation and temperature extremes affected Lxs and L2s more dramatically than the more resistant larvated eggs and infective larvae (Andersen et al.. 1967; Donald, 1968). Dry conditions that killed Lxs,
L2s and unembryonated eggs resulted in delayed development of embryonated eggs. They were able to survive for several months and hatch very guickly once the feces were moistened by rain (Furman, 1944). The size of the dung pat affected development of infective larvae with regard to differing levels of oxygen and humidity throughout the dung pat. The resulting variation in the time for L3s to develop within dung pats meant that larvae emerged from the feces over a long period of time (Silverman and Campbell, 1958). Numerous studies have reported successful overwintering of infective larvae of the genera Cooperia. Ostertaaia. Teladorsaaia, and Trichostronaylus in temperate climates, even in northern U.S. and Canada (Michel, 1968; Smith and 26 Archibald, 1968; Gibbs, 1979; Ayalew and Gibbs, 1973). In contrast, infective larvae of H. contortus were generally unable to overwinter on pasture.
NEMATOPHAGOUS FUNGI IN THE CONTROL OF PARASITIC NEMATODES
Early work Linford and colleagues were the first to investigate the use of predacious fungi in the control of pathogenic nematodes (Linford, 1937; Linford and Oliveira, 1938a, b; Linford and Yapp, 1939). Linford (1937) first reported a decrease in the infection of pineapple plants by the root- knot nematode after he added organic matter (chopped green pineapple leaves) to the soil. This amendment resulted in increased numbers of saprophytic nematodes followed by greater abundance of predacious soil hyphomycetes. When predacious fungi were added without organic matter, no effect was observed. He suggested that the added organic material promoted an increase in the number of free-living nematodes and this increased food source stimulated nematophagous fungi to develop at a rapid rate. The nonspecific trapping by the nematophagous fungi resulted in increased predation on pathogenic nematodes as well as free- living nematodes. Therefore, the numbers of free-living 27
nematodes was important in the control of parasitic
nematodes. The addition of cultured Dactvlella ellipsospora. a predacious trap-forming fungus, to sterile soil in pots seeded with pathogenic nematodes resulted in a reduction in the number of nematodes in treated pots compared to untreated pots (Linford and Oliveira, 1938a). Further studies demonstrated reduced damage to plants by root-knot nematodes when cultured ellipsospora was added with chopped green pineapple leaves to the soil (Linford and Oliveira, 1938b; Linford and Yapp, 1939). Adding the fungus alone had no effect. Peak activity of the fungus occurred early in the process of decomposition of the chopped leaves and declined quickly. The earliest work investigating the potential of nematode-destroying fungi in the control of animal parasites was completed at the Pasteur Institute in Paris, France. A series of studies documented the ability of certain species of predacious fungi (Arthrobotrvs oliqospora. Dactylella bembicoides. and fi. ellipsospora^ to kill the infective larvae of parasitic nematodes common in domestic animals including Stronayloides papillosum (Roubaud and Deschiens, 1939b), Bunostomum spp. (Roubaud and Deschiens, 1941a), Protostronaylus spp. (Roubaud and Deschiens, 1941b), and Dictyocaulus filaria (Deschiens, 1939). 28 Early experiments examining the effect of exposure to high levels of predacious fungi demonstrated no ill effects to either livestock or pastures (Deschiens, 1941). In one intriguing study, Roubaud and Deschiens (1941b) dusted one of two grassy plots with &. oliqospora. Dactvlella bembicoides. and fi. ellipsospora after artificially contaminating them with £. papillosum and Bunostomum spp. eggs. Two healthy sheep were placed on each plot for 35 days. Sheep that grazed the treated plot had no clinical signs of parasite infection, no eggs in the feces, and only a few specimens of Bunostomum spp. at necropsy. The two control sheep exhibited clinical signs of parasitism, high fecal egg counts, and high worm burdens at necropsy. Unfortunately, the small sample size precluded serious interpretation of the results. In the former USSR, Kornienko, Tendetnik and Charyev (1955, cited by Soprunov, 1958) studied the effects of culturing feces from a sick horse shedding strongylid eggs with each of the following predacious fungi: &. oliqosporaf A. dolioformis. Trichothecium qlobosporum. and Dactylaria brochopaqa. All species greatly reduced the number of infective larvae harvested from the samples compared to controls, but no statistical analysis was presented. In a titration study, they found that increasing concentrations of fungal spores had no effect on the results. They also 29 fed A. oliqospora to an infected donkey for five days and were able to isolate the fungus from feces from days 2 to 9. No adverse effects were associated with the feeding of the spores during the study or at necropsy. In an effort to control chronic hookworm infections in coal miners, Soprunov and Tendetnik (1953-1955, cited by Soprunov, 1958) broadcast spores of A. oliqospora and A. dolioformis throughout the mines once a year for three years. A level of safety associated with exposure to high levels of fungal spores was assumed because no fungal problems were observed among workers involved in cultivating the large vats of fungi and media, in volunteers receiving high levels of exposure, or with coal miners working in treated mines. Dramatic reductions in hookworm infections were observed during this treatment period. Parnell and Gordon (1963) described a fall to negligible levels in the number of infective larvae harvested from serial fecal cultures of two pen-reared sheep experimentally infected with Haemonchus contortus in
Australia. They attributed this effect to the activity of a nematophagous fungus, Acrostalaqmus (VerticilliunO spp. present in the feces. They suggested that the fungus had survived gut passage in the sheep because the cultures were made from fresh feces collected in bags attached to the sheep. In the former Czechoslovakia, Lysek (1963) placed 30 small piles of ascarid eggs on soil samples and noted that the eggs were destroyed by various species of fungi. Ascarid eggs incubated on soil samples at 18-22°C experienced a 21% infection rate by various fungi within two weeks. By five weeks, 45% of the samples were infected and
destroyed (Lysek, 1968).
Recent work
Arthrobotrys oliqospora: The majority of recent studies were done in Denmark and examined the potential of A. oliqospora for biological control of animal parasites. This fungus was chosen by Gronvold e£ al. (1985) because it was the most common nematode-destroying fungus in Danish soils (Shepherd, 1961). Gronvold et al- (1985) demonstrated in vitro that A. oliqospora effectively trapped and destroyed infective larvae of Cooperia spp. in cattle feces. The fungus rapidly trapped and killed Lxs, L2s, and free-living soil nematodes (Nansen e£ al* # 1986). Infective larvae were trapped efficiently, but required longer to be killed, presumably because of the protective effect of the sheath. Nansen et al* (1986) found that the fungus was able to capture but not penetrate the L3s of Cooperia oncophora. The Danish group found that pre-parasitic stages and soil nematodes induced traps equally well. Once traps had 31 been induced by the presence of nematodes, &. oliqospora indiscriminately trapped infective larvae of £. oncophora. C. curticei. H. contortus. Ostertaqia ostertagi. Dictvocaulus viviparus, Oesophaqostomuro dentatum. 0. quadr ispinulatum. Nematospiroides d-Ublns, equine cyathostomes as well as free-living soil nematodes (Nansen et al. . 1988). The level of trap induction appeared to be associated with nematode motility. Sluggish larvae like Dictvocaulus viviparus induced trap formation poorly. In a series of larval cultures in which increasing concentrations of &. oliqospora conidial spores were added to feces containing eggs of Cooperia spp., reduced numbers of infective larvae were observed after 13 days' incubation (Gronvold gt al., 1985). In contrast to the results of Kornienko gt al. (Soprunov, 1958), the level of reduction increased with increasing concentrations of conidial spores added. A concentration of at least 250 conidia per gram of feces was necessary to show a significant reduction. A concentration of 2500 conidia per gram of feces reduced the larval population by over 99%. When 10 artificially prepared cowpats containing C. oncophora were inoculated with &. oliqospora mycelia and conidia and placed on a clean pasture, an 86% reduction in the number of infective larvae harvested from surrounding herbage was observed compared to controls (Gronvold gt al., 32 1987). However, no quantification of the amount of fungus was possible. In a similar experiment, artificially prepared cowpats containing Q. ostertaai were inoculated with 2000 conidial spores per gram of feces of the same fungus and placed on pasture with control cowpats (Gronvold et al., 1988). Fewer
infective larvae were harvested from inoculated cowpats than from control cowpats. The reduction of herbage larval infectivity over three months ranged from 46-89%. Attempts to demonstrate &. olicrospora in the cow pats were made both by direct microscopic observation and by inoculation on cornmeal agar petri dishes. &. oliqospora was demonstrated in all inoculated cowpats and in none of the control cowpats.
A further study examined the effects of adding mycelia of A. oliqospora to cow pats on the development of infective larvae in the cowpats, on the infectivity of surrounding herbage, and the worm burdens of grazing calves (Gronvold et al.. 1989). A parasite-free pasture was divided into two comparable plots. At weekly intervals over a seven week period, control and fungus-inoculated feces containing 0. ostertaqi eggs were prepared. The feces were inoculated with fungal mycelia of A. oliqospora at a rate of 0.25 g/kg of feces. Control dung pats were placed on one plot and 33 fungus-inoculated dung pats were placed on the other plot.
Four calves were placed on each plot and allowed to graze for two months. There was a 42% reduction in the number of infective larvae in inoculated cow pats, a 50-71% reduction in herbage infectivity, and a 37% reduction in acquired worm
burdens of grazing calves. Hashmi and Connan (1989) discussed several unpublished studies on the effects of &. oliqospora on the development of infective larvae, but no details of the methods and experimental designs were provided. They reported that an
inoculum of 20 conidia per gram of feces in fecal cultures containing various species of parasitic nematode eggs
reduced infective larval yields by 40%. Under field conditions, 10 g of fungal mycelia per kg of calf feces containing C. oncophora resulted in an 86% reduction of infective larvae over four weeks. The addition of 50 conidia per gram of feces reduced infective larval yield by 82% over eight weeks. No significant further reduction was detectable after 8 weeks, even though the fungus was still isolated from fecal matter. When sheep feces containing H. contortus eggs were inoculated with 20 oliqospora conidia per gram of feces and scattered on a pasture, there was a marked reduction in the number of infective larvae harvested from the pasture (Hashmi and Connan, 1989). Similarly, conidia were added to 34 calf feces containing 0. ostertaai eggs in one study and C. oncophora in another study. Reductions in the infective larval populations of up to 72% were recorded as measured by pasture larval counts and the use of tracer calves. When sheep feces mixed with conidia were applied once at the beginning of the grazing season to pasture concurrently contaminated by infected sheep, larval counts of H. contortus and 2. circumcincta were equivocal, suggesting that fungal activity was only temporarily increased. When inoculated sheep feces were scattered at intervals through the season, reductions in the larval counts were seen. Hashmi and Connan (1989) also described a field trial where &. oliaospora conidia were fed regularly (8 million conidia per calf twice a week) to grazing calves infected with 0. ostertaai and C. oncophora. On a similar adjacent pasture, infected control calves were grazed. Reductions in pasture larval counts were 51% and 62% for Ostertaaia and Cooperia. respectively. In New Zealand, Murray and Wharton (1990) reported that A. oliaospora effectively caught and infected all three pre- parasitic stages of Trichostronaylus colubriformis. The invasion of L3s took 3.5 h while fungal hyphae invaded Lxs and L2s in 1.5 h. 35
Other fungal species: In France, Gruner gt al. (1985) fed 11 lambs allocated to three treatment groups 500 g of one of three nematode- destroying fungi, A. tortor. &. musiformis. and Dactvlaria Candida. cultured on millet seed. The lambs acted as their own controls. All three species of fungi were isolated from feces and reduced numbers of infective larvae (T. circumcincta and H. contortus^ were recovered from fecal cultures. The results have been questioned by Larsen et al. (1991) and Waller and Larsen (1993) because of irregularities between estimated egg counts and larval
counts from the cultures. Peloille (1991) fed 500 g of A. flaarans cultured on millet seeds to four sheep infected with T. circumcincta and collected feces every 24 h for five days. Four infected and untreated sheep supplied control feces. Fungus was isolated from treated feces during the first 48 hours only. Fecal larval cultures of feces from treated and control sheep were cultured for two weeks. Cultures of treated feces yielded fewer infective larvae than those of control feces, with the greatest reductions seen at 24 hours post-administration of the fungus. It was
unclear whether egg counts of the treated and control groups were similar at the commencement of the fecal cultures. 36
In vitro stress tests and in vivo survival tests: In Denmark, in vitro methods have been developed to identify the best fungal candidates for biological control investigations. A series of laboratory techniques designed to simulate passage of the fungi through the gastrointestinal tract of cattle were developed by Larsen gt al. (1991). Fifty-five field isolates were incubated in dilute rumen fluid at body temperature for 24 h as a primary stress test. All 21 surviving isolates were species that produced adhesive networks of traps. The surviving isolates were exposed to (1) synthetic saliva, (2) pepsin- hydrochloric acid solution, (3) trypsin solution for 4 h, (4) rumen fluid for 24 h, and (5) 24 h of rumen fluid followed by 4 h of pepsin-HCl solution, simulating effects of the abomasum and small intestine. Exposure to synthetic saliva, pepsin-HCl solution, and trypsin had no effect on fungal viability. Thirteen isolates of the genus Arthrobotrys survived the prolonged exposure to rumen fluid. Twelve isolates survived the rumen fluid + pepsin-HCL solution test. The predatory capacity of these 12 isolates was tested in a dung pat assay wherein feces from calves infected with a monoculture of 0. ostertaai were mixed with fungal material, formed into pats and incubated. Infective larvae were recovered by the Baermann technique three and four weeks 37 later. The reduction in the development of infective larvae ranged between 65% and 98% compared with controls. Larsen gt al» (1992) also tested 10 in vitro stress- selected Arthrobotrvs fungi for in vivo survival. Calves were fed nematophagous fungi on barley grains for four days. Feces were collected on days 4 and 5, and the viability of fungi after gut passage was tested by incubating barley grains from the feces on water-agar plates supplemented with antibiotics. Predacious capacity was determined in dung pat
assays and fecal cultures. Nine of the 10 species were re-isolated from barley grains after gut passage. Seven species exhibited high predacious capacity, reducing the number of infective larvae of 0. ostertaai by 61-93% in dung pat bioassays and 76-99% in fecal culture. There was good • correlation between results of the dung pat bioassay and fecal cultures. In
addition, these seven species of fungi exhibited similar predacity in dung pat assays after surviving the in vitro stress selection tests of Larsen et al. (1991). A single oliaospora isolate and two A. flaarans
isolates that survived both the in vitro stress test and in vivo test and exhibited a high trapping capacity against O. ostertaai larvae in fecal cultures and dung pat assays were fed to calves (Gronvold, et al., 1993b). Their feces, after mixing 1:1 with infected calf feces, were placed on clean 38 pasture and the resultant harvest of infective larvae
compared with control pats. No significant reduction was observed for &. oliqospora. but both flaqrans isolates reduced the number of infective larvae by 75-85%. In Australia, Waller and Faedo (1993) tested 94 catalogued (Centraalbureau Voor Schimmelcultures, Baarn, The Netherlands) species of nematophagous fungi for the capacity to (l) attract nematodes, (2) produce' larvicidal metabolites, and (3) reduce the number of infective larvae developing in fecal cultures. Nine genera produced chemo- attracting metabolites and nine genera exhibited ovicidal and/or larvicidal activity that appeared to be induced by the presence of nematodes. Six genera produced larvicidal and/or ovicidal substances without nematodes being present. Three genera reduced the percentage of infective larvae produced in culture by more than 80%: Arthrobotrys (7 spp.), Geniculifera (2 spp.), and Monacrosporium (2 spp.). A standard 1 cm diameter agar plug from cultures of these 11 species was added to increasing amounts of feces (5, 10, 20, and 50 g). Two species, &. oviformis and Geniculifera eudermata. eliminated infective larvae from all fecal cultures, indicating superior predacious activity. In a further study, Waller and Faedo (1993) added eight species of fungi demonstrating good predacious activity in culture to sheep fecal cultures containing H. contortus eggs 39 at various conidial concentrations (ranging from 1 to 1000 conidia per gram of feces). Significant reductions in larval counts occurred at concentrations as low as 10 conidia per gram of feces for A. oliaospora. A* oviformis. and G . eudermata. These eight species of fungi were cultured with feces from three sheep with comparable egg counts for H. contortus, T. circumcincta. and Trichostronaylus colubriformis. Although X. colubriformis consistently showed the lowest percent reduction and Teladorsaaia circumcincta the highest percent reduction, the differences were not significant (Waller and Faedo, 1993). The three fungal species, A*. oliaospora. A- oviformis. and G. eudermata. were subsequently cultured with feces of different fecal egg counts (varying from 100 to 22000 epg) at three different conidial concentrations (10, 100, 250 conidia per gram of feces). The percent larval reduction (approximate range, 40-100%) correlated with the conidial concentration, while no linear relationship was observed between larval reduction and egg count. Waller al. (1994) further applied the in vitro stress selection technique of Larsen et al. (1992) to mycelial and conidial fractions of eight species of fungi (A. iavanica. A. oliaospora. A- oviformis. A. polvcephala. £. boaoriensis. £. eudermata. 14* rutaeriense. |4. thaumasium) 39
at various conidial concentrations (ranging from 1 to 1000 conidia per gram of feces). Significant reductions in larval counts occurred at concentrations as low as 10
conidia per gram of feces for A* oliaospora. A. oviformis. and £. eudermata. These eight species of fungi were cultured with feces from three sheep with comparable egg counts for H- contortus. T. circumcincta. and Trichostronqylus colubriformis. Although 3?. colubriformis consistently showed the lowest percent reduction and Teladorsaaia circumcincta the highest percent reduction, the differences
were not significant (Waller and Faedo, 1993).
The three fungal species, A j. oliaospora. A* oviformis. and G. eudermata. were subsequently cultured with feces of different fecal egg counts (varying from 100 to 22000 epg) at three different conidial concentrations (10, 100, 250 conidia per gram of feces). The percent larval reduction (approximate range, 40-100%) correlated with the conidial concentration, while no linear relationship was observed between larval reduction and egg count. Waller e£ al. (1994) further applied the in vitro stress selection technique of Larsen et al. (1992) to mycelial and conidial fractions of eight species of fungi (A. iavanica. A. oliaospora. A* oviformis. A* polycephala. £. boaoriensis. £. eudermata. &. rutqeriense. JjL thaumasium) 40 that had exhibited superior larval trapping capabilities in the earlier screening studies (Waller and Faedo, 1993). Only limited survival of mycelia occurred for most fungal species, while most conidia survived well (Waller et al.
1994). &_•_ oliaospora. A- oviformis. G. eudermata. and M. thaumasium showed the greatest ability to survive these
conditions. In addition, in vivo survival studies in three sheep surgically fitted with abomasal and ileal cannulae were performed on three species of fungi that were prodigious conidia producers and had survived the screening tests well (A. oliaospora. A- oviformis. and G. eudermata). After oral dosing, viable conidia of all three species were present in the abomasum within an hour. Fecal samples were collected at 12, 24, 31, 48, 57, and 72 h post-administration. Fungal growth in feces was recorded once, in the 24 h fecal sample of A* oliaospora. Waller gt gl. (1994) also injected conidia into the abomasum of the cannulated sheep and took ileal samples every 2 h for 8 h, then hourly for 6 h, and finally every 2 h for 4 h. Fungi were isolated from ileal contents 4 h post-administration for A. oliaospora and C5. eudermata. while A* oviformis was isolated from all samples. Fecal samples were collected at 0, 9, 16, 19, 26, 29, 32, and 35 h post-dosing. Fungal growth was observed for A* oliaospora 41 and G. eudermata in the 9 h sample, whereas A. oviformis was
observed from all fecal samples. Fecal cultures of the 9 h samples resulted in substantial reductions in larval numbers. These results suggested that the in vitro stress tests developed by Larsen et al. (1991) were valuable screening techniques, but that the duration for fungal spore incubation was too long. Larsen et al* (1991) recommended 24 h rumen-fluid and 4 h pepsin-HCl solution incubation periods to simulate transit times. Waller et al* (1994) documented that at least in sheep, orally administered conidia appeared to travel in the fluid compartment of ingesta, resulting in decreased transit times. They estimated that conidia travelled through the rumen and abomasum in the greatest concentrations between 4 and 12 hours after oral dosing. The greatest flow though the small
and large intestines was estimated to take between 4 and 10 hours.
Commercial application: The commercial potential for biological control of parasitic nematodes using nematophagous fungi has been utilized for certain plant parasites. A product called Royal 300 containing A* robusta reduced the effect of mycophagous nematodes on mushroom production when applied at 42
the planting of mushroom spores (Cayrol e£ al., 1978). Another product, Royal 350, containing h. irregularis controlled a species of nematodes parasitic to horticultural crops, especially tomatoes (Cayrol and Frankowski, 1979). The nematode pests were not eliminated, but reduced in density, limiting crop damage to acceptable levels.
SUMMARY Nematode-destroying fungi exhibit a variety of methods to infect nematodes. Investigations concerning the potential for biological control have centered on predacious species, especially £. oliaospora. These studies examined the potential for biological control at three levels: (1) in fecal cultures under controlled laboratory conditions, (2) on pasture, and (3) in the animal. In general, predacious fungi trapped nematodes nonspecifically, suggesting that a single species may effectively control many species of parasitic nematodes. The free-living larval stages of a number of livestock parasites have been documented to be trapped and killed. These include the genera of Ancylostoma. Bunostomum. Cooperia. Dictyocaulus. Haemonchus. Nematodirus. Nematospiroides. Oesophaaostomum. Ostertaaia. Teladorsaqia. Trichostronqvlus. Stronqvlus. and gtrpngylbideg as well as the subfamily Cyathostominae. 43 No ill effects in people or animals, or to pastures have been associated with high level exposure to fungal mycelia or spores in limited studies.
Significant reductions in infective larval populations have been observed in numerous in vitro and field studies with nematophagous fungi. Isolated strains of several species of fungi fAcrostalaamus (VerticiIlium) spp., &. fla'arans. h- musiformis. &. oliaospora. A. oviformis. Dactylaria Candida. S. eudermata) have been shown to survive passage through the gut of sheep, cattle, horses, and donkeys. This ability was not shown to be a species characteristic as some isolates of the same species listed above have been unable to exhibit viability after being fed to animals, e.g. &. flaarans and £. oliaospora. In vitro screening tests have been developed to select species of fungi with good potential for biological control. Predatory activity was measured by observing significant reductions of infective larvae in culture, and the ability to survive gut transit was tested by exposure to simulated digestive solutions followed by in vitro culture. Promising candidates identified by these tests were then confirmed by in vivo and field testing, and good correlation was seen. However, rejected species of fungi were not tested to determine the ability of screening tests to accurately identify poor candidates. To evaluate the validity of screening tests, the proportion of true positives, false positives, true negatives, and false positives identified by the test compared with a "gold standard" needed to be determined. In the above studies, the proportion of true positives and false positives were determined, but the proportion of true negatives and false negatives were unknown. CHAPTER II. GROWTH AND PREDACIOUS ACTIVITY OF FOUR SPECIES OF NEMATOPHAGOUS FUNGI IN VITRO
A. CULTURE AND PREDACIOUS ACTIVITY
INTRODUCTION Techniques for culturing nematophagous fungi in the laboratory were studied and mastered. Growth
characteristics of fungi were compared on three different culture media. Potentially beneficial properties of fungi for use in parasite control were examined in four species of fungi. The properties examined were (1) amount of spore
production in culture and (2) nematode destroying activity of endoparasitic and trapping fungi.
MATERIALS AND METHODS Culture of free-living nonparasitic nematodes 1. Collection and cleaning of nematode eggs Free-living soil nematodes were used as a food source for predacious fungi cultured in the laboratory. They were harvested from garden soil using a Baermann apparatus (Figure 6). Gravid female nematodes were transferred to
45 46 Nigon's agar (Appendix D) in a 90 mm diameter sterile
plastic disposable Petri dish for the collection of eggs. Newly deposited eggs were suspended in approximately 10 ml of sterile distilled water and transferred to a 15 ml centrifuge tube. The suspension was centrifuged at 500g for 15 minutes and the supernatant discarded. The centrifugation and resuspension in sterile water was repeated twice. The rinsed eggs were transferred to fresh Nigon's agar, potato dextrose agar (PDA) and to water agar (WA) Petri dishes (Appendix D) and maintained at 22-25°C. Autoclaved peanut butter was placed on the WA plates as a source of nutrition for the developing nematodes five to
seven days later.
2. Maintenance of healthy populations of free-living nematodes Nematodes were transferred to fresh plates as needed to maintain healthy populations (approximately every seven to ten days) by scraping the contents of cultures onto a double layer of 4.5 X 8.5 inch wipes (KimWipe, Kimberly-Clark Corporation) supported by a coarse screen (3mm opening) covering a 145 mm diameter plastic Petri dish. Sterile distilled water was added until the water's surface touched the wipes. The nematode cultures were left in the water for approximately 6-12 hours to enable the nematodes to migrate 47 out of the culture medium, through the wipes, and into the water at the bottom of the Petri dish. The culture medium and wipes were discarded. The top layer of water was pipetted out of the Petri dish, leaving the bottom layer of nematodes undisturbed. The concentrated nematode suspension was pipetted into 50 ml test tubes and centrifuged at 500 g for 10 minutes. The supernatant was discarded and the nematodes resuspended in approximately 40 cc of sterile distilled water. This cycle of resuspension and centrifugation was repeated twice. The cleansed nematodes were transferred to new WA dishes and supplemented with autoclaved peanut butter after five to seven days. Cultures were examined daily. Sterile distilled water was added as needed to keep the cultures moist. Cultures exhibiting fungal contamination, introduced by non-sterile nematodes and by airborne spores, were discarded.
3. Harvest of free-living nonparasitic nematodes Free-living soil nematodes were harvested for feeding to fungi in the same manner as described above. The cleansed nematodes were resuspended in sterile distilled water at a concentration of approximately 500 nematodes per drop of suspension. 48 Culture of free-living stages of parasitic nematodes
Horse: Fecal samples were initially collected from thoroughbred horses at Woodburn Farm where it had been established that only cyathostome eggs were present (Herd and Gabel, 1990). In later studies, feces from pastured horses at The Ohio State University Finley Research Farm were used. These samples contained eggs of cyathostomes and Stronaylus vulaaris. Fecal samples were collected rectally and placed in clean plastic bags. They were kept in an ice cooler until transported to the laboratory for culturing. Sixteen-ounce brown glass honey jars were one third filled with fresh feces and gently packed. The lid was loosely placed on the mouth of the jar and the cultures were incubated at 26-28°C. The cultures were stirred to aerate and repacked daily. Sterile distilled water was added as need to keep the cultures moist. Using a Baermann apparatus, first stage larvae were harvested after 24 hours of incubation, second stage larvae were harvested after 3 days of incubation, and third stage larvae were harvested after 8 days of incubation.
Sheep: Feces were collected in clean canvas collection bags strapped to two 4-5 months old ram lambs grazing infective 49 pastures at the Ohio Agricultural Research and Development Center (OARDC), Wooster, Ohio. The feces were kept in an ice cooler until transported to the laboratory for culturing. Fecal cultures were performed as described above
and L3s, L2s , and L3s were harvested as described above.
Cattle: Feces from pastured heifers at The Ohio State Dairy Farm were collected rectally and placed in clean plastic bags and kept in an ice cooler until transported to the laboratory for culturing. Fecal samples were cultured as described above and L^s, L2s,
and L3s were harvested as described above.
Identification of larval stages The identification of first and second stage larvae of all genera were based on timing of the fecal cultures, not morphology. Eggs of most common strongylid parasites cultured in vitro at temperatures between 20-30°C have been documented to hatch to Lxs within 24 h and molt to L2s between 45-96 h and develop into L3s between 96-144 h (Andrews, 1933; Leland, 1967; Khan and Dorsman, 1978; Ogbourne and Duncan, 1985). However, infective larvae were identified by morphology and assigned to the appropriate genus based on criteria of Georgi (Bowman, 1995) and the 50 Manual of Veterinary Parasitological Laboratory Techniques (Ministry of Agriculture, Fisheries and Food, 1986).
Efforts to increase survival of first and second stage
larvae Newly harvested first and second stage larvae were placed on three plates each of WA, Nigon's agar, water agar
with antibiotics (TCC-WA), and fecal agar (FA) (Appendix D). For each larval stage, one plate of each medium received no further treatment. A second plate of each medium was streaked with a loop of live Escherichia coli as a supplemental food source. Several loops of heat-killed E. coli were added to the third culture plates as a supplemental food source. The plates were monitored every 12 hours for mortality of larvae.
Source of nematophagous fungi Four species of nematophagous fungi (Drechmeria coniospora. Haroosporium angulllulae, Ar.throbptrys oliaospora. and &. flaarans^ were supplied by Dr. G.L. Barron at the University of Guelph. Fungal cultures were grown on various nutrient agars described below. Each species was stored in a separate plastic box, placed in a large, unlit incubator at room temperature (22°-25°C) and humidified by a large flat pan of water on the bottom shelf. 51 The selection of fungal species was based on theoretical considerations of their ability to control pre-parasitic stages of animal parasites as discussed previously.
Culture and nematode-destroying activity of the four species of fungi Drechmeria coniospora 1. Culture
In order to maintain cultures of this endoparasitic fungus, free-living soil nematodes were infected with conidial spores that had been harvested from old cultures. The free-living soil nematodes were rinsed three times in sterile distilled water and placed on a TCC-WA plate. The antibiotics were added to control bacterial overgrowth. Conidial spores were harvested from old cultures of dead nematodes supporting fertile hyphae with approximately 10 ml of surfactant water (1-2 drops surfactant1 per 500cc sterile distilled water). The suspension of conidial spores was serially centrifuged at 500 g for 10 minutes and resuspended in sterile distilled water three times prior to deposition in fresh nematode cultures on TCC-WA. Contamination by
1 Acationox, Monoject Scientific Products, Division of Sherwood Medical, St. Louis, MO. 52 other fungi was a continuing problem with propagating D. coniospora because the host nematodes could not be sterilized. Contaminated cultures were eliminated and new
fungal cultures were continuously started.
2. Nematode-destroying activity Suspensions of fi. coniospora conidial spores were added to five TCC-WA Petri dishes, each of which held approximately 50-100 free-living nonparasitic nematodes which had been rinsed three times in sterile distilled water and placed on water agar plates. The dishes were examined at 3OX under a stereoscopic dissecting microscope to count live nematodes immediately after the addition of the conidia and four hours later to search for signs of adhesion. The percentage of live nematodes carrying adhesive conidia at the four hour observation period was recorded. The cultures were examined for infected nematodes every 24 h for five days. Infection was measured by observing the presence of conidiophores emerging from the nematode carcasses.
Harposporium anauillulae
1. Culture In order to propagate new cultures of H. anauillulae. infected nematodes with numerous infective conidial spores 53 were rinsed with approximately 10 ml of surfactant water.
The suspension of conidial spores was serially centrifuged at 500g for 10 minutes and resuspended in sterile distilled water three times prior to deposition in WA plates of cleansed free-living nematodes. Cultures contaminated by other fungi introduced by the free-living nematodes or airborne spores were discarded.
2. Nematode-destroying activity Suspensions of infective conidial spores were introduced into three WAA Petri dishes containing approximately 50-100 free-living nonparasitic nematodes. The cultures were examined daily for up to 10 days at 30X using a stereoscopic dissecting microscope for determination of infection rates.
Arthrobotrvs Qligggpgga 1. Culture A. oliaospora (University of Guelph #80) was grown on fresh nutrient agar-lined Petri dishes by the transfer of a few conidia from a single conidiophore at a trapping site if the fungus culture was contaminated, or of a one centimeter diameter plug of the agar from a pure culture. New cultures were inoculated every 4 weeks. Every two or three months, 4-5 day old cultures were fed nematodes and predatory 54 activity was observed. Stock cultures were grown on PDA agar slants, and when mycelial growth patches measured approximately 10-15 mm in diameter, the slants were refrigerated to maintain backup cultures.
To compare growth and conidia production on three different agar recipes, five Petri dishes each of PDA, dilute PDA (dPDA), and FA (Appendix D) were inoculated with 1 cm diameter plug of a two week old &. oliaospora culture. All cultures were incubated at 22-25°C in an unlit incubator. Mycelial growth and conidia production were monitored daily until the entire surface of all five samples of one nutrient agar was filled with dense mycelial growth. The level of growth on the other media were noted.
2. Nematode-destroying activity Fifty to 100 free-living non-parasitic nematodes were placed on four day old A. oliaospora dPDA cultures. The number of live nematodes were counted immediately. Every 24 hours, the number of nematodes trapped and killed were counted until all nematodes were dead. Approximately 50 horse and sheep LiS were added separately to five 5 to 7-day old A. oliaospora cultures on dPDA and examined at 30X magnification after 24 hours for signs of trap development, nematode capture and infection. Similar dPDA fungal cultures were set up for L2s and L3s, 55 except studies on L3s were observed every 24 h for three days. Due to the short survival times of Lxs and L2s, similar studies were done on L3s and L2s using pre-induced dPDA cultures. Pre-induced cultures were produced by introducing free-living non-parasitic nematodes to cultures two days prior to the addition of pre-parasitic larvae. After two days, free-living nematodes had induced fungal traps and had been trapped and consumed to such a degree that they were easily distinguished from pre-parasitic larvae.
Arthrobotrys flaarans
1. Culture A. flaarans (University of Guelph #126) was grown in disposable Petri dishes by transferring a one centimeter diameter plug from a two week old culture to fresh plates of PDA, dPDA, and FA as described for &. oligospora. Stock cultures were made on agar slants and after mycelial growth equalled 10-15 mm in diameter, the slants were refrigerated to maintain backup cultures.
2. Nematode destroying activity The ability of &. flaarans to form traps, capture and kill free-living larvae (LiS, L2s, and L3s) of sheep and 56 horse parasites was examined in vitro using cultures less than two weeks old grown on dPDA. After adding approximately 50-100 nematodes of the desired type to five dPDA cultures, each culture was observed every 24 hours for the development of traps and nematode capture at 3 OX magnification. Similar studies using pre-induced cultures were done on LiS and L2s as described above.
RESULTS
Culture of free-living non-parasitic nematodes Repeated attempts to culture free-living nematodes on Nigon's agar failed to establish healthy, growing populations, possibly due to inadeguate nutrition. Attempts
to develop populations on PDA also failed because of bacterial overgrowth. However, dense populations of nematodes were easily maintained and propagated using WA supplemented with autoclaved peanut butter. Fungal
contamination was rare once a closed population of nematodes had been established.
Culture of free-living stages of parasitic nematodes Larvae of the following parasites were cultured in abundance from the fecal samples: Horse - Cyathostominae? 57
Cattle - Cooperia and Ostertagia; Sheep - Haemonchus.
Teladorsagia. and Trichostronqvlus. Gvalocephalus (horse), Nematodirus (sheep, cattle), Oesophaqostomum (cattle), Poteriostomum (horse), Strongvloides (sheep), and stronqvlus vulgaris (horse) were obtained intermittently. Low numbers of LiS and L2s of all species of parasites were extracted with the Baermann apparatus, probably because they do not normally migrate out of fecal matter (Michel, 1985).
Efforts to increase survival of first and second stage larvae Virtually all larvae from horse samples were cyathostomes. Rarely, Stronqylus vulgaris larvae were observed. Sheep infective larvae were primarily
Trichostronqylus spp. with Haemonchus rarely observed. The LjS and L2s of all parasites died within 36 hours. Alterations in nutrient sources (different nutrient agars and addition of bacteria) failed to increase survival.
Culture and nematode-destroying activity of the four species of fungi Drechmeria coniospora Early in the six month observation period (extending from 12/4/91 to 7/1/92), fi. coniospora exhibited good parasitic activity against cultured free-living soil nematodes. Adhesion of the conidia occurred within minutes of contact. All nematodes acquired adhered conidia, but not all conidia adhered to nematodes. The numbers of conidia adhering to a single nematode varied from one to numerous
(too many to count) (Plate II). When only a few conidia adhered, they were usually found around the anterior end of the worm. Infection rates were undeterminable because nematodes reproduced during the observation period. Live females continued to produce viable eggs and dead infected females were observed with live larvae exiting the carcass. In the latter part of the observation period (6/17/91 to 7/1/92), £>. coniospora cultures exhibited dramatically decreased levels of infectivity. Initially, many infected nematodes were seen, but by the end of the period, no new infections were detected. Conidial adhesion to the nematode cuticle was observed throughout the observation period, but the numbers were much reduced toward the end of the observation period and no infection was associated with the adhesion of conidial spores. This fungus was eliminated from the study because of the failure to generate large numbers of conidial spores that could effectively infect nematodes. 59
Harposporium anauillulae The first signs of infection in nematodes by H. anauillulae appeared between six and nine days after exposure to conidia. However, prevalence of infection and the production of infective conidial spores were always low. Infection rates were undeterminable because the nematodes continued to reproduce during the observation period. This fungus was also eliminated from the study because of the failure to produce adequate numbers of conidia.
Arthrobotrys oliaospora and &. flagrant Potato dextrose agar (PDA) was far superior for hyphal growth and spore production compared with FA and dPDA for both &• oliaospora and A. flaarans. Dense lateral growth covered the PDA surface within five and seven days after inoculation of new cultures with conidia and within three and five days after inoculation with a one centimeter diameter plug of mature culture for A. oliaospora and A- flaarans. respectively. Growth on FA and dPDA plates were markedly less developed. All subsequent cultures for conidial production were grown on PDA. However, both species of fungi were cultured on dPDA for trapping studies because the sparse hyphal development optimized the ability to observe predation. Drying agar cultures prior to harvesting spores decreased the amount of medium that was 60 inadvertently collected with the spores, resulting in more concentrated spore suspensions. h. oliaospora cultures produced abundant conidia, but no chlamydospores were observed. By contrast, &. flaarans cultures were characterized by abundant chlamydospores intercalated along horizontal hyphae on and below the surface of the agar (Plate I), but conidial spores were rare. Chlamydospores were not induced to grow in the presence of nematodes. However, traps were observed developing immediately from conidia of A. oliaospora and A. flaarans (Plate III). Mycelial growth of A- flaarans was noticeably slower than for oliaospora on all three types of media. Cultures grown on PDA covered the entire surface of the agar by day 3 for A. oliaospora and by day 5 for A- flagrans.
A. oliaospora and A. flaarans both exhibited good ability to capture and kill LiS, L2s, and L3s of cyathostomes (horse) and Trichostronaylus spp. (sheep).
DISCUSSION Culture of free-living stages of parasitic nematodes Horse: These studies were done in the spring of 1992 (April through early June). Equine fecal samples containing high 61
numbers of cyathostome eggs were readily available. Several fecal culture samples from Finley Research Farm contained a single S. vulaaris larva, but overall the occurrence was too low to consider S. vulaaris as represented in the aliquots of larvae used in the study. The low level of S. vulaaris was consistent with other findings of a reduced prevalence or absence of £. vulaaris in horse feces (Lyons gt al.. 1981; Reinemeyer et al., 1984; Uhlinger, 1990; Herd and Gabel, 1990). The effect of the fungi were considered only for cyathostomes.
Sheep: Larvae in sheep feces were predominantly Trichostronavlus. Haemonchus was rarely observed at this time. This finding is consistent with the epidemiology of these two parasites wherein the longer-lived Trichostronavlus adults can overwinter and lay eggs in the gut while the short-lived Haemonchus adults die off (Courtney gt al., 1983, 1984; Waller and Thomas, 1981). Although hypobiotic worms of Haemonchus emerge in the spring, it was too early for their maturation and egg laying to contribute markedly to fecal egg counts. Thus, fungal predacious activity was considered only for Trichostronavlus. 62 Cattle: The highest bovine fecal egg counts were very low (mean <10 epg feces) so that few larvae were harvested at any one time. First and second stage larvae of all parasites suffered high mortalities (mean mortality = 57.5% (range 0- 100%) within 24 hours). This high mortality rate coupled with low numbers of cattle larvae virtually eliminated all
larvae before any fungal effect could be measured. However, Nansen el al. (1986) observed that &. oliaospora trapped and killed Lxs and L2s of £. oncophora efficiently, while Nansen et al. (1988) demonstrated that O. ostertaai and C. oncophora could induce trap formation and subsequently be entrapped and killed by &. oliaospora. Larsen et al* (1991) reported that oliaospora and &. flaarans were able to reduce the number of Q. ostertaai infective larvae when feces from infected cattle were incubated with spores from these two species of fungi. Therefore, it was assumed that cattle parasites were susceptible to predation by nematophagous fungi and thus should be included in further studies in this project.
Efforts to increase survival of first and second stage larvae High mortality rates were observed for Lxs and L2s from all livestock host species. This finding was consistent 63 with high mortality rates reported by Larsen gt al. (1991) for 0. ostertaai Lxs and L2s. In vitro cultivation of parasitic nematodes have been generally characterized by high mortalities (Smyth, 1990). Unknown environmental and nutritional factors may have been involved.
Culture and nematode-destroying activity of the four species of fungi Drechmeria coniospora A decrease in adhesion and infection by conidia of D. coniospora was observed in other studies (Waller, 1992, personal communication). This phenomenon may be associated with the culture technique. The conidial spores of D. coniospora have been shown to develop their adhesive knobs after they leave the conidiophore (Jansson gt al., 1984; Dijksterhuis et al. , 1991). In addition, only a few conidia located at the periphery of clusters of spores form adhesive knobs. In one study, an estimated 8% of D. coniospora conidia possessed adhesive knobs when grown on an agar surface (van den Boogert gt al., 1992). However, when spores were suspended in water, all conidia formed adhesive knobs after three days' incubation. The practice in these studies of harvesting conidia by flushing the conidiophore with surfactant water and then immediately adding the suspended conidia to nematode cultures may have exposed 64 nematodes to immature, nonadhesive conidia. Cultures were selected for conidial harvest when high numbers of conidia were observed on conidiophores. The conidia used in the present studies may have been too crowded for maturation and thus, fewer spores developed adhesive knobs. Van den Boogert al. (1992) hypothesized that maturation of spores was density-dependent and spores were only able to develop adhesive knobs after release from clumps on conidiophores. Incubation of harvested spores prior to use may result in improved adhesion and infection rates because the adhesive knobs were given time to develop (van den Boogert a t a l . , 1992).
Harposporium anauillulae The relatively long period between exposure to conidia of H. anauillulae and the appearance of infection in
nematodes was probably associated with delays in ingestion of the conidia. Jansson (1982a,b) documented that the mycelium of fi. anauillulae strongly attracted nematodes and thus increased the probability of ingestion of non attracting spores by luring feeding nematodes into the areas of greatest spore concentration. In general, the suspensions of conidia contained little mycelial components of the fungus. Therefore, there was little attractive force to aid in nematodes finding and ingesting the infective 65 spores. However, when fungal cultures were developed by placing a nematode carcass containing mycelium and spores in with washed free-living nematodes, infection rates still remained low. Therefore, H- anauillulae was a poor candidate for further study on biological control of
parasites.
Arthrobotrvs PligfiSP-OT.a and A. f3.agr.ans The results of growth studies on PDA, dPDA and FA determined that PDA was the medium of choice for producing the most spores in the least time. For studies requiring direct observations of interactions between fungi and nematodes, dPDA was the medium of choice due to the sparse growth pattern of predacious fungi paired with the transparency of the medium. Fecal agar (FA) was shown to be
superior for conidial production in seven species of predacious fungi (Waller et al., 1994), but in this study, both A- oliaospora and A* flaarans exhibited distinctly slower growth on FA compared with PDA. This phenomenon may have been associated with differences between the two studies, including different species of fungus (A- flaarans was not included in the Waller e£ al* study), different isolates of the same species (A* oliaospora), different sources of sheep feces that may vary in their nutritional components, and different media for comparison with FA 66 (cornmeal agar in the Waller at al., study and PDA in this study). Both A* oliqospora and A* flaarans displayed marked capabilities for entrapping and killing Lxs, L2s, and L3s of cyathostomes and Trichostronaylus. confirming the findings
of Roubaud and Deschiens (1939a), Pryadko and Osipov (1986);
Nansen et al- (1988), Murray and Wharton (1990), and Waller
and Faedo (1993).
SUMMARY The potential of two endoparasitic and two predacious species of nematophagous fungi for abundant in vitro culture and effective nematode predation was studied. Cultures of both endoparasitic isolates, JJ. coniospora and H. anauillulae. resulted in low numbers of infective conidia and limited rates of infection. They were consequently eliminated from further study. The two predacious species, A« oliaospora and A- flaarans. exhibited abundant growth and production of spores (conidia of A* oliaospora and chlamydospores of A- flaarans^ on potato dextrose agar. They demonstrated a strong ability to trap and kill all three larval stages of equine cyathostomes and ovine Trichostronavlus spp. 67
B. QUANTITATIVE STUDIES OF PREDACIOUS ACTIVITY; PRELIMINARY OBSERVATIONS
INTRODUCTION A series of three small quantitative studies were done to examine the ability of A* oliaospora and A. flaarans to reduce the number of infective larvae in fecal culture. These studies were needed to generate data for estimating sample sizes and concentrations of spore inoculations in larger studies designed with the statistical power to detect a 50% or greater reduction.
1. Effect of three fungal concentrations (spores-to-egg ratios) on the development of infective cyathostome larvae in vitro Feces from naturally-infected horses were cultured with spores from A» oliaospora and A- flaarans at three different spore-to-egg ratios to quantify the effect on the development of infective larvae.
MATERIALS AND METHODS Fungal cultures: Field isolates of Arthrobotrys oligospora (University of Guelph collection no. 80) and A> flaarans (University of 68 Guelph collection no. 126) were supplied by Dr. G.L. Barron and propagated on PDA in disposable Petri dishes as described earlier. New cultures were propagated by transferring a one centimeter diameter plug of mature (2-4 weeks old) culture onto each PDA plate.
Harvest of fungal spores: Two to four week old fungal cultures showing abundant spore production were dried for 24 hours in a clean air hood. Five ml of surfactant water were added to each culture and the surface was gently scraped with a spatula to suspend the spores. The suspension was pipetted into a clean test tube, sterile distilled water added to make a volume of 10 ml, and the fungal suspension centrifuged at 500g for 15 minutes. After centrifugation, the supernatant was discarded and the spore pellet resuspended in sterile distilled water. The centrifugation and resuspension was repeated twice. The final suspension was made by discarding the final supernatant and adding a small amount of sterile water. The number of spores per ml of suspension was calculated as the mean of at least five counts using a standard hemacytometer (Appendix E). 69 Fecal samples: Bulk fecal samples were collected from horses at Woodburn Farm. The feces were stored at 4°C and used within 10 days of collection. The feces were thoroughly mixed to randomly distribute the worm eggs. Five fecal egg counts were performed using the modified McMaster technique (Whitlock, 1948) with a sensitivity of 8 eggs per gram of feces (epg). The number of spores to be added to fecal samples was estimated from the mean of the five egg counts.
Experimental design: Three series of fecal cultures with increasing spore- to-egg ratios were set up: 0.1:1 (Series I), 1:1 (Series II), and 10:1 (Series III). For each series, bulk equine feces were thoroughly mixed to randomly distribute parasite eggs and divided into three equal portions. The appropriate volume of fungal spore suspension of each fungal species (A. oliaospora or A. flaarans) was added to a portion of feces to furnish the required spore-to-egg ratios for Series I, Series II, and Series III). The different concentrations of fungal suspensions resulted in differing volumes of spore suspensions. To compensate for this inequity, sterile distilled water was added until the final volume of liquid added to the fecal portions (including controls) was equal. For each treatment 70 group, five 10-gram samples were placed individually into small plastic cups with loosely fitted lids (15 samples per series) and incubated for 8 days at 26-28°C in an unlit incubator humidified by a large flat pan of water on the bottom shelf. The samples were stirred daily and gently repacked. Distilled water was added as needed to keep the cultures moist. Infective larvae were harvested using a Baermann apparatus, stained with iodine, identified and counted.
Statistical analyses: The larval counts for each species' culture were compared with that from the control in each series using a nonparametric test (Wilcoxon's rank sum). The fungi's abilities to effectively reduce larval numbers was assessed by calculating the percent reduction between each treatment and the control:
100% (^control " ^fungus )/ ^control ( )
RESULTS The mean cyathostome egg counts of the horse feces were 759 epg, 864 epg, and 735 epg for Series I, II, and III, respectively. All infective larvae belonged to the 71 Cyathostominae. The addition of fungal spores of both species significantly reduced (P<0.01) the number of cyathostome L3s at 1 spore/egg and 10 spores/egg (Table 1).
For &. oliaospora and &. flaarans. respectively, the percent reductions were 40.5% and 32.1% at a level of 0.1 spore/egg, 87.4% and 90.5% at 1 spore/egg, and 95.8% and 93.9% at 10
spores/egg.
DISCUSSION The results of these studies showed that &. oliaospora and &. flaarans significantly decreased the number of infective cyathostomine larvae developing in fecal culture. Reductions were observed in every series for both species of fungi. Significant differences between the treated samples and the control samples occurred when spore to egg ratios were 1:1 or higher (Table 1). These findings confirmed the work of Nansen gt al. (1988) showing that infective cyathostome L3s were able to induce trap formation, become entrapped and killed by &. oligospora. There appeared to be a threshold below which the number of spores per egg failed to effect significant mortality (below a 1:1 ratio, Table 1). Feces used in these experiments had moderate egg counts (735-864 epg) and low larval yields (28%, 6%, and 3% for Series I, II, and III, respectively). The low number of eggs resulted in feces 72 being inoculated with fewer spores. The fungal growth may have been too sparse to allow adequate larval contact. Gronvold gt al. (1988) reported that inocula of 1500 and 2000 spores of A. oliaospora per gram of feces significantly reduced Ostertaaia ostertaai L3s in culture. Waller and Faedo (1993) observed significant reductions of Haemonchus contortus L3s in fecal cultures with inocula as low as 10 spores per gram of feces. After converting the inocula in these studies to spores per gram of feces,
significant differences (P<0.01) between mean numbers of larvae of treatment and control groups were first seen at 864 spores per gram of feces (spg) (Series II) for both fungi. However, at a concentration of 76 spg (Series I), there was no significant difference between numbers of cyathostome L3s in the fungal treated samples and the controls (Table 1). These findings confirmed that pre-parasitic stages of parasites were able to induce effective predatory activity in &. oliaospora (Roubaud and Descazeaux, 1939; Soprunov, 1958; Nansen gt al., 1988; Murray and Wharton, 1990). A- flaarans was also reported to reduce the number of pre- parasitic larvae of Q. ostertaai (Larsen gt al., 1991, 1992; Gronvold gt al-/ 1993c). The reduction in cyathostome L3s seen in A- flaarans-treated cultures suggested that the predatory activity was non-specific as reported for other 73 species of predacious fungi (Nansen et al., 1986; Nansen et al.. 1988; Jansson and Nordbring-Hertz, 1980).
2. Effect of two fungal concentrations (spores-to-egg ratios) on the development of infective trichostrongylid larvae in vitro A second series of studies was undertaken to examine the ability of A* oliaospora and A- flaarans to reduce the number of ovine Trichostronavlus infective larvae in fecal culture. Feces from a naturally infected sheep were cultured at two different spore-to-egg ratios.
MATERIALS AND METHODS Harvest of fungal spores: Fungal spores from A* oliaospora and A- flaarans were produced and harvested as described above.
Fecal samples: Feces collected from a grazing lamb harnessed in a clean canvas fecal collection bag were stored at 4°C and used within two days of collection. The fecal pellets were broken up and the resulting sample was thoroughly mixed. Five fecal egg counts were performed using the modified McMaster technique (Whitlock, 1948) with a sensitivity of 16 74 epg to estimate the mean fecal egg count.
Experimental design: The first series of fecal cultures had a 1:1 spore-to- egg ratio (Series I); the second had a 10:1 ratio (Series II). For each series, the mixed feces were divided into three portions. The appropriate volume of spore suspension of each fungal species was added to one of two fecal portions to furnish the required spore-to-egg ratio. The third portion received only sterile distilled water as control. The different concentrations of fungal suspensions resulted in differing volumes of suspensions being added to the £. oliaospora and &. flaarans samples. To compensate for this inequity, sterile distilled water was added until the final volume of liquid added to each fecal portion (including controls) was equal. Five 10-gram aliquots from each fecal portion were placed individually into small plastic cups with loosely fitted lids (15 samples per series) and incubated for 8 days as described in the earlier study. The samples were stirred daily and gently repacked. Distilled water was added as needed to keep the cultures moist. Infective larvae were harvested using a Baermann apparatus, stained with iodine, identified and counted. 75 Statistical analysis: Larval counts for each species' culture were compared with that from the control in each series using a nonparametric test (Wilcoxon's rank sum). The fungi's abilities to reduce larval numbers was assessed by calculating the percent reduction between each treatment and
the control:
10 0 % (Xcontrol — XfungUB) / Xcont.rol ( 2)
RESULTS The mean egg count of the sheep feces was 266 epg for both series. All infective larvae belonged to the genus
Trichostrongylus. For A- oliaospora and h. flaarans. respectively, the percent reduction in L3s was 45.4% and 31.9% at a level of 1 spore/egg, and 49.9% and 54.0% at a level of 10 spores/egg. There were significantly (P<0.05) fewer L3s harvested from A- oliaospora-treated samples in both series compared to controls. A* flaarans-treated samples reduced the number of L3s significantly only at the 10 spores/egg level (Table 2).
DISCUSSION &. oliaospora and A. flaarans decreased the number of infective Trichostrongylus L3s developing in fecal culture. The percent reduction was lower than was observed for equine cyathostomes at the same ratios. Waller and Faedo (1993) reported that the percent reduction in L3s increased with the concentration of conidia in the feces, but no significant relationship was observed between fecal egg count and percent reduction of L3s. A comparison of the results between the two studies described here was complicated by differences in egg counts for the two studies. Because the egg count in the sheep trial was lower than in the horse trial, fewer spores were added to the
feces. The effective entrapment of larvae was dependent on the probability of its encounter with a fungal trapping organ.
The probability of this encounter was based on the volume of fecal material that a given amount of fungus had filled with trapping organs. Therefore, the amount of fungus added per gram of feces was deemed a more appropriate unit of measure. When the concentration of spores in these two studies was calculated in number of spores per gram of feces (Tables 1 and 2), the results showed that the number of spores per gram of feces was a more meaningful unit for measuring the number of spores to be added to feces. 77 3. Effect of three fungal concentrations (spores per gram of feces) on the development of infective cyathostome larvae in vitro
A third study was conducted to examine the effects of &. oliaospora and A. flaarans spores in three concentrations of spores per gram of cyathostome infected horse feces.
MATERIALS AND METHODS Harvest of fungal spores: Fungal spores from &. oliaospora and A. flaarans were produced and harvested as described above.
Fecal samples: Fresh horse feces from Woodburn Farm were stored at 4°C
and used within two days of collection. The samples were thoroughly mixed and five fecal egg counts (determined by the modified McMaster technique with a sensitivity of 8 epg) were used to estimate a mean egg count for the sample. The feces were divided into seven portions. Spores of either &. oligospora or &. flaarans were added to a portion of feces at concentrations of 50, 100 and 200 spores per gram of feces (spg). The last portion received only sterile, distilled water as the control sample. Care was taken that equal amounts of liquid was 78 added to each portion in the manner described earlier. For each of the seven samples, five 20-gram aliquots were placed individually into small plastic cups with loosely fitted lids and incubated for 8 days as described earlier. Larvae were harvested using a Baermann apparatus. The infective third stage larvae were stained with iodine, identified, and counted.
Statistical analysis: Larval counts for each species' culture were compared with that from the control in each series using a nonparametric test (Wilcoxon's rank sum). The fungi's abilities to reduce larval numbers was assessed by calculating the percent reduction between each treatment and the control:
10 0 % ( Xcontrol ^fiingua )/ ^control ( ^ )
RESULTS The mean egg count of the horse feces was 619 epg for all samples. All infective larvae belonged to the Cyathostominae. For A. oliaospora and A. flagrans. respectively, the percent reduction in L3s was 29.3% and 16.1% at 50 spg, 51.7% and 0% at 100 spg, and 52.1% and 39.9% at 200 spg. For A. oliaospora. significant 79 differences (P<0.05) were observed at treatment levels of 100 spg and 200 spg. For A. flaarans r a significant difference was seen at an inoculum of 200 spg compared to
controls (Table 3).
DISCUSSION The results based on number of spores added per gram of feces in both equine studies indicated that the percent reduction in the number of L3s developing in culture increased with increasing concentration of spores per gram
of feces (Table 4). For flaarans f reductions were observed in every test except the second equine study with 100 spg. However, significant differences between treated samples and control samples occurred only at levels of 200 spg and higher (Tables 1 and 3). Except for the 100 spg group, the percent reduction in larvae were comparable between the two fungal species. There appeared to be a threshold level of fungal matter in the fecal samples below which the fungi failed to cause a meaningful level (i.e., 50%) of mortality (below 100 spores of &. oliaospora per gram of feces and below 864 spores of A. flaarans per gram of feces in equine cyathostome infective larvae and below 2660 spg for both fungal species in ovine infective larvae, Tables 1, 2, and 80 3). Waller and Faedo (1993) reported that concentrations as low as 10 A* oliaospora spores per gram of feces significantly reduced the number of H* contortus infective larvae in fecal culture. Gronvold gt al. (1988) reported that inocula of 1500 and 2000 spores of A. oliaospora per gram of feces significantly reduced 0. ostertaai infective larvae on herbage surrounding cowpats on pasture. The effect of fecal egg count on the reduction of infective larvae appeared negligible. Disparate fecal egg counts (266 epg and 619 epg) treated with similar spore concentrations (266 spg and 200 spg respectively) resulted in similar reductions (52.1% and 45.4%, respectively) (Tables 2 and 3). The ability to detect significant differences was absent with the lower fecal egg count and present with the higher fecal egg count. The number of spores per gram of feces was positively associated with the level of larval reduction. As the number of spores increased, the percent of reduction increased exponentially (Figure 7). Estimates from this graph indicated that marked increases in the percent reduction of infective larvae accompanied increases in the concentration of spores in culture to a certain point (approximately 900 spores spg). Further increases in spore inoculation resulted in only slightly greater reductions. 81 Because of the nonspecificity of entrapment exhibited
by these fungi, the results of the second equine pilot study were used to derive estimates for the concentration of fungi added to cattle and sheep for the larger subsequent in vitro study. Cattle fecal samples were not available in high enough numbers for a pilot investigation. A reduction in the number of infective larvae in fecal culture of at least 50% was arbitrarily chosen as the lowest difference to be detected in future studies. The results of the equine and ovine studies in which the reduction was approximately 50% were used to calculate a sample size of five for a larger study (Table 5) (Daniel, 1987).
SUMMARY The results of quantitative studies of predacious activity indicated that further studies into the potential of h. oliaospora and flaarans were warranted. Both species exhibited significant activity against the development of equine and ovine infective larvae in fecal culture. Increasing the concentrations of spores increased the percent reduction of infective larvae. High fecal egg counts were desirable for detecting significant differences in the data. 82 C. QUANTITATIVE STUDIES OF PREDACIOUS ACTIVITY: MAJOR OBSERVATIONS
INTRODUCTION This study investigated the effect of two predacious
fungi, A- oliqospora and A- flaarans. on the development of infective larvae from naturally-infected horses, sheep, and cattle. The effects of fecal egg counts and concentrations of fungal spores on the number of L3s harvested from fecal cultures were observed. The time period of predacious activity was studied and the most appropriate time period
for fecal culture was determined.
MATERIALS AND METHODS Culture and harvest of fungal spores:
Spores of A» oliaospora and A- flaarans were cultured on PDA and harvested as described in the pre] iminary studies. The concentrations of the resulting suspensions were determined using a hemacytometer as described earlier. The spores were used within five hours of harvest.
Collection of fecal samples: 1. Horses Horses on pasture at Finley Farm were identified as high and low egg shedders by three separate fecal egg 83 counts. Fecal samples were collected per rectum or, if the horse was observed to defecate, from the top of the dung pile where feces had not touched the ground. The feces were kept in a chilled cooler until transported to the laboratory where they were stored in a refrigerator at 4° C. Feces were used within 48 hours of collection. Fecal samples from these animals were mixed as needed to result in a high egg
count (1230 epg) and low egg count (411 epg) samples.
2. Sheep Grazing lambs at OARDC were identified as high and low shedders by three separate fecal egg counts. Fecal samples were collected as described in the preliminary studies. The feces were kept in a chilled cooler until transported to the laboratory where they were stored in a refrigerator at 4° C. Fecal samples from these animals were mixed as needed to produce a high egg count (1800 epg) and low egg count (608 epg) samples.
3. Cattle Twelve 6-14 month old grazing heifers of The Ohio State University Dairy were sampled every 2-4 weeks starting June 2 and fecal egg counts performed. Five heifers were identified as moderate to high shedders by mid-August. Samples were collected per rectum or, if the heifer was 84 observed to defecate, from the top of the dung pile where feces had not touched the ground. Feces were kept in a chilled cooler until transported to the laboratory where they were stored in a refrigerator at 4° C. Because the number of eggs shed varied widely day to day and overall the egg counts were chronically low, cattle feces with adequate
fecal egg counts were created artificially by harvesting parasite eggs from bulk feces (as described below) and adding the concentrated suspensions of eggs to fresh feces. The addition of harvested parasite eggs to fresh feces resulted in a high egg count sample of 707 epg for both fungal species' cultures, and low egg count samples of 298 epg for &. oliaospora and 234 epg for &. flaarans.
Recovery and concentration of parasite eggs from cattle feces: a. Sedimentation technique Parasite eggs in fecal samples were recovered and concentrated by washing approximately 2 kg portions of feces with 4 liters of cold tap water through a coarse #18 (1 mm opening) sieve and then a fine #35 (0.5 mm) sieve into a plastic bucket. The resulting suspension was allowed to settle for 30 minutes. The supernatant was siphoned off. The sediment was re-sieved, settled and siphoned until the rinsing water was clear. The resulting suspension was 85 poured into one-liter graduated cylinders, gently stirred to
dislodge eggs adhering to the column sides, and allowed to settle for three hours. The supernatant was siphoned off. The egg count of the sediment was estimated using the McMaster technique. The sediment was added as needed to
fresh cattle feces. b. Flotation technique A second technique was a modification of a technique by Reinecke (1990) wherein the washed sediment attained from the serial washings described above was poured into clean 150 cm2, canted neck polystyrene tissue culture flasks (Corning Glass Works, Corning, NY). Saturated sugar solution was added until a positive meniscus was formed. The lid was screwed on, avoiding air bubbles. The bottle was laid on a flat surface for 30 minutes, allowing the eggs to float up and adhere to the inner, upper surface of the flask. The sugar suspension was slowly poured out to avoid fluid currents that might disturb eggs on the surface of the flask. The sludge remaining on the bottom of the flask was rinsed out with a plastic wash bottle, taking care to avoid splashing. Ten ml of cold tap water was added and the flask capped and shaken to resuspend the eggs. The concentration of eggs in suspension was estimated using an inversion McMaster slide (with the grid on the bottom of the chamber). 86
Preparation of feces: Fecal samples were thoroughly mixed by hand in a large plastic tub for 20 minutes to randomly distribute the
parasite eggs. Five egg counts were performed on randomly selected fecal samples using a modified McMaster technique that is sensitive to 8 eggs per gram of feces in horses and 16 gram per feces in cattle and sheep to estimate the mean fecal egg count of the mixed samples.
Preparation of fungal spores: A. olioospora and A- flaarans were cultured as described earlier. Prior to harvesting spores for the studies, fungi were tested for predacity by placing 1 cm diameter disks of mature fungal cultures (2-4 week old for A. oliaospora; 4-6 week old for A- flaarans) on water agar dishes. After 24 hours, approximately 500 free-living nematodes were added. The cultures were observed after 24 hours for abundant trap formation (a subjective judgement) and entrapment of nematodes. Spores were harvested as described earlier. Aliquots of spore suspension were added as needed to attain the desired concentration in each fecal sample. 87 Fecal culture preparation: Horses: Approximately 3.5 kg of feces from the high egg shedding horses were mixed thoroughly in a large tub for 20 minutes. The mean of five fecal egg counts was used to estimate the sample's fecal egg count. An egual amount of
feces was collected from the low shedders and treated in a like manner. From each mixed sample, five 500 g aliquots of feces were weighed out and each placed in a clean, large plastic bowl. For the high egg count sample, the volume of suspension of each species of fungus required to result in an estimated 400 spg of feces when added to 500 g of feces was placed in a separate test tube. Twice that amount was delivered to additional test tubes, resulting in 800 spg concentration of fungal spores when added to 500 g of feces. Sterile distilled water was added to the 400 spg test tubes until the volumes of fluid of both suspensions of spores were equal. Four of the five 500 g aliquots were each mixed with a measured volume of fungal spores to result in a 400 and an 800 A- oligospora spg sample, and a 400 and an 800 flaarans spg sample. The fifth aliquot received an equal volume of sterile distilled water as the control. The low egg count sample was similarly divided into 5 groups: a 400 and an 800 &. oliaospora spg sample, a 400 and 88 an 800 A* flaarans spg sample, and a control sample. After the addition of the spore suspensions, all samples were again mixed thoroughly for 15 minutes. From each 500 g sample, fifteen 20 g aliquots of feces were each lightly packed into a 104 ml capacity plastic cup and loosely
covered with a plastic cap. The cups were placed in a humidified incubator at 24° C for a maximum of 24 days. Each sample was stirred daily and gently repacked. Sterile distilled water was added as needed to keep the cultures moist, not saturated. To minimize the potential for fungal contamination, the preparation of feces was done sequentially: first, controls were set up, followed by A. flaarans samples. Then the bench was cleaned, glassware and equipment washed, and all surfaces wiped down with 70% isopropyl alcohol. The A* oliaospora samples were set up last.
Sheep: Approximately 3.5 kg of fecal pellets with high egg counts and 3.5 kg with low egg counts were each placed in a plastic bowl. The pellets were broken up and thoroughly mixed for 20 minutes. The two groups of feces were each divided into five 500 g portions and each was inoculated with a species of fungus as described for horses. To each of the resulting 10 groups, 25 g of sterile vermiculite was 89 added to help aerate the fecal culture. Each sample was again mixed thoroughly for 15 minutes. Fifteen 21 g (20 g of the original feces and 1 gram of vermiculite) aliquots from each sample were lightly packed into plastic cups and covered loosely. The cups were kept in a humidified incubator set at 24° C for a maximum of 24 days. Each sample was stirred daily and gently repacked. Sterile water was added as needed to keep the cultures moist, but not saturated. The same precautions as described for the preparation of horse samples were followed to minimize the risk of fungal contamination.
Cattle: The high egg count series was set up by harvesting eggs from approximately 20 kg of feces collected from four heifers during their peak shedding period. Five kg of feces with a fecal egg count of 707 epg was produced by the techniques described earlier. The feces were placed in a clean plastic tub and thoroughly mixed for 30 minutes. Five
500 g samples were each placed in a large, clean plastic bowl. Then 200,000 and 400,000 &. oligospora spores in suspension were each added to one of two samples resulting in a concentration of 400 and 800 spg of feces, respectively. In a similar manner, A. flagrans spore suspensions were each added to one of two other samples, 90 resulting in like concentrations of 400 and 800 spg of feces. The fifth sample was the fungus-free control. Sterile distilled water was added as needed to make the amount of liquid added to each sample of feces equal. Twenty-five g of sterile vermiculite was added to each 500 g aliquot to help aerate the fecal culture. Each sample was remixed for 15 minutes. From each 500 g sample, fifteen 21 g (20 g of the original feces plus 1 g of vermiculite) aliquots of feces were gently packed into plastic cups and loosely covered. The cups were placed in a humidified incubator at 24° C for a maximum of 24 days. Each sample was stirred daily and gently repacked. Sterile water was added as needed to keep the cultures moist, not saturated. Low fecal egg count samples were created in a manner similar to that described for the high fecal egg count sample. Feces for the &. oliaospora and &. flaarans cultures had an egg count of 284 epg and 234 epg, respectively. Each fecal sample was divided into three 500 g aliquots and fungal spore suspensions were added to two of the three portions to make concentrations of 400 and 800 spores per gram of feces for each fungal species. The third samples were the controls. Each sample was mixed and fecal cultures set up and incubated as described above. 91 Harvest, enumeration, and identification of infective nematode larvae: Samples were incubated for three weeks. Every eight days, the infective larvae from five randomly selected samples from each group were harvested. The contents of each culture were placed in a double thickness of
cheesecloth and placed in a Baermann apparatus for 24 hours. Larvae at the bottom of the column of water were collected and stored in 5% formaldehyde in individually labelled vials. At counting, excess formaldehyde was pipetted out of the vial to concentrate larvae. One drop of Lugol's iodine2 was added to stain the larvae. The suspension of larvae was mixed and 3-4 drops were placed on a standard microscope slide. A coverslip was placed on the suspension. The first 100-200 larvae were counted and identified to the genus level. Identifications were based on criteria of the Central Veterinary Laboratory Weybridge Manual (Ministry of Agriculture, Fisheries, and Food, 1977) and Georgi (Bowman, 1995). The remaining larvae were enumerated only.
2Lugol/s iodine: 4g potassium iodide dissolved in 200 ml distilled water. 2g iodine added to solution and stirred until dissolved. Solution stored in brown glass bottle away from light. 92 Estimates of the total number of larvae of each genus were calculated. Seven cultures were contaminated by high numbers of free-living nematodes and their vials contained excessive numbers of nematodes. In these cases, the first 100 infective larvae were identified while counting the number
of L3s in 10% of the sample. If fewer than 100 L3s were identified, a second aliguot was examined. An estimate of the total number of L3s of each genus in the sample was calculated based on these proportions. The uncounted balance of nematodes in the sample was saved for further enumeration if needed.
Statistical analysis: Larval counts from each species' culture were compared with that from the control using a nonparametric test
(Wilcoxon's rank sum test). The samples contaminated with high numbers of free-living non-parasitic nematodes were not included. The fungi's abilities to reduce larval numbers was assessed by calculating the percent reduction between each treatment and the control:
100% * (Xoont.rol — X(unguB)/ Xcontrol (4) 93
RESULTS Predacious activity of fungi in pure culture over time: No obvious differences were observed in the ability of A. oliaospora and A. flaarans to form traps, capture, and kill free-living nematodes over the four month period during which the fungi were cultured for spores to add to fecal cultures.
Concentration of parasite eggs from cattle feces: Both techniques resulted in an approximately 10-fold concentration of eggs. The sedimentation technique resulted in considerable fecal material in addition to the eggs. The flotation technique resulted in cleaner suspensions of eggs. However, greater numbers of eggs were recovered per unit of time with the sediment technique and most of the eggs were harvested using this method. There was a loss of approximately 49% of parasite eggs associated with processing (Table 6).
Fecal cultures: sample size and genera A sample size of five cultures per sampling time was derived from preliminary study data in order to detect reductions of at least 50% or more (Table 5). A comparison of the genera present in control samples at each sampling time indicated that an eight day culture period was adequate 94 for equitable representation of all genera (Table 7). The proportion of the total number of L3s represented by each genus in all samples remained essentially unchanged over the
three week incubation period.
1. Horse study Overall, cyathostomes comprised 98.1% of the total infective larvae harvested (ranging from 86.3% to 100%). Few non-cyathostome infective larvae were present in the study (Table 7). Nonparametric analysis on cyathostome L3s and repeated on total parasitic L3s (cyathostomes plus large strongyles) yielded virtually identical results (Tables 8
and 9).
Arthrobotrvs oligospora Low fecal egg count cultures: For A. oliaosporar no significant differences between the number of infective larvae harvested from treated and control low egg count cultures were seen at 400 spg during the three week incubation (Table 8). An inoculation of 800 spg resulted in a significant difference only after three weeks' incubation. The percent reduction in the number of
L3s was well below 50% for the first two weeks of culture, but increased to 55.4% for the 400 spg inoculum group and 87.0% for the 800 spg inoculum group by day 24 (Figure 8). 95 High fecal egg count cultures: High egg count fecal cultures showed significant differences between the number of infective larvae from A. oliaospora treated and control samples at both levels of inoculum and for all three sampling periods (Table 8). The percent reductions were markedly higher than those observed for low fecal egg count cultures and ranged between 88.4% and 98.3%. The percent reduction increased over time (Figure 8).
Arthrobotrys flaarans Low fecal egg count cultures: For A- flaarans. significant differences in the number of infective larvae between treated and control cultures were observed for all three time periods at both concentrations of spores (Table 8). The percent reduction in L3s ranged between 60.1% and 96.5%. In general, the percent reduction in L3s increased with time (Figure 9). Cultures showed initial low reductions followed by noticeably higher reductions by 16 days.
High fecal egg count cultures: Significant differences in the number of L3s harvested from treated and control cultures were observed for all three sampling times at both 400 spg and 800 spg inoculation 96 level (Table 8). The percent reduction in L3s ranged between 92.7% and 98.7%. In general, reductions were high by 8 days with minor increases at 16 and 24 days (Figure 9).
2. Sheep study
Haemonchus. Trichostrongvlus. Teladorsagia. and Strongyloides spp. were consistently present. Low numbers
of Oesophagostomum and Nematodirus were occasionally seen. Nonparametric analyses of Haemonchus. Trichostrongylus. Teladorsagia. and total infective larvae showed similar results (Tables 10, 11, 12, 13). The percent reduction in infective larvae were generally higher for &. flagrans-treated cultures than for A. oligospora-treated cultures (Tables 10, 11, 12, 13). Feces with high egg counts inoculated with the same concentration of fungi as feces with low egg counts resulted in greater reductions of all larvae with both species of
fungi (Figures 10, 11).
Arthrobotrys oligospora Low fecal egg count cultures: The effect on the development of all infective larvae in fecal cultures with low egg counts showed variable reductions (ranging from 3.2% to 69.3%) and the differences between the numbers of L3s harvested from treated and 97 control cultures were significant only for the eight-day samples inoculated with 800 spg of feces (Table 10).
High fecal egg count cultures: Fecal cultures with high egg counts exhibited consistently higher reductions for all samples (ranging from
49.2% to 94.3%) (Figure 10). Significant differences in the number of L3s harvested from treated and control high egg count cultures were detected at both concentrations of
oliaospora in the eight-day samples from Haemonchus. Teladorsagia. Trichostronavlus and Stjcongyloi.des, and total parasites (the sum of all infective larvae) (Tables 10, 11,
12, 13).
Arthrobotrvs flaarans Low fecal egg count cultures: The percent reduction in the number of L3s harvested from treated and control cultures ranged from 63.5% to 83.5%
for total L3s (Table 10). Within individual genera, the reductions ranged from zero to 97.2% (Tables 11, 12). Significant differences were observed in the 8-day samples at 400 and 800 spg inoculations for Haemonchus. Trichostronaylus and the total infective larval counts (Tables 10, 11, 12). The difference in the number of Teladorsagia larvae harvested from treated and control 98 samples were significant at 8 days and 24 days for the 800 spg inoculation only (Table 13).
High fecal egg count cultures: High egg count cultures experienced greater reductions in Haemonchus and the total infective larval counts than low egg count cultures (Figure 11). Significant reductions in the number of L3s harvested occurred in the 8-day samples at both 400 spg and 800 spg of h. flaarans for Haemonchus and Teladorsagia (Tables 11, 13). Trichostronaylus larvae exhibited significantly fewer numbers in treated cultures at day 24 for the 400 spg inoculation and at both day 8 and day
24 for 800 spg inoculation (Table 12). Total infective larvae exhibited significantly fewer numbers in treated cultures at day 8 and day 24 for 400 spg inoculation, but only at day 8 for the 800 spg inoculation (Table 10).
3. Cattle study Cooperia and Ostertaaia were present in moderate to high numbers. Trichostronaylus occurred in lower numbers, and Oesophaaostomum and Nematodirus occurred episodically. The results from nonparametric tests on Cooperia. Ostertaaia. and total infective larvae were similar (Table 14, 15, 16). 99 Arthrobotrys oliaospora Low fecal egg count cultures: Treatment with oliaospora spores of low egg count feces resulted in low reductions in the total number of L3s harvested compared with controls (Table 14). Cooperia L3s experienced low to moderate percent reductions when inoculated with 800 spg and the difference was significant on day 24 (Table 15). Low reductions and no significant differences were observed when the inoculation was 400 spg. Ostertaaia L3 reductions were widely disparate compared with controls at both inoculation levels (Table 16). With an inoculation of 400 spg, the numbers harvested from treated cultures were significantly greater than controls at day 24. With an 800 spg inoculation, no significant differences were observed. On day 8, the treated cultures had more larvae than the controls.
High fecal egg count cultures: The samples at both inoculation levels showed effective
reductions (more than 50%) in the total number of L3s harvested from treated cultures compared with controls except for the first sample (Figure 12). The differences were significant at days 16 and 24 for both inoculation levels. Cooperia and Ostertagia constituted the greatest proportion of the total infective larvae and they exhibited 100
similar results to the total infective larvae. Significant differences were observed in Cooperia at days 16 and 24 with a low inoculation and at day 24 with the high inoculation. Ostertaaia L3s were significantly reduced at day 16 and 24 for both inoculation levels. A 65.3% increase in the number
of L3s was observed on day 8 at 400 spg.
Arthrobotrvs flagrans Low fecal egg count cultures: The numbers of Cooperia. Ostertaaia. the total infective larvae inoculated with 400 and 800 spg were
reduced at all three sampling times (Tables 14, 15, 16). Significant differences in the number of total infective larvae harvested from treated and control samples were found on day 8 and day 24 for 400 spg inoculation and on day 8 for 800 spg inoculation (Table 14). For Cooperia larvae, significant differences were observed in day-8 samples at the 400 spg inoculation, and in day-8 and day-24 samples at the 800 spg inoculation level (Table 15). There were significantly fewer Ostertaaia L3s in day-8 and day-24 samples at the 400 spg inoculation level, and in day-8 only at the 800 spg inoculation level (Table 16). 101
High fecal egg count cultures: Greater percent reductions in infective larvae were observed in the high egg count cultures than in low egg count cultures (Figure 13). The percent reductions were 69.3% - 87.0%, 69.1% - 93.3%, and 67.9% - 89.5% in Cooperia. Ostertaaia. and the total larval counts, respectively. Significantly fewer infective larvae were harvested from all treated samples examined (Tables 14, 15,
16).
DISCUSSION Concentration of parasite eggs from cattle feces: The egg concentration techniques used for producing fecal samples with higher egg counts were highly labor- intensive. Processing 20 kg of feces took approximately 10 hours. An estimated 49% of the total number of eggs in feces were lost during the processing. It was unknown if the genera of parasite eggs may have suffered differential losses during the processing. For example, the large size of Nematodirus eggs may have resulted in greater retention in the fecal matter, while the smaller eggs were rinsed through the sieves. The slurry or suspension of eggs added to the bulk feces increased the fluid component of the feces, which may more closely approximate the consistency of some parasitized animals shedding high numbers of eggs. 102 Fecal cultures: Culture techniques for the identification of infective larvae have estimated time periods 7 to 12 days for the development of L3s at room temperature (Henriksen and Korsholm, 1983; Roberts and O'Sullivan, 1949; Bowman, 1995; Manual of Veterinary Parasitological Laboratory Techniques, 1971). Genera present after 24 days of incubation were the same as those present after 8 days in approximately the same numbers. These results indicated that future experiments investigating the potential of isolates of fungi to reduce the number of infective larvae in fecal cultures require only an 8 day incubation period. The essentially unchanged proportion of the total number of nematodes represented by each genus of parasitic nematodes suggested the nonspecificity of mortality effects associated with fungal activity and time. The finding of nonspecificity was consistent with the observations of Linford (1937), Jansson and Nordbring-Hertz (1980), and Nansen et ai. (1988).
1. Horse study The cyathostomes were the dominant group of horse parasites identified in the fecal larval cultures. These results supported similar reports on the decline of large strongyles and the increase in the prevalence of 103 cyathostomes (Lyons et al., 1981; Reinemeyer et &1. , 1984; Reinemeyer, 1988; Uhlinger, 1990; and Herd and Gabel, 1990).
The effects of Arthrobotrys oliaospora and A. flaarans: At low fecal egg counts, the ability of A- oliaospora to reduce the number of infective larvae in treated fecal cultures was inferior to A- flaarans during the first 2 weeks of culture (Figure 14). However, by day 24, an 800 spg inoculation resulted in L3 reductions comparable to that observed for A> flaarans. while a 400 spg inoculation remained low. These results suggested that A- oliaospora
required a longer period of growth before it was capable of entrapping larvae to any great degree. Several factors could effect such a situation. Slower growth of A* oliaospora compared with A- flaarans would result in longer time periods for the fungus to develop a mycelial mass substantial enough to allow contact with larvae and their metabolic secretions/excretions. Traps induced by the
presence of metabolic substances have been shown to increase the attractiveness of the fungus to nematodes (Field and Webster, 1977; Jansson, 1982a). Thus, once the larvae had induced traps, more larvae would be attracted to the fungus, trapped and killed. However, in vitro culturing showed the induction of traps by A- oliaospora to be equal or better than induction by A* flagrans. 104 The markedly higher reduction of L3s in high egg count cultures inoculated with oliqospora suggested that a threshold level of nematode/nematode products was necessary to stimulate high trapping activity. It apparently took two weeks for larvae to secrete/excrete products in sufficient quantities to induce abundant trapping networks that killed larvae at detectable levels. By contrast, Waller and Faedo (1993) found no significant linear relationship between fecal egg count and percent reduction. However, Gronvold (1989) reported that increased L3s induced greater numbers of traps, suggesting a positive correlation between fecal egg counts and percent reduction in L3s. The preliminary studies described earlier showed no relationship between fecal egg counts and percent reductions of L3s, but this may have been due to the limited observations (see DISCUSSION section, p. 80).
2. Sheep study In contrast to the preliminary study, the dominant nematode species harvested from fecal cultures were Haemonchus. Trichostronaylus. and Teladorsagia. Feces from this study were collected in late July, by which time populations of all species of parasites had an opportunity to become established in the flock and pasture. Although all three genera were present in higher numbers in the high 105 egg count fecal samples, Haemonchus and Teladorsagia eggs were present in much higher numbers than Trichostronaylus eggs. This difference was probably related to the high fecundity of Haemonchus and Teladorsagia compared with
Trichostronaylus.
The effects of Arthrobotrvs oliaospora and A- flaarans; A pattern similar to that in the horse study was observed in sheep. A reduction in the total number of L3s harvested from A* oliaospora-treated cultures of at least 50% did not occur until after 8 days, whereas 8-day old A- flaarans-treated cultures already exhibited reductions exceeding 60% (Figure 15), indicating that predacious activity in A* flaarans was stimulated more quickly than in A. oliaospora. The consistency of the results in horses and sheep suggested that the growth and predacious activity behavior of these isolates differ in fecal culture compared to pure monoculture on nutrient media. A pattern of L3 reduction observed for 400 spg and 800 spg A* oliaospora-treated low egg count cultures compared to high egg count cultures suggested that high reductions occurring within the first 8 days required either high numbers of fungal spores or high numbers of eggs or both (Figure 18). However, no additive effect was seen - i.e., 800 spg inoculum in high egg count cultures experienced no 106 greater reductions than the 400 spg inoculum in high egg count cultures. For the first 8 days, percent reductions of the low and high fecal egg count groups were similar for 800 spg low fecal egg count cultures. These findings suggested that &. oliaospora has some kind of threshold level for stimulation of effective predatory activity (greater than 50% reduction) that can be fulfilled by either more fungi being present to increase the
probability of contact with nematode/nematode products or by more nematodes being present to contact fungal material. Once this threshold has been reached, additional stimulation has limited effect on the predatory activity. In &. flaarans. all the groups showed effective predatory activity (high percent reduction) after 8 days' incubation suggesting that this fungus has a lower threshold for stimulation of
effective predatory activity (Figure 15).
3. Cattle study The finding of Cooperia. Ostertaaia and Trichostronaylus as the dominant genera was consistent with other studies in this area (Herd and Heider, 1985; Herd et al., 1987). Statistical analyses were performed on total larvae, Cooperiaf and Ostertaaia because their consistently high numbers ensured more confidence in the results. Trichostronaylus. Nematodirus. and Oesophaaostoroum were 107 present in such low numbers that minor reductions of only a few larvae resulted in major differences that were not
biologically significant.
The effect of Arthrobotrys oliaospora and A* flaarans; A pattern similar to that observed in the horse and sheep studies was seen in cattle. The total number of infective larvae experienced greater reductions in high egg count compared with low egg count cultures; whereas differences between two levels of inoculation were negligible (Figure 19). These findings indicated that the stimulatory effect of larvae was more important to predatory
activity of fungi than the amount of fungi initially present. A possible explanation was that fungi were able to produce mycelial networks of essentially equal density at both inoculation levels, but the presence of nematodes at high levels induced the formation of more trapping organs and greater nematode mortality. As observed in horses and sheep, flaarans treatment resulted in greater reductions in the number of L3s that developed in culture compared to A. oliaospora.
SUMMARY The effect of two species of nematophagous fungi (A. oliaospora and &. flaarans1 on the development of infective 108 larvae of horse, sheep, and cattle nematodes were examined in vitro. In general, A. flaarans exhibited superior predatory activity causing greater reductions in numbers of infective nematode larvae and quicker onset of activity
compared with &. oliaospora. Feces with higher fecal egg counts promoted greater predacious activity, while the concentration of the fungal inoculum had a negligible effect.
D. QUANTIFICATION OF FUNGI IN FECES INTRODUCTION Although several in vivo studies on feeding nematophagous fungi to animals have reported the dose (Waller and Faedo, 1994; Gronvold et al. , 1993; Hashmi and Connan, 1989), no techniques have been developed to measure the amount of fungus excreted in the feces of fungus-fed animals. It would be of great interest to estimate the number of propagative units (propagules) in the feces of fungus-fed animals as a measure of a fungus' ability to survive gut transit. In the present study, attempts to develop such a method were based on a technique developed by Reed and Muench (1938) for estimating fifty percent endpoints (EID60) in biological procedures such as titration of sera or infective virus particles. A modification of the EID50 technique was used to estimate the number of fungal 109 propagules present in feces. Initial studies compared concentrations of spores suspended in water as estimated by
the EIDbo technique to hemacytometer counts. In subsequent studies, EID50 estimated fungal propagules in fecal samples of a horse, sheep, and cow were compared to hemacytometer
counts.
EIDso method Gaddum (1933) showed that the endpoint of serum or viral titrations at which 50 percent of test animals react was less affected by small chance variations than any other endpoint. The best method of determining these endpoints was the use of large numbers of animals at dilutions near the 50 percent reaction level. Reed and Muench (1938) presented a practical method to determine this endpoint which used a relatively small number of dilutions of small sample size. The use of a range of critical dilutions created the effect of using larger sample sizes than were actually included at these dilutions. Two assumptions critical to this method were that a culture positive at a weaker dilution would be positive for all stronger dilutions and similarly, all cultures negative at a strong dilution would be negative for all weaker dilutions. All positive cultures in a series of dilutions would be summed from the bottom up in the cumulative positive column, 110
and all negative cultures would be summed from the top down in the cumulative negative column (Appendix F). Percent positives were calculated as ______cumulative positive______(5) cumulative positive + cumulative negative In order to determine the 50 percent endpoint, the proportional distance (PD) was calculated as
______50% ~ percent positive below 50%______(6) percent positive above 50% - percent positive below 50%
PD was added to the exponent associated with the percent below 50%, and the 50 percent endpoint was calculated as the base number used for dilution raised to the summed exponent.
Estimation of spore concentrations in water
MATERIALS AND METHODS Spores harvested from A. oliaospora and A- flaorans were suspended in aliquots of sterile distilled water. The concentration of spores in each suspension was estimated as the mean of at least 5 counts using a hemacytometer. In the first test of each fungal species, each suspension was diluted to produce a series of 5-fold dilutions (range 5° to 5‘5). In a second series of tests, each suspension was Ill diluted to produce a series of 10-fold dilutions (range 10'2 to 10“7). For each dilution, one ml of suspension was placed in each of four 60 X 15 mm Petri dishes containing PDA. After five days' incubation at 24° C, approximately 500 cultured soil nematodes were added to each plate. Each plate was examined for fungal growth weekly for 6 weeks under 60X magnification. Fresh soil nematodes were added to the plates every two weeks. Plates were designated presumptive positive if fungal spores and/or traps of the appropriate morphology were observed and as negative if no fungal structures were detected. All positive cultures were subcultured on fresh PDA plates and classified as positive if the appropriate spore morphology and trapping organs were observed. Plates were definitively categorized as positive only if both the original culture and the subculture were positive.
RESULTS The modified EIDso technique usually overestimated the number of spores per ml (spm) present in suspension as determined by hemacytometer counts (Table 17). When the dilution factor was base 5, spore concentrations estimated as 50 spores per ml of suspension (spm) by hemacytometer counts were 48 and 625 spm using the EID5Q method for A. oliaospora and flaarans. respectively. 112
When the dilution factor was base 10, EID5„ dilutions of &. oliaospora spore suspension estimated as 700 spm by the hemacytometer method did not bracket the 50% endpoint.
Therefore, the estimated EIDso concentration >106 spm. Similarly, when the hemacytometer estimate was 150 spm, the estimate was >106 spm by the EIDso method. £. flaarans spore suspension estimated as 150,000 spm by the hemacytometer method was estimated as 31,623 spm by the EIDso method.
DISCUSSION The method for harvesting the spores of both species of fungi resulted in suspensions that contained hyphal elements as well as spores. Fragments of hyphae as well as spores were able to propagate new fungal growth. Thus, the EIDBO technique assessed fungal growth initiated by all propagules (spores and hyphal fragments). The hemacytometer method counted only spores. The discrepancy between the two estimates may be due to the different kinds of propagules detected in each technique. Thus, hemacytometer counts underestimated the number of propagules in suspension and thus were of limited value for documenting the accuracy of the EID50 estimates. If fungal elements other than spores survived gut transit and contributed to new fungal growth, they must be included in estimates of the number of propagules in 113 excreted feces from fungus-fed animals. Theoretically, the EID50 technique was potentially useful for estimating the
number of propagative fungal units in feces, but only relative numbers in a series of dilutions were comparable. It was not possible to compare the results of the dilution method with a direct enumeration of the number of spores in the suspension.
Estimation of spore concentration in feces of horse, sheep, and cow
MATERIALS AND METHODS Spores from &. oliaospora and &. flaarans cultures were harvested separately and the concentration of spores in each suspension was estimated as the mean of at least five counts using a hemacytometer. The suspensions were diluted to give concentrations that differed by factors of ten. Each suspension was added to 5 g of fresh feces collected from an adult horse, sheep, and cow (Table 18, 19, 20). Each inoculated fecal sample was thoroughly mixed and a series of 10-fold dilutions was produced. For each dilution, one ml of fecal suspension was placed in each of four Petri dishes containing PDA. 114 After five day's incubation at 24° C, approximately 500 cultured soil nematodes were added to each plate. Each plate was examined for fungal growth weekly for six weeks under 60X magnification. Fresh soil nematodes were added to the plates every two weeks. Plates were designated presumptive positive if fungal spores and/or traps of the appropriate morphology were observed and as negative if no fungal structures were detected. All positive cultures were subcultured on PDA plates and classified as positive if the appropriate spore morphology and trapping organs were observed. Plates were categorized as positive only if both the original culture and the subculture were positive.
RESULTS Horse: Estimates of the number of propagules per gram of feces (ppg) by the EID5Q method were greater than spores counts by the hemacytometer method by a factor of ten or more (Table 18). Estimates of uninoculated samples were zero for both species of fungi. In four cases, the 50 percent endpoint exceeded the maximum dilution level used in the EID50 method. In the &. oligospora trials, a hemacytometer estimate of
1000 was estimated as greater than 10,000 by the EID60 method. In the &. flaarans trials, hemacytometer estimates of 20,000 and 100,000 were estimated as greater than one 115
million by the EIDso method. In one case, the EID5Q estimate was lower than the hemacytometer count: 100,000 and 200,000, respectively. Sheep: Estimates of the number of propagules by the EIDso method were generally higher than the number of spores determined by the hemacytometer method (Table 19). Uninoculated samples were zero for both species of fungi. For &. oligosporar hemacytometer estimates of 100, 1000, and 10.000 were estimated as 1778, 3162, and 27,542, respectively by the EID50 method. A hemacytometer count of 100.000 was 4169 by the EIDso method. For A- flaarans. hemacytometer estimates of zero, 100 and 1000 were zero, 10 and 2630, respectively, by the EID5Q method.
Cattle: The hemacytometer estimates were consistently lower than the EIDso in all trials except for uninoculated samples when both estimates were equal (Table 20). For A. oligospora. hemacytometer estimates of zero, 100, 1000, 10,000, and 100,000 were zero, 1000, 16,218, > 100,000, and > 100,000, respectively, by the EID50 method. For A. flaarans. hemacytometer estimates of zero, 100 and 1000 were zero, 263, and >100,000, respectively, by the EID5Q method. 116
DISCUSSION The higher estimates derived from the EID5Q method compared with hemacytometer counts was expected as explained on pages 112-113. However, the sheep and cattle inoculations were made from a single suspension of harvested fungi. If the assumption that proportional amounts of propagules were in each dilution, then a strong correlation between the estimates was expected - not in absolute numbers but in relative amounts. It was not possible to determine this from the results. Because the levels of propagules in the fecal samples were much higher than expected, the dilution series were frequently too limited to bracket the 50 percent endpoint. In these cases, a lower limit of the 50% endpoint was recorded (Table 18, 19, 20). This problem resulted in an inability to differentiate between 1000 and 10,000 A. oligospora ppg in horses and between 10,000 and 100,000 &. oligospora ppg in cattle. For A. flagrans. this problem resulted in the inability to distinguish between 2000 and 200,000 spg in horses, and 1000, 10,000, and 100,000 spg in cattle. The problems in choosing the appropriate number of dilutions needed to bracket the 50% endpoint made this technique a crude semiquantitative measure of the number of propagules in fecal samples. Lower inoculations were generally estimated as lower in the EID50 method and negative controls were always negative. For this study, the technique served as a crude estimator of the number of fungal elements in feces. The utility of this technique lay in its ability to estimate of relative amounts of propagules in feces excreted at different times from different species. CHAPTER III.
PREDACIOUS ACTIVITY OF ARTHROBOTRYS OLIGOSPORA AND A. FLAGRANS IN VIVO
A. DOSING TRIALS OF FUNGI IN HORSES. SHEEP AND CATTLE
INTRODUCTION A preliminary study was done to investigate the ability of A- oligospora conidia and &. flaarans chlamydospores to survive gut passage in horses and ruminating sheep and calves. For each species of livestock and each species of fungus, a trial was done to determine a) an oral dose of fungal spores that resulted in detectable levels of spores in the feces, and b) the excretion pattern of a single dose of fungi over a 48 hour period.
MATERIALS AND METHODS Culture and harvest of spores: A. oligospora conidial spores were produced from cultures grown on PDA and harvested as described earlier.
118 A. flaarans was cultured on barley grains as described by Larsen al. (1992). Two hundred grams of whole barley grains were rinsed three times in sterile distilled water. The barley grains were then placed in 200 ml of distilled water and autoclaved at 120°C under 30 pounds of pressure for 25 minutes. The cooked barley grains were inoculated with five l centimeter diameter plugs from two to four weeks old pure cultures grown on PDA and cultivated at room temperature (22-26°C) for two weeks. The barley cultures were shaken twice a week. All cultures were subcultured on PDA and tested for predacious activity and characteristic morphology of spores and spore-forming structures. A small amount of sterile distilled water was added to barley cultures. After thorough mixing, two grams of A- flaarans barley slurry were homogenized with 8 ml of water using a 15 ml capacity Tenbroeck tissue grinder. The number of chlamydospores per ml of original slurry was estimated as the mean of five counts using a hemacytometer after adjusting for the additional 8 ml of water.
Dosing and excretion of spores: The concentration of fungus present in excreted feces was the important consideration. The pattern of excretion of orally fed fungus was unknown. The pattern of spore concentration in feces that would be most dilute occurred 120 when they were uniformly distributed throughout the digesta. In order to estimate an oral dose for each species of livestock, the amount of feces produced in 24 h by each experimental animal was measured and a dose calculated that would result in 3000 spores per gram of feces excreted if all spores were uniformly distributed throughout the digesta and survived gut transit. The initial dose for each animal was approximately 3000 spores g"1 produced in 24 h.
Horse: A 232 kg adult pony mare was placed in a pen and her 24 h fecal output collected and weighed just prior to dosing to aid in determining the dose. The pony was given 3.0 X 107 A. flaarans conidial spores mixed with cracked corn and molasses over a two hour period. Feces were collected rectally just before administration of the fungal slurry and every 12 h thereafter for 48 h. The number of viable fungal units per gram of feces was estimated using the modified EID50 method described in the quantification section. Feces of each collection were plated onto PDA plates in 10-fold dilutions of 0, 10'1, 10"2, 10-3, 10-4, 10"5. Approximately 500 soil nematodes were added after five days and the cultures were observed weekly for traps and spore production for six weeks under 60X magnification. Fresh soil nematodes were added to each plate every two weeks. All positive cultures 121 were subcultured on PDA for confirmation based on morphology of spores, spore-producing structures, and trap-formation
induced by nematodes. Seven days after the administration of the A. flaarans slurry, the pony was dosed with 3.0 X 107 A- oligospora spores mixed with cracked corn and molasses. Fecal samples were collected and plated on PDA as described above. Positive cultures were subcultured for confirmation as
described above.
Sheep: A 65 kg adult ewe was placed in a pen with a companion ewe and her 24 hour fecal production was collected and weighed to aid in determining the fungal dose. The ewe was dosed with 7.6 X 106 A. oligospora conidial spores. Feces were collected just before dosing and every 12 h thereafter for 48 h by harnessing the ewe in a clean canvas fecal bag. The feces were collected and plated on PDA as described in the horse section. Seven days after the administration of the conidia, the ewe was placed in a clean pen and dosed with 7.6 X 106 A. flaarans chlamydospores. Feces were collected at the same intervals and plated on PDA as described above. This procedure was repeated three weeks later with the dose doubled to 1.5 X 107 spores for both species of fungus 122 when no fungi were detected in feces after the initial dose level. Fecal samples were collected and plated on PDA as described above.
Cattle: A 4 month old Jersey bull calf was placed in a pen with his dam and his feces were collected for 12 h prior to dosing. From this amount, the 24 h fecal production was estimated. The calf was dosed with 1.8 X 107 A- oligospora conidial spores and fecal samples collected per rectum at 12 h intervals starting just before fungal dosing and ending 48 h later. A second 3.5 month old Jersey bull calf in a separate pen with his dam was dosed with 1.8 X 107 A* flaarans chlamydospores and feces were collected at the same intervals as described above. Feces were plated on PDA as described above. Three weeks later, a 4 month old jersey bull calf was dosed with 3.6 X 107 A- oligospora conidia when no fungal units were detected in feces collected after the initial dose. Fecal samples collected per rectum at 12 h intervals starting just before fungal dosing and ending 48 h later. 123
RESULTS Horse: In the mare's modified EID50 quantification fecal cultures, viable &. oligospora propagules were detected at
12 and 24 h after administration and the estimated excretion level was 14 and 3 units g"1 feces, respectively. An estimated excretion rate of 5, 10, 3 and zero units of A. flaarans propagules g_1 feces was observed at 12, 24, 36 and 48 h, respectively, after administration (Table 21).
Sheep: All plates in the quantification cultures of feces collected after the initial fungal dose were negative. When the dose was doubled, viable &. oligospora propagative units were excreted at estimated rates of >10,000, 6500, 1000, and 100 units g_1 feces at 12, 24, 36, and 48 h post administration, respectively. Viable &. flaarans propagules were excreted at 100, 200, zero and zero units g'1 feces at 12, 24, 36 and 48 h post administration, respectively (Table
21).
Calves: No A- oligospora was cultured from feces when dosed at the initial level. When the dose was doubled, viable fungal units were detected only at 12 h post administration, 124 excreted at an estimated 10 units g'1 feces. However, A. flaarans propagules were detected when administered at the initial lower dose. At 12, 24, 36 and 48 h post administration, excretion rates of ,A. flaarans were estimated as zero, zero, 47 and 316 units g-1 feces, respectively (Table 21).
DISCUSSION The initial dose based on a theoretical uniform excretion pattern of 3000 spores excreted g-1 feces produced in 24 h by each animal resulted in detectable amounts of &. oligospora in the pony and &. flaarans in the pony and calf. When the dose was based on 6000 spores excreted g_1 feces produced in 24 h, A- oligospora was cultured from excreted feces in both the ewe and calf and flaarans from the ewe. Waller et al. (1994) orally dosed sheep with 1.2 X 106 A. oligospora conidia and subsequently cultured fungi from excreted feces 24 h later. Assuming that the sheep in that experiment produced 2.5 kg feces in 24 h, the dose was based on 480 conidia excreted g-1 feces. Gronvold et a!. (1993b) dosed calves with an estimated 10® A. flaarans chlamydospores and documented a significant reduction in the number of infective larvae in herbage. Assuming that the 24 h fecal production of the calves was 8.0 kg, the dose was based on 1.25 X 105 spores excreted g"1 feces. However, they 125 mixed these feces with an equal amount of fungus-free feces, effectively halving the theoretical maximum amount of fungus to 62,500 spores excreted g"1 feces. An initial dose of fungus based on a theoretical uniform excretion pattern of 3,000 spores g'1 feces was chosen as likely detectable in feces, and feasible for culture and harvest.
Waller et al. (1994) suggested that spores were small enough to be suspended in the fluid phase of the digesta (rather than the particulate phase) which allowed orally administered conidia to pass through the gastrointestinal tract of sheep in approximately 24 h. Argenzio e£ al. (1974) demonstrated that an orally administered fluid marker passed through the gastrointestinal tract of a 160 kg pony after 12 h with the greatest amount at 24 h. The results of this study indicated that the greatest excretion of spores in the pony and ewe was between 12 and 36 h, supporting the hypothesis that spores were carried in the
fluid phase of digesta. In general, fungal material survived gut transit through the horse better than ruminants. These differences were likely due to the damaging effects of rumen transit in the latter. Waller al. (1994) reported that fungi administered orally to sheep were isolated from abomasal digesta with greater frequency than from feces. When the rumen was bypassed and fungi were administered directly into 126 the abomasum, fungi were isolated from ileal digesta and feces with egual freguency. The more complex digestive processes of the forestomachs in ruminants appeared to destroy greater amounts of the fungi compared with the non ruminant system of the horse. A. oligospora spores appeared to survive gut transit in the calf less well than in the adult sheep. Although the calf was ruminating, it was penned with its dam and had the opportunity to suckle. Clotting milk in the abomasum may have captured spores, effectively removing them from the fluid phase and lengthening the time spores were exposed to the destructive environment of low pH and digestive enzymes. This phenomenon may explain the increased time period for detection of fungi in feces after administration of A- flaarans (36-48 h, Table 21). Whereas the more delicate A. oligospora conidia may have been destroyed, thicker coated chlamydospores of A* flaarans were able to survive increased exposure to low pH and digestive enzymes. Doubling the number of A« oligospora conidia administered to the calf may have allowed some conidia to escape the clotted milk and continue through the gut in the fluid phase. 127 B. EFFECT OF FUNGAL DOSING OF LIVESTOCK ON MIGRATION OF INFECTIVE NEMATODE LARVAE TO PASTURE
INTRODUCTION The effect of orally administered A. oligospora and A* flaarans spores on the development and migration of infective larvae from feces of horses, sheep, and cattle onto pasture was examined. Artificial dung pats of feces
from fungus-fed animals and control feces were placed on a clean pasture. The efficacy of fungi to kill nematodes was assessed by comparing pasture larval counts on herbage surrounding treatment and control pats four and eight weeks later.
MATERIALS AND METHODS Fungal spores: A. oligospora conidial spores were grown on PDA and harvested as described earlier. A* flaqrans chlamydospores were grown on barley grains and prepared as described in the dosing trial.
Horse studies: Dosing animals and collecting feces Five adult mares at the Finley Research Farm were placed in individual stalls and approximately 2 kg of 128 control feces were collected from each either rectally or from the top of the fecal pile immediately after defecation. The samples were stored in an ice cooler for transport to the laboratory. Fungal dosing was then initiated. Attempts to orally dose each mare with a suspension of approximately 9.9 X 107 &. oligospora spores by dose syringe were abandoned when the horses became unruly and fungal suspensions were spit out. The suspensions were subsequently added to enough cracked corn and sweet feed to make the mixture palatable and the five horses were kept in their stalls for approximately one hour until the mixtures were totally consumed. Twenty-four hours after fungus administration, approximately 2 kg of treated feces were collected from each mare and transported to the laboratory in an ice cooler. Three days after the administration of A. oligospora spores, three mares from the A. oligospora experiment and two new mares were each fed approximately 10.1 X 107 A. flagrans chlamydospores mixed with cracked corn and sweet feed as described above. Two mares were replaced because they were temperamental and difficult to handle. Twenty- four hours later, 2 kg fecal samples were collected from each mare and handled as described above. 129 Collection of parasite eggs by the Visser filter technique Three donor horses identified as shedding high numbers of parasite eggs at the Finley Research Farm were placed in individual stalls and feces were collected as described above. The feces were placed in an ice cooler and transported to the laboratory for harvest of parasite eggs by the Visser filter technique of Reinecke (1992) as described below: Approximately 1 kg of feces was placed in a large plastic bowl and mixed with cold water to produce a slurry.
The slurry was washed through a series of three sieves with mesh sizes of 1 mm, 0.5 mm, and 0.25 mm, respectively, using approximately 5 1 of cold water and the liquid collected in a 5.5 1 plastic bucket, including excess water squeezed out of the fecal solids. The fecal solids were rewashed through the three sieves using another 5 1 of cold water into a second bucket. Washed fecal solids were discarded and the suspensions were allowed to settle for 30 minutes. The top 4 1 of water was siphoned from each bucket and discarded. The remaining two liters of suspended eggs and fecal material were filtered through a Visser filter apparatus (Figure 17). This apparatus consisted of two filters, a 110 micron filter nested inside a 25 micron filter. The sieved fecal suspension was poured into the inner 110 micron filter and vigorously washed with a strong jet of cold water. The 130 washing continued until the efflux was clear. The sediment in the 110 micron filter was discarded, and the sediment caught by the 25 micron filter was washed into a graduated beaker. The eggs harvested from the bulk fecal sample were then allowed to settle in the beakers for approximately 3 h. The clear liquid layer of the column was discarded and the egg-containing sediment reserved. The fecal egg count of the sediment was estimated as the mean of five counts using the McMaster technique sensitive to 8 epg.
Preparation of control and treated feces Control feces were the pooled fecal samples collected immediately prior to administration of fungal suspensions. Treated feces were the pooled fecal samples collected 24 h after fungus administration. Each pooled sample was thoroughly mixed and the egg count estimated as the mean of at least five counts. Suspensions of known concentrations of harvested parasite eggs were added to produce samples with mean fecal egg counts of approximately 1000 epg. Sterile distilled water was added as needed to ensure that equal volumes of liquid were added to each sample. The samples were again thoroughly mixed. Eight 1 kg aliquots each of treated and control feces were placed in individual plastic bags, labelled and stored in an ice cooler for transport. A 5 g aliquot of each treated sample 131 and each control (four samples total) was set aside for quantification of propagative units as described in the quantification study.
Placement of dung pats on pasture A one acre pasture of mixed grass, broadleaf weeds, alfalfa, and clover that had not been grazed for two years was selected for placement of the artificial dung pats. To confirm the pasture's clean status, grass samples were collected and processed for infective larvae recovery using a modification of the methods of Taylor (1939) and Lancaster (1970) (Appendix G). Four days before placement of feces, the pasture was mowed. Two grids of 16 sites spaced 2.1m apart were staked and numbered, one for A. oligospora and one for A. flagrans. Each site within a grid was randomly allocated by the flip of a coin to receive either a treated or control dung pat. An approximately 20 cm diameter, 10 cm high cone-shaped 1-kg mass of hand-shaped fecal balls was placed at each stake. The fecal balls were formed to simulate natural horse dung. During the eight week experimental period, gridded plots were watered with a standard sprinkler for 30 minutes if no rain had occurred for two consecutive days. 132 Collection of herbage and harvest of pasture larvae: Four weeks after placement of feces on pasture, herbage in a 1413.7 cm2 area defined by a semicircle with a radius of 30 cm from the center of the fecal mass was clipped level to the ground, placed in a labelled plastic bag and stored in an ice cooler for transport to the laboratory. Larvae
were harvested from the herbage as described in Appendix G. The herbage samples were dried in a drying oven and weighed. Eight weeks after placement, the second semicircle of the surrounding herbage was collected and processed as described
above. Collection and processing of herbage, enumeration, and identification of larvae were done blinded with respect to their treated or control status.
Sheep studies: Dosing animals and collecting feces Five adult ewes were penned in a large box stall and size 44 men's jockey briefs were fitted over their hindquarters (Plate IV). Feces for control samples were collected over a period of three hours, placed in individual plastic bags, and stored in an ice cooler for transport to the laboratory. Each ewe was orally dosed by syringe with a suspension of approximately 1.5 X 107 A. oligospora spores. Twenty-four hours later, the ewes were again fitted with clean jockey briefs and feces for treated samples were collected and 133 handled as described above. Three days later, five different adult ewes were penned
and outfitted in clean jockey briefs. Control feces were collected and handled as described above. Each ewe was then dosed by syringe with a suspension of approximately 1.5 X 107 flaarans chlamydospores. Twenty-four later, fecal samples were collected from each ewe and handled as described above.
Collection of parasite eggs by the Visser filter technique Two donor lambs at OARDC, Wooster, Ohio were harnessed in fecal collection bags for approximately 6 hours. The collected feces were placed in an ice cooler and transported to the laboratory. The sheep feces were processed for parasite eggs as described on pages 129-130.
Preparation of control and treated feces The pooled control feces and pooled treated feces were each thoroughly mixed and their egg counts estimated as the mean of at least five counts. Suspensions of harvested parasite eggs were added to produce samples with mean egg counts of approximately 1000 epg. Sterile distilled water was added as needed to ensure that equal volumes of liquid were added to each sample. The samples were again thoroughly mixed. Eight 100 g aliquots of each fecal sample were placed in individual plastic bags, labelled and stored in an ice 134 cooler for transport to the Finley Research Farm. A 5 g aliquot of each treated sample and each control (four samples total) was set aside for quantification of propagative units as described in earlier.
Placement of dung pats on pasture For each species of fungus, a grid of 16 sites spaced 2.1 m apart were staked and numbered. Each site was randomly allocated to either the treated or control group. A 10 cm diameter, 2 cm high 100 g mass of soft sheep feces was placed at each site. The fecal samples were watered with a standard sprinkler for 30 minutes if no rain had occurred for two consecutive days.
Collection of herbage and harvest of pasture larvae Four weeks after placement of feces on pasture, herbage in a 1413.7 cm2 area defined by a semicircle with a radius of 30 cm from the center of the fecal mass was clipped level to the ground, placed in a labelled plastic bag and stored in an ice cooler for transport to the laboratory. Larvae were harvested from the herbage as described in Appendix G. The herbage samples were dried and weighed. Eight weeks after placement, herbage from the second semicircle was harvested and processed as described above. 135 Cattle studies: Dosing animals and collecting feces Five 4-6 month old Jersey calves at The Ohio State University dairy barn were penned and feces collected by inducing defecation by gently stroking the dorsal aspect of the rectum with a gloved finger. Feces were collected in labelled plastic bags. Approximately 2 kg of feces per calf were collected over a 4-6 h period. The samples were stored in an ice cooler for transport to the laboratory. Each calf was dosed by syringe with a suspension of approximately 10® spores of A. oliaospora. Twenty-one hours later, the calves were penned and feces collected over a 4-6 hour period as described above. Seven days later, control feces were collected from the first five calves to enter the holding pen: three of the same Jersey calves and two new Holstein calves. The calves were dosed with approximately 10® A- flaarans chlamydospores. Treated feces were collected and handled as described for A- oliaospora.
Collection of parasite eggs by the Visser filter technique Fecal samples from donor Holstein heifers on pasture at the Marion Prison Farm, Marion, Ohio were collected and egg counts performed. High shedding heifers were identified. Two days before fungal dosing, the high shedding heifers at the prison farm were placed in concrete stalls that had been 136 scrubbed out prior to their entry. Approximately 25 kg of feces were collected over a 2-3 h period by shovelling the freshly defecated feces off the floor with a clean shovel. The feces were kept in a cool area of the barn and transported in an air conditioned truck cab to the laboratory. The feces were processed as described above over a 36-38 h period. Approximately 150 g of feces were cultured for identification of nematodes present in the sample to make sure that soil nematode eggs were not seriously contaminating the donor parasite eggs.
Preparation of control and treated feces Pooled control fecal samples were thoroughly mixed and the egg count estimated as the mean of at least five counts. All harvested parasite eggs were added to the fecal samples to achieve maximum egg counts. Sterile distilled water was added as needed to ensure that equal volumes of liquid were added to each sample. The samples were again thoroughly mixed. Eight 1-kg aliquots of each fecal samples were placed in individual plastic bags, labelled and stored in an ice cooler for transport to Finley Research Farm.
Placement of dung pats on pasture Three days before placement, two plots were mowed and a 4X4 grid of 16 sites spaced 2.1 m apart was staked and 137 numbered on each plot. Each site was randomly allocated to receive either treated or control feces. An approximately 20 cm diameter, 10 cm high 1-kg dung pat was placed at each stake. The fecal samples were watered with a standard sprinkler for 30 minutes if no rain occurred for two consecutive days.
Collection of herbage and harvest of pasture larvae Four and eight weeks after placement of dung pats on pasture, herbage were collected and infective larvae harvested as described for horses and sheep.
Statistical analysis The null hypothesis for this study was that the number of L3s harvested from treated sites (i.e., sites receiving feces collected from fungus-fed animals) was equal to or greater than the number of L3s harvested from control sites. The alternative hypothesis was that the number of L3s from treated sites was less than the number harvested from control sites. The difference between the number of infective larvae harvested from control and treated sites within a plot were compared using a nonparametric one-sided Wilcoxon's rank sum test. Percent reduction in the number of L3s harvested from treated sites compared to control sites was calculated as:
100% (Xcontrol — Xfungus)/ XcontroX (7) 138
RESULTS Horse studies: Attempts to dose mares with A- oliaospora resulted in an estimated 10% loss of fungal suspension in two of the five doses administered. There was considerable variability in the amount of corn and sweet feed added to make the fungal suspensions palatable. The unit of measurement was a plastic scoop and the amount ranged from two to six scoops. Administration of &. flaarans resulted in no obvious losses of the total dose, however, generally greater amounts of corn and sweet feed was needed to make the mixture palatable. In order to achieve fecal egg counts of at least 1000 epg, approximately 1 kg of the concentrated egg suspension was added to each treated and control sample. The final egg counts were 928 epg for treated feces and 934 epg for control feces, and1033 epg for treated feces and 1000 epg for control feces for &. oliaospora and &. flaarans. respectively. Cyathostomes comprised 99.9% of L3s observed. In the h. oliaospora plot, a significant reduction (p < 0.025) in the number of L3s harvested from treated sites compared to control sites was observed at four weeks, but not at eight weeks. The mean number of larvae harvested was 139 2348.0 and 1454.0 at four weeks, and 2007.0 and 2917.0 at eight weeks from control and treated sites, respectively (Table 22). The mean dry weight of herbage was 38.7 g and 39.0 g at four weeks and 44.0 g and 44.5 g at eight weeks for control and treated sites, respectively. The number of larvae per kg herbage harvested from treated sites was significantly reduced (p < 0.025) compared to control sites at four weeks, but not at eight weeks. The mean number of L3s/kg herbage was 61,340 and 37,360 at four weeks and 45,010 and 67,581 at eight weeks on control and treated sites, respectively. The percent reduction per unit area surrounding treated dung pats was
38.1% and 0% at four and eight weeks, respectively. The percent reduction per kg of herbage was 38.6% and 0% at four and eight weeks, respectively (Figure 18). In the flaarans grid, a similar pattern emerged. Significantly (p < 0.01) fewer L3s per unit area were harvested from treated sites compared to control sites at four weeks, but not at eight weeks. The mean number of L3s was 778.0 and 349.0 at four weeks and 606.0 and 503.0 at eight weeks from control and treated sites, respectively (Table 22). The mean dry weight of the herbage was 39.0 g and 37.1 g at four weeks and 48.6 g and 45.4 g at eight weeks for control and treated sites, respectively. The number of L3s per kg herbage harvested from treated sites was significantly reduced (p < 0.025) at four weeks, but not at eight weeks. The mean 140 number of L3s/kg herbage was 19,917 and 9913 at four weeks and 13,322 and 11,642 at eight weeks from control and treated sites, respectively. The percent reduction in the number of larvae per unit area surrounding treated dung pats was 55.1% and 17.0% at four and eight weeks, respectively. The percent reduction in the number of larvae per kg of herbage was 52.8% and 11.1% at four and eight weeks, respectively (Figure 22). The number of propagative fungal units in the feces was estimated as zero for both control and treated samples in the A. oliaospora study, and zero and 2754 spg for control and treated samples, respectively, in the A* flaarans study (Table 23).
Sheep studies: The dosing of sheep and collection of feces were attained without problem. The estimated egg counts of sheep feces supplemented with harvested parasite eggs were 1145.6 epg for both treated and control samples in the A- oliaospora study and 1104.0 epg for treated and 1209.6 epg for control samples in the A. flaarans study. Infective larvae harvested from herbage belonged to the genera Haempncjtius , Trichostronavlus. and Teladorsaaia. Haemonchus comprised 93.2% of all L3s. In the A- oliaospora study, a significant reduction (p < 0.04) in the number of L3s harvested from treated sites compared to controls was observed at four weeks but not at 141 eight weeks. The mean number of larvae was 346.1 and 160.6 at four weeks and 270.6 and 107.4 at eight weeks from control and treated sites, respectively (Table 24). The mean dry weight of the herbage was 21.8 g and 21.5 g at four weeks and 33.1 and 34.0 g at eight weeks from control and treated sites, respectively. The number of larvae per kg herbage was
significantly reduced at four weeks, but not at eight weeks. The mean number of L3s per kg herbage was 17,959 and 7744 at four weeks and 7129 and 3208 at eight weeks from control and treated sites, respectively. The percent reduction in the number of larvae per unit area surrounding treated dung pats was 53.5% and 60.4% at four and eight weeks respectively. The percent reduction in the number of larvae per kg of herbage was 52.9% and 61.4% at four and eight weeks, respectively
(Figure 22). In the A. flaarans study, the mean number of larvae was 194.0 and 124.0 at four weeks and 128.0 and 51.2 at eight weeks from control and treated sites, respectively (Table 24). Reductions observed in the number of L3s harvested were not significant. The mean dry weight of the herbage was 29.5 g and 25.6 g at four weeks and 26.3 g and 28.2 g at eight weeks from control and treated sites. The mean number of infective larvae per kg herbage was 6876 and 4644 at four weeks and 5032 and 1921 at eight weeks from control and treated sites, respectively. The reductions observed in the number of L3s 142 harvested were not significant. The percent reduction in the number of larvae per unit area surrounding treated dung pats was 36.1% and 60.0% at four and eight weeks, respectively. The percent reduction in the number of larvae per kg of herbage was 26.3% and 62.7% at four and eight weeks, respectively (Figure 22). The number of propagative fungal units in the feces was estimated as zero and 316 spg for control and A. oliaospora treated feces, respectively, and zero and 8 spg for control and A- flaarans treated feces, respectively (Table 23).
Cattle studies: The collection technique for bulk donor feces presented a potential for serious contamination with eggs from non- parasitic free-living nematodes. Fecal cultures of the suspension of harvested eggs resulted in the development of infective larvae belonging to the genera of Cooperia, Ostertaaia. Trichostronaylus. Nematodirus . and Oesophaaostomum. Together, Cooperia and Ostertaqia comprised over 90% of the total infective larvae. No free-living nematodes were observed. The mean egg count of feces were 1036.8 and 1062.4 for control and treated samples, respectively, in the oliaospora study, and 1254.4 and 1177.6, respectively, in the A. flagrans study. In the A>. oliaospora study, there was an overall decrease in the total number of larvae harvested from all sites at four weeks compared to all other collections. The mean total number of L3s was 7.6 from control sites and 4.0 from treated sites and although a reduction in the number of L3s harvested from treated compared with control sites was observed, it was not significant. At eight weeks, a significant reduction (p < 0.025) in the total number of L3s harvested at treated sites were observed. The mean total number of L3s was 1237.0 and 775.0 from control and treated sites, respectively (Table 25). The mean dry weight of herbage was 32.1 g and 23.9 g at four weeks and 19.5 g and 18.4 g at eight weeks from control and treated sites, respectively. No significant reductions were observed in the number of L3s per kg herbage harvested from treated sites. The mean number of larvae per kg herbage was 166 and 153 at four weeks and 55,802 and 38,968 at eight weeks for control and treated sites, respectively. The percent reduction in the number of larvae per unit area surrounding treated dung pats was 47.4% and 37.3% at four and eight weeks, respectively. The percent reduction in the number of larvae per kg herbage was 29.3% and 33.6% at four and eight weeks, respectively (Figure 22). In the A- flaarans study, significant reductions in the number of infective larvae harvested from treated sites were observed at both four (p < 0.05) and eight weeks (p < 0.004). 144 The mean number of larvae was 343.0 and 23.3 at four weeks and 412.0 and 113.6 at eight weeks from control and treated samples, respectively (Table 25). The mean dry weight of herbage was 40.7 g and 43.2 g at four weeks and 40.6 g and 33.0 g at eight weeks from control and treated sites, respectively. A significant reduction (p < 0.05) in the number of L3s per kg herbage was observed at four weeks but not at eight weeks. The mean number of larvae per kg herbage was 7911 and 569 at four weeks and 9696 and 3384 at eight weeks from control and treated sites, respectively. The percent reduction in the number of larvae per unit area surrounding treated dung pats was 93.2% and 72.4% at four and eight weeks, respectively. The percent reduction in the number of larvae per kg herbage was 93.6% and 66.1% at four and eight weeks, respectively (Figure 22). Quantitative assay estimated the presence of fewer than one spore g-1 feces for A. oliaospora and 3 spores g'1 feces for A- flaarans feces (Table 23). The actual level of fungal excretion was higher for the following reason. To achieve fecal egg counts of approximately 1000 epg, cattle feces were mixed with an equal amount of processed feces containing concentrated parasite eggs, but no fungi. Therefore, assayed feces contained only half the concentration of fungal elements occurring in the freshly excreted feces. 145 DISCUSSION Horses studies: Horses were dosed at a rate of approximately 4000 spores g_1 of feces produced in 24 h. However, the amount of fungus administered may have differed from the calculated dose for two reasons. Firstly, A. oliaospora losses that occurred when the first two mares were being dosed could not be accurately measured. Therefore, the amount of fungus actually fed to those two mares may be more (if less than 10% was actually lost) or less (if more than 10% was actually lost) than what was calculated. Secondly, an unknown amount of both A. oliaospora and A. flaarans may have adhered to the wooden surface of the mangers in which the fungus-corn-sweetfeed mixtures were fed. However, extra molasses spread on the manger successfully encouraged mares to lick the manger out. In addition, a reduction in the concentration of excreted fungal material occurred when the feces from mares were diluted by the sediment containing parasite eggs at a ratio of 10:1 (9.1% dilution). Therefore, the reduction in the number of L3s observed in the herbage surrounding treated sites was considered a conservative estimate. The similarity between the percent reduction in L3s per unit area and per kg herbage was due to the nearly equal weights of herbage from treated and control sites. However, the number of L3s per kg herbage was a more 146 meaningful way to assess the effect of feeding fungi to livestock because the acquisition of infection from pasture was dependent on the amount of herbage consumed, not by the area grazed. The length of the sward as well as the density of plant growth would have a major impact on the potential
intensity of infection for animals grazing the same size area. In vitro studies with both species of nematophagous fungi indicated that equal concentrations of fungal spores in feces with high egg counts were similar in their ability to reduce the number of L3s developing in culture. However, the results of the pasture study indicated that A. flaarans was superior to A- oliaospora in its ability to reduce the number of L3s developing in fecal pats and migrating onto the surrounding herbage in horses and cattle (Figure 22). Greater gut survival in horses may have been involved, supporting findings in the dosing study described earlier. Comparisons between the results of the A. oliaospora and the A. flaarans studies were made with the recognition that the initial doses of fungal elements may have been unequal. A. flaarans suspensions included mycelium as well as spores, while A. oliaospora suspensions were primarily conidial spores. Waller et al. (1994) observed that survivability of mycelial structures was poor in the sheep; the ability of mycelia to survive gut transit in the horse has not been studied. If mycelia components were able to survive gut 147 passage in the horse, then the amount of A. flaarans fed to each animal was considerably higher than the amount of A. oliaospora. contributing to the increased reductions observed for flaarans.
Sheep studies: Sheep were dosed at a higher rate than horses (approximately 6000 spores g'1 of feces produced in 24 h) as dictated by the preliminary dosing trial. The dosing of the fungal suspensions went smoothly, thus the amount of fungus that each ewe ingested was considered essentially equal. Approximately 100 g of a suspension containing harvested parasite eggs from donor sheep were added per kg to each of the 4 bulk samples (A. oliaospora treated feces and its control, A. flaarans treated feces and its control), resulting in a 9.1% dilution. Therefore, the observed effect on L3 migration onto herbage should be considered a conservative result. The small amount of feces making up the fecal masses was done to mimic the amount of feces normally produced at one time by sheep. Consequently, fewer eggs were deposited on the pasture that could develop into L3s compared with the horse and cattle studies. The decrease in numbers of potential L3s in the sheep study also resulted in greater variability in the different samples. Therefore, the power of the study to detect differences of the magnitude estimated in the pilot 148 study was decreased. The p values were close enough to 0.05 to suggest that modest increases in sample size may have been sufficient (Table 24). Patterns of fungal activity were different in the sheep study compared with horse and cattle studies. A. oliaospora
and A* flaarans exhibited equal efficacy in reducing the number of L3s that developed and migrated onto herbage surrounding treated sheep dung pats. In the horse and cattle studies, A- flaarans showed superior ability in reducing L3 numbers. For both species of fungus, greater reductions in
L3s occurred at eight weeks than at four weeks in the sheep study, while greater reductions occurred at four weeks in horse and cattle studies. Factors associated with the size and shape of the dung pats may have contributed to the different behavior of nematophagous fungi in sheep feces. More variable moisture and temperature levels in the smaller sheep fecal masses may have resulted in decreased numbers of larvae to develop. Fewer nematodes may have resulted in decreased trapping activity as suggested by the results in Chapter II. However, by eight weeks, recruitment of soil nematodes into the dung pat may have induced greater trapping activity. Assuming that the fungi act at or near the surface of the dung pat (J. Gronvold, personal communication, 1992), the surface area of the smaller sheep dung pat (a cylinder with a base diameter of 149 10 cm and a height of 2 cm) was approximately 303.7 cm2 for a maximum of 100,000 larvae, resulting in a potential stimulatory effect of 330 larvae/cm2 of dung surface. In the larger dung pats of the horse and cattle studies, the lateral surface area of a cone with an estimated base diameter and height of 20 cm and 10 cm, respectively, was 444.3 cm2 for a maximum of 1,000,000 larvae, resulting in a greater potential stimulatory effect of 2251 larvae/cm2 of dung surface.
Cattle studies: Heifers were dosed at a rate of approximately 10,000 spores per gram of feces produced in 24 h. The relative low fecal egg counts of all heifers resulted in the feces from the experimental animals fed fungi being mixed 1:1 with the sediment of small particle fecal solids containing harvested eggs from donor heifers. Therefore, the concentration of fungal propagules in the artificially formed dung pats was halved. In the A. oliaospora study, the extremely low harvest of L3s from both treated and control sites at four weeks was unexpected. The relatively large mass of the dung pat and the regular watering of the plots made massive mortality of larvae due to high temperature and/or dehydration unlikely. In addition, the number of L3s from the half circle of surrounding herbage harvested at eight weeks from the same 150 sites were at levels more in keeping with other collections, suggesting that L3s were not killed in the half circle of the surrounding herbage area sampled four weeks earlier. The low yield four week collection of A. oliaospora in the bovine study (10/14/94) occurred within a week of the four week collection of A. flaarans for that same study (10/21/94) and the eight week collections of A. oliaospora and A. flaarans in the ovine study (10/12/94 and 10/15/94, respectively) (Table 26). The numbers of L3s harvested at these times were of similar magnitudes and all were much greater than the low yield four-week A- oliaospora sample indicating that there were no weather-related conditions causing the difference. Overall, the performance of A- flaarans was superior to A* oliaospora in reducing the number of L3s harvested from surrounding herbage. This effect was probably due to increased numbers of A. flaarans spores surviving gut transit.
SUMMARY Propagative fungal elements of A- oliaospora and A- flaarans survived gut transit through horses, sheep, and cattle. In horses and sheep, both species of fungi were similar in their ability to reduce the numbers of infective larvae that migrate onto herbage. In cattle, A* flaarans demonstrated superior ability to reduce the number of infective larvae compared to A* oliaospora. 151 More pronounced reductions observed at four weeks compared with eight weeks in the horse and cattle studies were expected because mortality due to environmental factors normally dilutes the effect of predation over time. In sheep, however, eight week reductions were greater than those seen at four weeks for both species of fungi. This result was attributed to decreased trapping activity associated with fewer nematode larvae in a small fecal mass. However, this effect was likely overcome by recruitment of soil nematodes into the dung pat by eight weeks. Over 200 species of nematophagous fungi have been described and isolated from soils world-wide in many different ecological conditions. Studies to date have demonstrated good efficacy in reducing pasture infectivity without signs of toxicity. The horse, cattle and sheep results of the present studies all suggested that the use of nematophagous fungi has considerable potential as an important component of sustainable parasite control strategies. Appendix A Tables
152 Table I . Mean number of infective cyathostome larvae (L3) recovered from 5 10-g samples of horse feces mixed with 0 .1, I , or 10 spores of Arthropotrys oligospora (Ao) and A. flagrans (Af) per parasite egg.
Series 1 Series II Series III
Ao Af Control Ao Af Control Ao Af Control
# eggs/g feces 759 759 759 864 864 864 735 735 735
# spores/egg 0.1 0.1 0 1 1 0 10 10 0
# spores/g feces 76 76 0 864 864 0 7350 7350 0
mean #L3s 1265 1443 2125 66 50 524 9 13 213
standard deviation 480 386 660 35 13 221 4 6 32
% reduction8 40.5 32.1 — 87.4 90.5 — 95.8 93.9 —
1P W ilcoxon b .09 .09 — .01 .01 — .01 .01 —
8 Percent reduction of L3s in treated samples compared with controls.
b Significance level for Wilcoxon’s rank sum statistic. Table 2. Mean number of infective larvae (L3) recovered from 5 10-g samples of sheep feces mixed with 1 or 10 spores of Arthrobotrvs oliaospora and A. flaarans per parasite egg.
Series I Series II
Ao Af Control Ao Af Control
# eggs/g feces 266 266 266 266 266 266
# spores/egg 1 1 0 10 10 0
# spores/g feces 266 266 0 2660 2660 0
mean #L3s 137.2 171.2 251.4 143.2 131.4 285.6
standard deviation 30.5 53.4 92.7 78.3 35.8 77.6
% reduction8 45.4 31.9 — 49.9 54.0 —
— — *P Wilooxon b .02 .21 .04 .01
8 Percent reduction of L3s in treated samples compared with controls.
b Significance level for Wilcoxon’s rank sum statistic. Table 3. Mean number of infective cyathostome larvae (L3s) recovered from 5 20-g fecal samples cultured with 50,100, or 200 fungal spores per gram (spg) of horse feces.
Arthrobotrvs oliaospora study Arthrobotrvs flaarans study
50 sdq 100 SDQ 2 0 0 sdo 50 spa 1 0 0 spg 2 0 0 sdci Treated Treated Treated Control Treated Treated Tested Control
# eggs/g feces 619 619 619 619 619 619 619 619
# spores/egg .08 .16 .32 0 .08 .16 .32 0
# spores/g feces 50 100 2 0 0 0 50 1 00 2 0 0 0 mean # Las 746.0 510.0 505.6 1054.8 700.6 872.4 502.0 834.0 standard deviation 239.9 123.7 136.6 227.0 117.8 50.4 110.1 153.2
% reduction* 29.3 51.7 52.1 ----- 16.1 0 39.9 --
P b — — ' WBcoxan .06 .01 .01 .14 1 .0 .0 2 '
a Percent reduction of L3s in treated samples compared with controls.
b Significance level for Wilcoxon’s rank sum statistic. Table 4. Percent reductions in the number of infective larvae (L3) recovered from equine fecal cultures with different concentrations of Arthrobotrvs oligospora and A. flagrans.
Percent reduction # spores/g feces # epg A. oliaospora A. flaarans
50 619 29.3 n.s. 16.1 n.s.
76 759 40.5 n.s. 32.1 n.s.
100 619 51.7 ** 0 n.s.
200 619 52.1 ** 39.9*
864 864 87.4 ** 90.5 **
7350 735 95.8 ** 93.9 **
n.s. P > .05 using a Wilcoxon’s rank sum test. * P < .05 using a Wilcoxon’s rank sum test. ** P < .01 using a Wilcoxon’s rank sum test. 156 Table 5. Sample size determination for in vitro studies.
2 2 2 (s )(z *Z ) c t 1-a/2 1-p n =
/ - - \ 2 ( X - X ) C t
Equine studv Ovine studv Control Treated Control Treated Control Treated Control Treated mean number of L3s 1055.0 505.6 1055.0 510.0 285.6 143.2 285.6 131.4 standard deviation 227.0 136.6 227.0 123.7 77.3 78.3 77.3 35.8 estimated sample size(n) 2 ------2 — 5 3
X,. Mean number of L3s recovered from control samples. X Mean number of L3s recovered from treated samples. s2c Variance of control mean. s2, Variance of treated mean. Z1kj/2 Standard normal statistic where a = 0.05. 157 Z,.p Standard normal statistic where p = 0.2. 158
Table 6. Percent loss of parasite eggs associated with sedimentation technique for harvesting eggs from large quantities of feces.
Initial number of parasite eggs in 20 kg bulk fecal sample:
20 kg feces X 211 eggs/g feces X 1000g/kg = 4,220,000 eggs
Number of parasite eggs in resulting sediment after processing:
1 kg fecal sediment X 2160 eggs/g feces X 1000g/kg = 2,160,000 eggs
Percent loss:
4,220,000 -2,160,000 ______X 100% = 48.8% 4,220,000 159
Table 7. Percent of total infective larvae recovered from control fecal cultures comprised by different genera.
______Horse _ feces ______
High egg count samples Low egg count samples
day 8 day 16 day 24 day 8 day 16 day 24
Arthrobotrvs olioospora
Cyathostomes 98 99 99 100 100 98
Gyalocephalus 1 1 0 0 0 0
Poteriostomum 0 0 0 0 0 1
Strongvlus 1 0 1 0 0 1
A. flaorans
Cyathostomes 98 99 99 100 100 98
Gvalocephalus 1 1 0 0 0 0
Poteriostomum 0 0 0 0 0 1
Strongvlus 1 0 1 0 0 1 160
Table 7 (continued)
______Sheep feces ______
High egg count samples Low eoo count samples
day 8 day 16 dav 24 day 8 dav 16 dav 24
A. olioospora
Haemonchus 46 43 58 16 18 20
Trichostronovlus 4 6 15 64 72 74
Teladorsaoia 6 1 9 2 2 1
Stronovloides 44 50 17 14 5 4
Nematodirus 0 0 1 0 0 0
Oesophaoostomum 0 0 0 4 3 1
A. flaorans
Haemonchus 46 43 58 16 18 20
Trichostronovlus 4 6 15 64 72 74
Teladorsaoia 6 1 9 2 3 1
Stronovloides 44 50 16 14 5 4
Nematodirus 0 0 2 0 0 0
OesoDhaoostomum 0 0 0 4 2 1 161
Table 7 (continued)
Cattle feces
Hiah eaa count samDles Low eaa count samDles
dav 8 dav 16 dav 24 day 8 dav 16 dav 24
A. oliaospora
CooDeria 74 61 63 80 75 74
Ostertaaia 22 36 31 19 16 15
Trichostronovlus 4 3 5 1 9 5
Nematodirus 0 0 1 0 0 0
Oesophaaostomum 0 0 0 0 0 7
A. flaarans
Cooperia 74 61 63 51 76 80
Ostertaaia 22 36 31 39 14 15
Trichostronovlus 4 3 5 10 10 5
Nematodirus 0 0 1 0 0 0
OesoDhaaostomum 0 0 0 0 0 0 Table 8. Mean number of total infective larvae (L3) recovered from low (411 epg) and high (1230 epg) egg count horse feces mixed with 0, 400 or 800 spores of Arthobotys oligospora (Ao) and A. flagrans (Af) per gram of feces (spg)-
400 spg 800 spg 0 spg (control)
Days of culture 8 16 24 8 16 24 8 16 24
Ao low FECa
sample size 5 3 4 5 5 3 4 3 5
mean # L3s 1008.3 670.0 663.0 951.9 1006.2 193.7 1039.5 1064.0 1488.0
standard deviation 129.6 245.0 185.9 220.6 121.3 69.2 106.8 236.0 724.0
1p W ilco xo n b 0.54 0.19 0.06 0.90 1.00 0.05
% reduction' 3.1 37.0 55.4 8.5 5.5 87.0
Ao high FEC
sample size 5 5 5 5 5 5 5 5 5
mean #L3s 373.9 151.1 87.6 346.1 128.0 66.2 3231.0 5476.0 3789.0
standard deviation 156.6 49.2 23.7 46.3 38.6 18.7 617.0 992.0 942.0
p 1 W ilco xo n 0.01 0.01 0.01 0.01 0.01 0.01
% reduction 88.4 97.2 97.7 89.3 97.7 98.3 Table 8 (continued)
400 spg 800 spg 0 spg (control)
Days of culture 8 16 24 8 16 24 8 16 24
Af low FEC
sample size 5 5 4 3 4 5 4 34
mean # L3s 318.0 147.4 109.2 415.0 187.7 52.2 1039.5 1064.0 1488.0
standard deviation 42.4 76.8 72.1 192.0 187.9 33.6 106.8 236.0 724.0
□ ' W ilcoxon 0.02 0.04 0.03 0.05 0.05 0.02
% reduction 69.4 86.1 92.7 60.1 82.3 96.5
Af high FEC
sample size 5 5 5 5 5 5 5 5 5
mean #L3s 236.2 209.2 193.6 204.1 107.6 49.0 3231.0 5476.0 3789.0
standard deviation 174.6 42.4 126.5 79.8 14.3 19.3 617.0 992.0 942.0
p 1 W ilcoxon 0.01 0.01 0.01 0.01 0.01 0.01
% reduction 92.7 96.2 94.9 93.7 98.0 98.7 Table 8 (continued) a Fecal egg counts estimated with McMaster technique sensitive to 8 eggs per gram feces (epg). b Significance level of the Wilcoxon’s rank sum test statistic for the comparison of #L3s recovered from treated fecal cultures compared to controls. c Percent reduction in #L3s recovered from treated fecal cultures compared to controls. Table 9. Mean number of infective cyathostome larvae (L3) recovered from low (411 epg) and high (1230 epg) egg count horse feces mixed with 0, 400 or 800 spores of Arthrobotrvs oliaospora (Ao) and A. flagrans (Af) per gram of feces (spg).
400 spg 800 spg 0 spg (control)
Days of culture 8 16 24 8 16 24 8 16 24
Ao low FEC
sample size 5 3 4 5 5 3 4 3 4
mean # L3s 1000.7 662.0 659.5 942.0 1002.4 191.4 1030.7 1045.0 1447.0
standard deviation 130.6 245.0 187.3 218.5 121.3 69.8 103.8 222.0 698.0
p 1 W ilcoxon 0.54 0.19 0.06 0.90 1.00 0.05
% reduction0 2.9 36.7 54.4 8.6 4.1 86.8
Ao high FEC
sample size 5 5 5 5 5 5 5 5 5
mean #L3s 370.2 116.6 85.8 338.2 108.0 61.8 3176.0 5396.03736.0
standard deviation 159.3 22.1 22.7 48.7 29.8 19.1 595.0 938.0 937.0
p 1 W ilcoxon 0.01 0.01 0.01 0.01 0.01 0.01
% reduction 88.4 97.8 97.7 89.4 98.0 98.3 165 Table 9 (continued)
400 spg 800 spg 0 spg (control)
Days of culture 8 16 24 8 16 24 8 16 24
Af low FEC
sample size 5 5 4 3 4 5 4 3 4
mean # L3s 315.4 146.0 107.0 411.0 185.0 51.6 1030.7 1045.0 1447.0
standard deviation 42.1 76.5 71.2 191.0 187.1 32.4 103.8 222.0 698.0
p ' W ilco xo n 0.02 0.04 0.03 0.05 0.05 0.02
% reduction 69.4 86.0 92.6 60.1 82.3 96.4
Af high FEC
sample size 5 5 5 5 5 5 5 5 5
mean #L3s 225.0 185.3 188.2 196.2 81.4 47.6 3176.0 5376.0 3736.0
standard deviation 159.0 36.6 127.3 76.2 3.0 18.0 595.0 938.0 937.0
p ' W ilcoxon 0.01 0.01 0.01 0.01 0.01 0.01
% reduction 92.9 96.6 95.0 93.8 98.5 98.7
cr> CTi Table 9 (continued) a Fecal egg counts estimated with McMaster technique sensitive to 8 eggs per gram feces (epg). b Significance level of the Wilcoxon’s rank sum test statistic for the comparison of #L3s recovered from fecal cultures compared to controls. c Percent reduction in #L3s recovered from treated fecal cultures compared to controls. Table 10. Mean number of total infective larvae (L3)recovered from low (608 epg) and high (1800 epg) egg count sheep feces mixed with 0, 400, or 800 spores of Arthrobotrys oligspora (Ao) and A. flagrans (Af) per gram of feces (spg)-
400 spg 800 spg 0 spg (control)
Days of culture 8 16 24 8 16 24 8 16 24
Ao low FECa
sample size 5 5 5 5 5 5 5 5 3
mean # L3s 1081.0 156.3 199.2 681.0 130.4 260.6 1117.0 425.0 507.0
standard deviation 297.0 82.5 111.8 277.0 45.1 97.8 238.0 397.0 318.0
P b r W ilcoxon 0.68 0.68 0.14 0.04 0.68 0.37
% reduction0 3.2 63.2 60.7 39.0 69.3 48.6
Ao high FEC
sample size 5 5 5 5 5 5 5 4 5
mean #L3s 3538.0 264.8 72.0 3437.0 289.8 129.2 6970.0 2386.0 1270.0
standard deviation 573.0 104.5 49.5 790.0 196.3 28.8 844.0 2010.0 1421.0
D r W ilcoxon 0.01 0.18 0.09 0.01 0.11 0.14
% reduction 49.2 88.9 94.3 50.7 87.9 89.8 168 Table 10 ( continued)
400 spg 800 spg 0 spg (control)
Days of culture 8 16 24 8 16 24 8 16 24
Af low FEC
sample size 5 5 5 5 5 5 5 5 3
mean # L3s 404.9 155.0 110.0 258.5 170.2 83.7 1117.0 425.0 507.0
standard deviation 107.8 69.2 37.2 61.9 72.7 34.0 238.0 397.0 318.0
p r W ilcoxon 0.01 0.40 0.04 0.01 0.68 0.04
% reduction 63.8 63.5 78.3 76.9 60.0 83.5
Af high FEC
sample size 5 5 5 5 5 4 5 4 5
mean #L3s 1545.0 179.5 84.3 1313.0 284.4 72.8 7050.0 2386.0 1270.0
standard deviation 378.0 61.4 89.5 519.0 86.3 27.0 903.0 2010.0 1421.0
p * W ilcoxon 0.01 0.04 0.09 0.01 0.18 0.18
% reduction 78.1 92.5 93.4 81.4 88.1 94.3 169 Table 10 (continued) a Fecal egg counts estimated with McMaster technique sensitive to 8 eggs per gram feces (epg). b Significance level of the Wilcoxon’s rank sum test statistic for the comparison of #L3s recovered from treated fecal cultures compared to controls.
0 Percent reduction in #L3s recovered from treated fecal cultures compared to controls. Table 11 Mean number of infective Haemonchus larvae (L3) recovered from low (608 epg) and high (1800 epg) egg count sheep feces mixed with 0, 400 or 800 spores of Arthrobotrvs oliaospora (Ao) or A. flaarans (Af) per gram of feces.
400 spg 800 spg 0 spg (control)
Days of culture 8 16 24 8 16 24 8 16 24
Ao low FEC
sample size 5 5 5 5 5 5 5 5 3
mean # L3s 135.8 17.1 18.5 131.5 17.2 42.7 182.1 88.0 78.0
standard deviation 36.0 13.5 14.7 38.0 15.4 19.4 79.0 82.9 46.0
□ 1 W ilcoxon 0.40 0.40 0.07 0.53 0.53 0.37
% reduction 25.4 80.6 76.3 27.8 80.5 45.3
Ao high FEC
sample size 5 5 5 5 5 5 5 4 5
mean #L3s 1530.0 84.7 31.7 1365.0 91.2 47.6 3186.0 1044.0 853.0
standard deviation 514.0 36.6 26.8 411.0 88.7 17.8 460.0 823.0 1005.0
p 1 W ilcoxon 0.01 0.18 0.14 0.01 0.07 0.14
% reduction 52.0 91.9 96.3 57.2 91.3 94.4 1 7 1 Table 11 (continued)
400 spg 800 spg 0 spg (control)
Days of culture 8 16 24 8 16 24 8 16 24
Af low FEC
sample size 5 5 5 5 5 5 5 5 3
mean # L3s 72.7 18.1 8.5 39.8 24.3 21.8 182.1 88.0 78.1
standard deviation 25.3 15.2 5.8 12.3 19.3 8.3 79.0 82.9 46.0
D * W ilcoxon 0.02 0.53 0.04 0.01 0.40 0.07
% reduction 60.1 79.4 89.1 78.1 72.4 72.1
Af high FEC
sample size 5 5 5 5 5 4 5 4 5
mean #L3s 497.0 35.7 45.0 395.0 56.2 29.2 3186.0 1044.0 853.0
standard deviation 245.0 14.3 61.4 161.0 21.9 21.1 460.0 823.0 1005.0
□ r W ilcoxon 0.01 0.02 0.14 0.01 0.18 0.18
% reduction 84.4 96.6 94.7 87.6 94.6 96.6
to Table 11 (continued) a Fecal egg counts estimated with McMaster technique sensitive to 8 eggs per gram feces (epg). b Significance level of the Wilcoxon’s rank sum test statistic for the comparison of #L3s recovered from treated fecal cultures compared to controls. c Percent reduction in #L3s recovered from treated fecal cultures compared to controls. Table 12 Mean number of infective Trichostronavlus larvae (L3) recovered from low (608 epg) and high (1800 epg) egg count sheep feces mixed with 0, 400 or 800 spores of Arthrobotrvs oHgospora(Ao) or A. flaarans (Af) per gram of feces (spg).
400 spg 800 spg 0 spg (control)
Days of culture 8 16 24 8 16 24 8 16 24
Ao low FEC
sample size 5 5 5 5 5 5 5 5 3
mean # L3s 626.0 115.1 164.8 341.0 88.8 202.0 708.0 293.0 387.0
standard deviation 146.0 52.8 93.0 162.0 38.2 85.5 151.0 301.0 270.0
p r W ilcoxon 0.40 0.68 0.14 0.02 0.40 0.37
% reduction 11.6 60.7 57.4 51.9 69.7 47.8
Ao high FEC
sample size 5 5 5 5 5 5 5 4 5
mean #L3s 117.6 14.4 16.0 170.4 18.6 39.3 252.8 66.4 56.9
standard deviation 49.0 13.2 8.9 43.9 7.5 11.4 54.5 42.8 37.2
p 1 W ilcoxon 0.01 0.04 0.09 0.06 0.07 0.30
% reduction 51.9 52.0 71.9 32.6 72.0 30.9 174 Table 12 (continued)
400 spg 800 spg 0 spg (control)
Days of culture 8 16 24 8 16 24 8 16 24
Af low FEC
sample size 5 5 5 5 5 5 5 5 3
mean # L3s 174.5 114.0 82.4 83.8 98.7 31.2 708.0 293.0 387.0
standard deviation 60.2 52.1 34.5 17.7 39.0 8.6 151.0 301.0 270.0
p ' W ilcoxon 0.01 0.40 0.04 0.01 0.53 0.04
% reduction 75.4 61.1 78.7 88.2 66.3 91.9
Af high FEC
sample size 5 5 5 5 5 4 5 4 5
mean #L3s 105.0 20.9 7.4 78.9 17.8 7.2 252.8 66.4 56.9
standard deviation 146.0 4.9 3.7 252.8 7.4 4.6 54.5 42.8 37.2
p * W ilcoxon 0.14 0.07 0.09 0.01 0.07 0.11
% reduction 58.5 68.5 87.0 68.9 73.2 87.3 5 7 1 Table 12 (continued)
3 Fecal egg counts estimated with McMaster technique sensitive to 8 eggs per gram feces (epg). b Significance level of the Wilcoxon’s rank sum test statistic for the comparison of #L3s recovered from treated fecal cultures compared to controls. c Percent reduction in #L3s recovered from treated fecal cultures compared to controls. Table 13 Mean number of Teladorsaoia infective larvae (L3) recovered from low (608 epg) and high (1800 epg) egg count sheep feces mixed with 0, 400 or 800 spores of Arthrobotrvs oliaspora (Ao) or A. flaarans (Af) per gram of feces (spg).
400 spg 800 spg 0 spg (control)
Days of culture 8 16 24 8 16 24 8 16 24
Ao low FEC
sample size 5 5 5 5 5 5 5 5 3
mean # L3s 19.6 0.6 0.7 31.3 0.8 1.1 17.1 6.8 7.1
standard deviation 12.1 0.9 1.0 16.8 1.3 1.1 4.1 7.8 3.9
p ‘ W ilcoxon 0.83 0.17 0.04 0.06 0.17 0.04
% reduction 0 91.2 90.1 0 88.2 84.5
Ao high FEC
sample size 5 5 5 5 5 5 5 4 5
mean #L3s 101.0 10.1 8.3 164.0 26.1 9.8 395.0 24.2 9.8
standard deviation 106.0 4.5 4.7 163.0 20.3 5.5 141.0 34.9 5.5
p 1 W ilcoxon 0.02 1.00 1.00 0.06 0.81 0.83 7 7 1
% reduction 74.5 58.3 15.3 58.6 0 0 Table 13 (continued)
400 spg 800 spg 0 spg (control)
Days of culture 8 16 24 8 16 24 8 16 24
Af low FEC
sample size 5 5 5 5 5 5 5 5 3
mean # L3s 30.7 1.9 0.8 4.1 0.2 0.2 17.1 6.8 7.1
standard deviation 17.7 2.6 1.3 1.6 0.4 0.4 4.1 7.8 3.9
p * W ilcoxon 0.21 0.35 0.04 0.01 0.08 0.04
% reduction 0 72.1 88.7 76.1 97.1 97.2
Af high FEC
sample size 5 5 5 5 5 4 5 4 5
mean #L3s 55.4 14.4 3.6 81.7 9.6 5.5 395.0 24.2 9.8
standard deviation 44.5 6.5 2.0 64.4 5.8 3.7 141.0 34.9 5.5
p ' W ilcoxon 0.01 0.90 0.01 0.01 1.00 0.54
% reduction 86.0 40.5 67.4 79.3 60.3 43.9 178 Table 13 (continued)
a Fecal egg counts estimated with McMaster technique sensitive to 8 eggs per gram feces (epg).
b Significance level of the Wilcoxon’s rank sum test statistic for the comparison of #L3s recovered from treated fecal cultures compared to controls.
c Percent reduction in #L3s recovered from treated fecal cultures compared to controls. Table 14. Mean number of total infective larvae (L3) recovered from low (298 epg Ao, 234 epg Af) and high (707 epg) egg count cattle feces mixed with 0, 400, and 800 spores of Arthrobotrys oligospora (Ao) or A. flagrans (Af) per gram of feces (spg).
400 spg 800 spg 0 spg (control)
Days of culture 8 16 24 8 16 24 8 16 24
low FECa
sample size 5 5 5 4 5 5 5 5 5
mean # L3s 307.0 181.0 275.2 318.0 161.1 272.0 361.0 208.0 455.0
standard deviation 183.0 135.0 60.2 53.4 63.9 104.0 177.0 181.0 115.0
Pr W ilcoxon b 0.68 0.83 0.02 0.71 0.21 0.06
% reduction0 15.0 13.0 39.5 11.9 22.5 40.2
Ao high FEC
sample size 5 5 5 5 5 5 5 5 5
mean #L3s 511.0 123.5 267.0 340.0 233.0 253.0 509.0 507.8 923.0
standard deviation 106.0 39.4 150.0 224.0 162.0 149.0 147.0 86.4 433.0
p 1 W ilco xo n 1.00 0.01 0.02 0.14 0.04 0.02
% reduction -0.4 75.7 71.1 33.2 54.1 72.6 Table 14 (continued)
400 spg 800 spg 0 spg (control)
Days of culture 8 16 24 8 16 24 8 16 24
Af low FEC
sample size 5 5 5 5 5 5 5 5 4
mean # L3s 100.7 117.8 78.4 118.0 108.9 67.6 248.4 180.5 225.0
standard deviation 11.8 73.7 46.0 52.5 74.5 62.3 70.6 35.6 91.5
p 1 W ilcoxon 0.01 0.21 0.04 0.01 0.21 0.06
% reduction 59.5 34.7 65.2 52.5 39.7 70.0
Af high FEC
sample size 5 5 5 5 4 5 5 5 5
mean #L3s 163.6 79.1 153.0 85.1 72.7 97.2 509.0 507.8 923.0
standard deviation 67.3 61.5 104.0 27.1 37.0 89.0 147.0 86.4 433.0
p 1 W ilcoxon 0.01 0.01 0.01 0.01 0.02 0.01
% reduction 67.9 84.4 83.4 83.2 85.7 89.5
oo Table 14 (continued)
3 Fecal egg counts estimated with McMaster technique sensitive to 8 eggs per gram feces (epg). b Significance level of the Wilcoxon’s rank sum test statistic for the comparison of #L3s recovered from treated fecal cultures compared to controls. c Percent reduction in #L3s recovered from treated fecal cultures compared to controls. Table 15 Mean number of infective Cooperia larvae (L3) recovered from low (298 epg Ao, 234 epg Af) and high (707 epg) egg count cattle feces mixed with 0, 400 or 800 spores of Arthrobotrvs oliaospora (Ao) or A. flaarans (Af) per gram of feces (spg).
400 spg 800 spg 0 spg (control)
Days of culture 8 16 24 8 16 24 8 16 24
Ao low FEC
sample size 5 5 5 4 5 5 5 5 5
mean # L3s 231.0 139.0 217.5 189.0 127.2 133.5 266.0 147.0 221.1
standard deviation 154.0 102.0 50.1 57.2 51.9 39.8 121.0 43.1 51.9
□ ' wilcoxon 053 1.00 0.83 0.39 0.30 0.02
% reduction 13.2 5.4 1.6 28.9 13.5 65.6
Ao high FEC
sample size 5 5 5 5 5 5 5 5 5
mean #L3s 338.0 87.5 202.0 266.0 170.0 199.0 401.0 302.2 578.0
standard deviation 109.0 22.3 108.0 191.0 127.0 118.0 105.0 67.9 244.0
p 1 W ilcoxon 0.30 0.01 0.04 0.14 0.09 0.04
% reduction 15.7 71.0 65.1 33.7 43.7 65.6 183 Table 15 (continued)
400 spg 800 spg 0 spg (control)
Days of culture 8 16 24 8 16 24 8 16 24
Af low FEC
sample size 5 5 5 5 5 5 5 5 4
mean # L3s 51.7 83.2 65.9 58.9 77.1 47.8 125.0 135.8 180.0
standard deviation 5.0 61.2 38.8 24.4 50.8 41.0 27.2 32.4 78.2
p r W ilcoxon 0.01 0.14 0.07 0.01 0.14 0.04
% reduction 58.6 38.7 63.4 52.9 43.2 73.4
Af high FEC
sample size 5 5 5 5 4 5 5 5 5
mean #L3s 123.0 61.1 119.9 54.2 51.2 74.9 401.0 302.2 578.0
standard deviation 47.2 44.7 85.6 18.0 18.6 65.4 105.0 67.9 244.0
p ' W ilcoxon 0.01 0.01 0.01 0.01 0.02 0.01
% reduction 69.3 79.8 79.3 86.5 83.1 87.0 4 8 1 Table 15 (continued)
3 Fecal egg counts estimated with McMaster technique sensitive to 8 eggs per gram feces (epg). b Significance level of the Wilcoxon’s rank sum test statistic for the comparison of #L3s recovered from treated fecal cultures compared to controls. c Percent reduction in #L3s recovered from treated fecal cultures compared to controls. Table 16 Mean number of infective Ostertaaia larvae (L3) recovered from low (298 epg Ao, 234 epg Af) and high (707 epg) egg count cattle feces mixed with 0, 400 or 800 spores of Arthrobotrvs oliaospora (Ao) or A. flaarans (Af) per gram of feces (spg).
400 spg 800 spg 0 spg (control)
Days of culture 8 16 24 8 16 24 8 16 24
Ao low FEC
sample size 5 5 5 4 5 5 5 5 5
mean # L3s 63.9 25.6 43.0 120.2 28.2 103.8 77.2 57.0 166.1
standard deviation 23.3 19.4 8.0 12.6 14.6 50.4 45.2 18.6 61.6
p ' W ilcoxon 0.83 0.06 0.01 0.18 0.06 0.14
% reduction 17.2 55.1 74.1 -55.7 50.5 37.5
Ao high FEC
sample size 5 5 5 5 5 5 5 5 5
mean #L3s 167.3 28.2 53.8 59.7 50.2 43.0 101.2 191.0 271.0
standard deviation 35.1 18.4 38.6 28.2 33.3 25.8 51.3 101.0 149.0
p * W ilcoxon 0.06 0.02 0.01 0.21 0.02 0.01
% reduction -65.3 85.2 80.1 41.0 73.7 84.1 6 8 1 Table 16 (continued)
400 spg 800 spg 0 spg (control)
Days of culture 8 16 24 8 16 24 8 16 24
Af low FEC
sample size 5 5 5 5 5 5 5 5 4
mean # L3s 42.0 24.4 10.3 48.7 18.1 14.6 98.6 25.4 32.6
standard deviation 8.7 12.4 7.1 23.8 15.1 14.1 39.5 10.6 13.7
□ 1 W ilcoxon 0.01 1.00 0.04 0.04 0.30 0.18
% reduction0 57.4 3.9 68.4 50.6 28.7 55.2
Af high FEC
sample size 5 5 5 5 4 5 5 5 5
mean #L3s 31.3 12.8 28.4 23.5 17.7 18.9 101.2 191.0 271.0
standard deviation 26.4 13.9 18.4 6.3 16.5 22.7 51.3 101.0 149.0
p ' W ilcoxon 0.04 0.01 0.01 0.01 0.02 0.01
% reduction 69.1 93.3 89.5 76.8 90.7 93.0 7 8 1 Table 16 (continued) a Fecal egg counts estimated with McMaster technique sensitive to 8 eggs per gram feces (epg). b Significance level of the Wilcoxon’s rank sum test statistic for the comparison of #L3s recovered from treated fecal cultures compared to controls. c Percent reduction in #L3s recovered from treated fecal cultures compared to controls. Table 17. Estimated number of spores suspended in water by the modified EID50 method and by direct count using a hemacytometer.
Arthrobotrys # pos # neg Estimated # spores/ml oliaospora dilution plates plates EID50 hemacytometer
5° 4 0 5'1 4 0 5'2 2 2 5-3 1 3
5-* 0 4 5'5 0 4 48 50
102 4 0 10‘3 4 0 10-4 4 0 10‘5 4 0 10-6 3 3 > 106 700
102 3 1 10’3 3 1 10-4 4 0 105 4 0 105 4 0 189 10-6 3 1 > 106 150 Table 17 (continued)
Arthrobotrvs # pos # neg Estimated # spores/ml flaarans dilution plates plates EIDS0 hemacytometer
5° 4 0 5'1 4 0 5'2 4 0 5'3 2 2
5-* 2 2 5'5 2 2 625 50
10'3 3 1 10-4 2 2 10'5 2 2 10-6 1 3 107 0 4 1045 1.5 x 105 190 Table 18. Estimated number of spores/g equine feces by the modified EID^ method and by direct count using a hemacytometer.
Arthrobotrvs oliaospora Arthrobotrvs flaarans # pos # neg Estimated # spores la # pos # neg Estimated # spores/a dilution plates plates EID50 hemacytometer dilution plates plates EID^ hemacytometer
10° 0 4 10° 0 4
10-1 0 4 10'1 0 4
10’2 0 4 10'2 0 4
10-3 0 4 0 0 10'3 0 4 0 0
10° 4 0 10'331
10" 4 0 10-4 4 0
10-2 1 3 10'5 4 0
10"3 1 3 10-6 3 1 >106 2 x 104
10" 1 3
10-5 1 3 10'3 4 0
10-6 1 3 3.6 x102 10 10" 4 0
10 s 4 0
10° 4 0 10-6 3 1 >106 105
10" 4 0
10'2 2 2 10'3 3 1 191 10'3 2 2 10" 4 0 Table 18 (continued)
Arthrobotrvs oliaospora Arthrobotrvs flaarans # pos # nea Estimated # SDores/a # d o s # nea Estimated # SDores/a dilution plates plates EID^ hemacytometer dilution plates plates EIDS0 hemacytometer
10-4 2 2 10 s 2 2
10 s 1 3 1.8 x 1 0 3 102 10-6 1 3
10'7 0 4 10s 2 x10s
10° 4 0
10'1 4 0
10'2 2 2
10‘3 3 1
10-4 4 0 >104 103
10° 4 0
10'1 4 0
10'2 1 3
10’3 4 0
10-4 4 0
10 s 2 2 2.8 x104 104 192 Table 19. Estimated number of spores/g ovine feces by the modified EID^ method and by direct count using a hemacytometer.
Arthrobotrvs oliaospora Arthrobotrvs flaarans # pos # nea Estimated # spores/a # d o s # nea Estimated # SDores/a dilution plates plates EID^ hemacytometer dilution plates plates EID^, hemacytometer
10° 0 4 10° 0 4
10-1 0 4 10'1 0 4
10'2 0 4 1 0 2 0 4
10'3 0 4 10-3 0 4
10-4 0 4 0 0 10^ 0 4 0 0
10° 4 0 10° 0 4
10-1 3 1 10‘1 4 0
ICC2 4 0 10-2 1 3
10'3 2 2 10-3 0 4
10-4 2 2 1.8 x103 102 10-4 0 4 10 102
10° 4 0 10° 0 4
10’1 4 0 1 0 1 4 0
10'2 4 0 10'2 2 2
10-3 2 2 1 0 3 4 0
10-4 0 4 3.2 x103 103 10-4 4 0 193 Table 19 (continued)
Arthrobotrvs oliaospora Arthrobotrvs flaarans # pos # neg Estimated # spores/a # pos # neg Estimated # spores/a dilution plates plates EIDk, hemacytometer dilution plates plates EIDS0 hemacytometer
10i-5 2.6 x 103 103
10° 1 3
10'1 4 0
10'2 4 0
10'3 4 0
10-4 4 0
10 s 2 2 2.8 x 104 104
10° 1 3
10'1 4 0
10-2 4 0
10-3 4 0
10-4 2 2
10 s 1 3
10-6 0 4 4.2 x 103 105 194 Table 20. Estimated number of spores/g bovine feces by the modified EID^ method and by direct count using a hemacytometer.
Arthrobotrys oliaospora Arthrobotrvs flaarans # pos # neg Estimated # spores/g # pos # neg Estimated # spores/a dilution plates plates EID^ hemacytometer dilution plates plates EID^ hemacytometer
10° 0 4 10° 0 4
10" 0 4 10" 0 4
10-2 0 4 10-2 0 4
10-3 0 4 10-3 0 4
10" 0 4 0 0 10" 0 4 0 0
10° 0 4 10° 1 3
10" 4 0 10" 1 3
10'2 4 0 10-2 4 0
10'3 2 2 10-3 4 0
10" 2 2 10" 0 4 2.6 x 102 102
10'5 2 2 103 102
10° 0 4
10° 0 4 10" 4 0
10-1 4 0 10'2 4 0
10'2 4 0 10'3 4 0 t-> VO 10-3 4 0 10" 4 0 Table 20 (continued)
Arthrobotrvs oliaospora Arthrobotrvs flaarans # pos # neg Estimated # spores/g # pos # neg Estimated # spores/g dilution plates plates EID, 50 hemacytometer dilution plates plates EID, 50 hemacytometer
10* 3 1 10I-5 > 10s 103
10'5 3 1 1.6 x 104 103
10° 0 4
10'2 4 0
1 0 3 4 0
10-4 4 0
10'5 4 0 > 105 104
10° 0 4
10'1 4 0
10’2 4 0
10-3 4 0
10^ 4 0
10'5 4 0 > 105 105 196 Table 21. Estimated number of viable fungal elements excreted per gram of feces from fungal-fed horses, sheep, and cattle sampled every 12 hours for 48 hours after dosing.
Horse SheeD Cattle
A. oliaosDora A. flaarans A. oliaospora A. flaarans A. oliaospora A. flaarans
to 0 0 0 0 0 0
*12 14 5 >104 102 10 0
^24 3 10 6.5 x 103 2 x 102 0 0
^36 0 3 103 0 0 47
tl8 0 0 102 0 0 3.2 x 102
# spores/dose 3 x 107 3 x 107 1.5 x 107 1.5 x 107 3.6 x 107 1.8 x 107 Table 22. Mean number of infective larvae (L3) per kg herbage surrounding treated and control equine dung pats.
Arthrobotrvs oliaosDora studv Arthrobotrvs flaarans studv 4 weeks 8 weeks 4 weeks 8 weeks Treated Control Treated Control Treated Control Treated Control sample size 8 8 8 8 8 8 8 8 mean #L,s semicircle 1454.0 2348.0 2917.0 2007.0 349.0 778.0 503.0 606.0
Mean herbage dry wt(g)a 39.0 38.7 44.5 44.0 37.1 39.0 45.4 48.6
Mean #L,s kg herbage 37,360 61,340 67,581 45,010 9913 19,917 11,642 13,322
standard deviation 16,398 23,226 15,536 15,769 8282 6195 4664 8408
% reduction15 38.6 — 0 — 52.8 — 11.1 —
median #L,s kg herbage 33,444 65,859 66,901 38,901 7491 19,733 10,954 12,599
— — — — 1P w ilcoxon C <0.025 >0.10 <0.025 >0.10
Mean dry weight of herbage harvested from a 30 cm radius semicircle around dung pat.
6 Percent reduction in number of L3s/kg herbage recovered from treated sites compared with control sites.
c Significance level of the Wilcoxon’s rank sum test statistic for the comparison of #L3s/kg herbage recovered from treated sites 8 9 1 compared with control sites. 199
Table 23. Estimated number of fungal spores excreted per gram of feces immediately prior to and 24 h after dosing horses, sheep and cattle.
Spores per gram of feces
Arthrobotrys oligospora A. flagrans
Horse
treated* 0 2754
control15 0 0
Sheep
treated 316 8
control 0 0
Cattle
treated <1 3
control 0 0 a Fecal sample collected 24 h after dosing. b Fecal sample collected before dosing. Table 24. Mean number of infective larvae (L3) per kg herbage surrounding treated and control ovine dung pats.
Arthrobotrvs oliaosDora studv Arthrobotrvs flaarans studv 4 weeks 8 weeks 4 weeks 8 weeks Treated Control Treated Control Treated Control Treated Control
sample size 8 8 8 8 8 7 8 8
mean #L,s semicircle 160.6 346.1 107.4 270.6 124.0 194.0 51.2 128.0
Mean herbage dry wt(g)a 21.5 21.8 34.0 33.1 25.6 29.5 28.2 26.2
Mean #L„s kg herbage 7744.0 17,959.0 3208.0 7129.0 4644.0 6876.0 1921.0 5032.0
standard deviation 8906.0 12,849.0 2319.0 6882.0 3005.0 6117.0 1790.0 3867.0
% reduction15 52.9 — 61.4 — 26.3 — 62.7 —
median #L„s kg herbage 4947.0 17,344.0 2676.9 4843.2 4623.0 6014.8 1302.7 5659.5
— — — 1P w ilcoxon c <0.025 <0.063 — >0.10 <0.075
Mean dry weight of herbage harvested from a 30 cm radius semicircle around dung pat.
b Percent reduction in number of L3s/kg herbage recovered from treated sites compared with control sites.
0 Significance level of the Wilcoxon’s rank sum test statistic for the comparison of #L3s/kg herbage recovered from treated sites 200 compared with control sites. Table 25. Mean number of infective larvae (L3) per kg herbage surrounding treated and control bovine dung pats.
Arthrobotrvs oliaosDora studv Arthrobotrys flaarans studv 4 weeks 8 weeks 4 weeks 8 weeks Treated Control Treated Control Treated Control Treated Control sample size 8 8 8 8 8 8 8 8 mean #L,s semicircle 4.0 7.6 775.0 1237.0 23.3 343.0 113.6 412.0
Mean herbage dry wt(g)a 23.9 32.1 18.4 19.5 43.2 40.7 33.2 40.6
Mean #L„s kg herbage 153.0 166.0 38,968.0 55,802.0 569.0 7911.0 3384.0 9696.0 standard deviation 170.0 228.0 39,227.0 34,587.0 357.0 8512.0 2183.0 6097.0
% reduction*" 29.3 — 33.6 — 93.6 — 66.1 — median #L,s kg herbage 86.5 101.4 21,531.0 59,407.0 594.0 5155.0 2521.8 8161.8
p1 W ilcoxon c >0.10 — >0.10 — <0.05 — >0.10 —
Mean dry weight of herbage harvested from a 30 cm radius semicircle around dung pat. b Percent reduction in number of L3s/kg herbage recovered from treated sites compared with control sites. c Significance level of the Wilcoxon’s rank sum test statistic for the comparison of #L3s/kg herbage recovered from treated sites compared with control sites. 2 0 2 Table 26. Dates of dung pat placement on pasture and herbage collection.
fungus 4 week x8 week species placement collection collection
Equine study:
A. oligospora 8/25/94 9/22/94 10/20/95
A. flagrans 9/1/94 9/29/94 10/27/94
Ovine study:
A. oligospora 8/17/94 9/14/94 10/12/94
A. flagrans 8/20/94 9/17/94 10/15/94
Bovine study:
A. oligospora 9/16/94 10/14/94 1 I/I 1/94
A. flagrans 9/23/94 10/21/94 11/18/94 Appendix B Figures
203 Figure 1. Capture organs of predacious fungi a. stalked adhesive knobs b. adhesive knobs c. elongated adhesive knobs d. adhesive stalks e. solitary non-constricting rings f. linear array of adhesive rings g. three-dimensional network of adhesive rings h. constricting rings
204 205
Figure 1. Figure 2. Endoparasitic fungi Top: Adhesive spores sticking to the anterior end of nematode.
Middle: Infected nematode filled with hyphae. Dark chlamydospores are intercalated along the hyphae. Fertile spores have pierced the body wall and produced conidia to be released into the enviroment.
Bottom: Anterior aspect of nematode with ingested spore lodged in esophagus. Germination of the spores has begun.
206 fo o vj
Figure 2. Figure 3. Egg-attacking fungus. Hyphae grow toward egg in environment, penetrate the shell, and fill the egg while absorbing its contents.
208 209
Figure 3. Figure 4. Drechmeria coniospora a. Nematode with adhesive conidia attached on the cuticle. b. Early infection of nematode with incipient hyphal growth. c. Conidial spores released into the environment. d. Mature infection of nematode with interior hyphae having filled the carcass. Fertile hyphae have emerged from the body and produced conidia. 2 1 0 2 1 1 Figure 4. Figure 5. Harposporium anauillulae a. Early germination of ingested conidia lodged in esophagus of nematode. b. Nematode carcass filled with hyphae. Fertile hyphae have emerged from the body and produced conidia. c. Close up of mature infection. Dark, thick-coated chlamydospores are intercalated along the hyphae. 2 1 2 0 0 Figure 6. Baermann apparatus Glass funnel is attached to a conical test tube by rubber tubing. 214 215 (f) H s tz /n/'/BA/' Figure 6. Figure 7. Pooled data on fecal cultures inoculated with Arthrobotrys olicrospora and flaarans from two preliminary equine studies. 216 90- 60 - Percent Reduction 30 - ** 0 1500 3000 4500 6000 7500 Number of fungal spores per gram of feces Figure 7. 217 Figure 8. Percent reduction of total infective larvae and cyathostomes in Arthrobotrys oliaospora-treated equine feces cultured for 8, 16, and 24 days. 400 low group had a low fecal egg count and received an inoculum of 400 spores per gram of feces. 800 low group had a low fecal egg count and received an inoculum of 800 spores per gram of feces. 400 high group had a high fecal egg count and received an inoculum of 400 spores per gram of feces. 800 high group had a high fecal egg count and received an inoculum of 800 spores per gram of feces. 218 % reduction % reduction 100 100 80 60 40 20 I 1 W m . nn? n n 400 low 800 low 400 high 800 high 400 low 800 low 400 high 800 high treatment group treatment group ■ I day 8 I H ] day 16 CZZ day 24 day 8 day 16 .... [ I day 24 Total infective larvae Cyathostomes Figure 8. Figure 9. Percent reduction of total infective larvae and cyathostomes in Arthrobotrys flaarans-treated equine feces cultured for 8, 16, and 24 days. 400 low group had a low fecal egg count and received an inoculum of 400 spores per gram of feces. 800 low group had a low fecal egg count and received an inoculum of 800 spores per gram of feces. 400 high group had a high fecal egg count and received an inoculum of 400 spores per gram of feces. 800 high group had a high fecal egg count and received an inoculum of 800 spores per gram of feces. 220 % reduction % reduction 100 100 400 low 800 low 400 high 800 high 400 low 800 low 400 high 800 high treatment group treatment group Hi day 8 H H day 16 d day 24 HU day 8 HH day 16 CZH day 24 Total infective larvae Cyathostomes F i g u r e 9 . Figure 10. Percent reduction of total infective larvae, Haeroonchus. Trichostrongvlus. and Teladorsagia larvae in Arthrobotrvs ol icrospora-treated ovine feces cultured for 8, 16, and 24 days. 400 low group had a low fecal egg count and received an inoculum of 400 spores per gram of feces. 800 low group had a low fecal egg count and received an inoculum of 800 spores per gram of feces. 400 high group had a high fecal egg count and received an inoculum of 400 spores per gram of feces. 800 high group had a high fecal egg count and received an inoculum of 800 spores per gram of feces. 222 223 % reduction % reduction 100 100 400 low 800 low 400 high 600 high 400 low 800 low 400 high 800 high treatment group treatment group ■ ■ day 8 M l day 16 I I day 24 ■ ■ d a y 8 i day 16 CD day 24 Total Infective larvae Haemonchus % reduction % reduction too |------1001------ 80 -- treatment group treatment group day 8 KWXWN day 18 I I day 24 day 8 ESS day 18 I ,...l day 24 Trichostrongylus Teladorsagia Figure 10. Figure 11. Percent reduction of total infective larvae, Haemonchus. Trichostrongvlus. and TelfrflQrsagia larvae in Arthrobotrys flaarans-treated ovine feces cultured for 8, 16, and 24 days. 400 low group had a low fecal egg count and received an inoculum of 400 spores per gram of feces. 800 low group had a low fecal egg count and received an inoculum of 800 spores per gram of feces. 400 high group had a high fecal egg count and received an inoculum of 400 spores per gram of feces. 800 high group had a high fecal egg count and received an inoculum of 800 spores per gram of feces. 224 225 % reduction % reduction 1001------ treatment group treatment group ■ i day 8 ESS day18 C H I day 24 day 8 fSSS day18 □ □ day 24 Total Infective larvae Haemonchus % reduction % reduction 1 0 0 1------1001------ 4000 low 8000 low 4000 high 8000 high treatment group treatment group day 8 KWNWN day 18 I I day 24 day 8 M day 18 C D day 24 Trichostrongylus Teladorsagia Figure 11. Figure 12. Percent reduction of total infective larvae, Coppgclfl and Qs.tgct.ft.qlft larvae in Arthrobotrvs oligospora-treated bovine feces cultured for 8, 16 and 24 days. 400 low group had a low fecal egg count and received an inoculum of 400 spores per gram of feces. 800 low group had a low fecal egg count and received an inoculum of 800 spores per gram of feces. 400 high group had a high fecal egg count and received an inoculum of 400 spores per gram of feces. 800 high group had a high fecal egg count and received an inoculum of 800 spores per gram of feces. 226 227 % reduction 100 400 low 800 low 400 high 800 high ■ I day 8 KM day 16 I -) day 24 Total infective larvae % reduction % reduction 100 100 JLu 400 low 800 low 400 high 800 high 400 tow 800 low 400 high 000 high treatment group treatment group M day 8 ESS day M CZD day 24 I day a day 18 I__ I day 24 Cooper ia Ostertagia Figure 12. Figure 13. Percent reduction of total infective larvae, Cooperia and Qstertaqifl larvae in Arthrobotrvs flagrans-treated bovine feces cultured for 8, 16 and 24 days. 400 low group had a low fecal egg count and received an inoculum of 400 spores per gram of feces. 800 low group had a low fecal egg count and received an inoculum of 800 spores per gram of feces. 400 high group had a high fecal egg count and received an inoculum of 400 spores per gram of feces. 800 high group had a high fecal egg count and received an inoculum of 800 spores per gram of feces. 228 229 % reduction 100 400 low 8 0 0 low 400 high 800 high ■ i day 8 E H day 18 CZD day 24 Total infective larvae % reduction % reduction 100 I ) low aoo low 400 low aoo low 400 low 100 low 400 high aoo 1 treatmant group treatment group I day a M O day 24 I day 0 day 10 I— I day 24 Cooper ia Ostertagia Figure 13. Figure 14. Percent reduction of total infective larvae in fungal-treated equine feces cultured for 8, 16 and 24 days. 400 low group had a low fecal egg count and received an inoculum of 400 spores per gram of feces. 800 low group had a low fecal egg count and received an inoculum of 800 spores per gram of feces. 400 high group had a high fecal egg count and received an inoculum of 400 spores per gram of feces. 800 high group had a high fecal egg count and received an inoculum of 800 spores per gram of feces. 230 Arthrobotrys oligospora A. flagrans 100 100 80 60 40 20 400 low 800 low 400 high 800 high B H day 8 H H day 16 d i day 24 UNJ F i g u r e 1 4 . Figure 15. Percent reduction of total infective larvae in fungal-treated ovine feces cultured for 8, 16 and 24 days. 400 low group had a low fecal egg count and received an inoculum of 400 spores per gram of feces. 800 low group had a low fecal egg count and received an inoculum of 800 spores per gram of feces. 400 high group had a high fecal egg count and received an inoculum of 400 spores per gram of feces. 800 high group had a high fecal egg count and received an inoculum of 800 spores per gram of feces. 232 Arthrobotrys oligospora A. flagrans U)PvJ U) Figure 16. Percent reduction of total infective larvae in fungal-treated bovine feces cultured for 8, 16 and 24 days. 400 low group had a low fecal egg count and received an inoculum of 400 spores per gram of feces. 800 low group had a low fecal egg count and received an inoculum of 800 spores per gram of feces. 400 high group had a high fecal egg count and received an inoculum of 400 spores per gram of feces. 800 high group had a high fecal egg count and received an inoculum of 800 spores per gram of feces. 234 Arthrobotrys oligospora A. flagrans u>to Figure 17. Visser filter apparatus. 100 micron diameter pore filter nests inside a 25 micron diameter pore filter. Strongyle eggs collect within the larger outer filter which is harvested through the stopcock valve. 236 237 Figure 17. Figure 18. Percent reduction of total infective larvae harvested from herbage surrounding fungus- treated feces at 4 and 8 weeks after placement of treated and control dung pats from horses, sheep, and cattle. 238 % reduction % reduction % reduction 1 00------ A. oUgoapora A. flagmn* A. ollgoapora A. flagrant A. flagrant I w i ll 4 ^ w N k 8 Horse Sheep Cattle F i g u r e 1 8 . Appendix C Plates 240 Plate I. Chlamydospores intercalated along the length of hyphae of Arthrobotrys flaarans grown in culture. Scale equals 40 microns 241 242 Plate I. Plate II. Cyathostome infective larva with numerous Drechmeria coniospora conidia adhered to the cuticle. 243 244 Plate II. O f ' * * " Plate III. Several Arthrobotrvs oliaospora spores with curving cells forming rings (arrow). 245 Plate III. v v A ; \ to Plate IV. Sheep fitted with size 44 jockey briefs for fecal collection during a 1-2 h period. 247 Appendix D. Procedures for making nutrient media used in culturing. 249 250 Nigon's agar (NA) 1. 0.75g MgS04, 0.75g K2HP04, 2.75g NaCl, 3.0g KN03, 2. 5g peptone, l.Og lethicin, 15.Og Bacto-agar (Difco Laboratories, Detroit, MI, USA) was added to 1 1 distilled water. 2. The water was heated while stirring until the solutes were dissolved. 3. The solution was autoclaved under 30 lbs of pressure at 120° C for 25 minutes. 4. After cooling to approximately 40° C, the solution was poured into sterile Petri dishes in a laminar flow clean air hood and allowed to cool. 5. The NA dishes were stored at 4° C until used for cultures. Potato dextrose aaar (PDA) 1. 18.5 g of potato dextrose agar (Difco Laboratories, Detroit, MI, USA) was added to 500 ml distilled water. The water was heated while stirring until the powder was dissolved. 2. The solution was autoclaved under 30 lbs of pressure at 120° C for 25 minutes. 3. After cooling to approximately 40° C, the solution was poured into sterile Petri dishes in a laminar flow clean air hood and allowed to cool. 4. The PDA dishes were stored at 4° C until used for cultures. 251 Diluted potato dextrose aaar (dPDA) 1. 9.0 g of potato dextrose agar and 5.0 g Bacto-agar (Difco Laboratories, Detroit, MI, USA) was added to 500 ml distilled water. 2. The water was heated while stirring until the powder was dissolved. 3. The solution was autoclaved under 30 lbs of pressure at 120° C for 25 minutes. 4. After cooling to approximately 40° C, the solution was poured into sterile Petri dishes in a laminar flow clean air hood and allowed to cool. 5. The dPDA dishes were stored at 4° C until used for cultures. Water aaar (WA) 1. 5 g of dehydrated Bacto-agar (Difco Laboratories, Detroit, MI, USA) was added to 500 cc distilled water. 2. The water was heated while stirring until the powder was dissolved. Then the solution was autoclaved under 30 lbs of pressure at 120° C for 25 minutes. 3. After cooling to approximately 40° C, the solution was poured into sterile Petri dishes in a laminar flow clean air hood and allowed to cool. 4. The WA dishes were stored at 4° C until used for cultures. Water aaar + antibiotics (TCC-WA) 1. Water agar solution was prepared as described above. 2. After cooling to approximately 40° C, 30 mg of chlortetracycline was added. 252 3. The solution was swirled to mix thoroughly, poured into sterile Petri dishes in a laminar flow clean air hood and allowed to cool. 4. The TCC-WA dishes were stored at 4° C until used for cultures. Fecal aaar (FA) 1. 50 mg of sheep feces were filtered through 75 um sieve with 500 ml of distilled water. 2. 5 g of Bacto-agar (Difco Laboratories, Detroit, MI, USA) was added and the filtrate was autoclaved under 30 lbs pressure at 120° C for 25 minutes. 3. After cooling the media, it was poured into sterile Petri dishes in a laminar flow clean air hood and allowed to gel. 4. The FA dishes were stored at 4° C until used for cultures. 4 Appendix E. Method of estimating the number of spores per ml in suspension. 253 254 Gridded hemacytometer chamber Volume of each gridded chamber is 0.9 microliters 1. Spore suspension was mixed thoroughly. 2. Both hemacytometer chambers were charged with the spore suspension. 3. The number of spores present in the grid of a chamber was counted. A second count was done of the second grid. 4. Steps 1-3 were repeated until 5 counts had been completed. 5. Mean number of spores were calculated. 6. The estimated number of spores per ml of suspension = mean number of spores/chamber X 1000 microliters/ml • 0.9 microliters/chamber Appendix F. Calculation of estimated numbers of fungal propagules in feces from cultures of serial dilutions of feces. 255 256 CALCULATION OF ESTIMATED NUMBERS OF FUNGAL PROPAGULES IN FECES. 1. 9 ml of sterile distilled water was added to 1 g of feces to make the 1CT1 dilution. 2. The suspension was mixed well and 1 ml added to 9 ml of sterile distilled water to make the 10"2 dilution and so on to prepare athe desired series of dilutions. 3. l ml of each dilution suspension was placed on 4 nutrient agar plates and cultured as described in the text. 4. Number of positive plates observed for each dilution were recorded. 5. Number of negative plates were recorded. 6. Cumulative positive plates were summed from the bottom up and the cumulative negative plates were summed from the top down. 7. The ratio of the number of cumulative positive plates to the total number of plates (i.e., number of positive plates + number of negative plates) was recorded. This ratio was then converted into a percentage. Example: # pos #neg cumul cumul Dilution plates olates pos nea total % EPS 10° 4 0 12 0 12/12 100 10'1 4 0 8 0 8/8 100 10"2 3 1 4 1 4/5 80 1(T3 1 3 1 4 1/5 20 10"4 0 4 0 8 0/8 0 257 The 50 % endpoint was between 80% and 20%. To interpolate between these two limits, proportionate distance (PD) was calculated. PD = % above 50 - 50 % above 50 - % below 50 = 80 - 50 80 - 20 = 30 60 = 0.5 50% endpoint = 10*"b = dilution that on average gives 1 propagule per aliquot suspension (i.e., 1 ml) * exponent of the upper limit dilution (i.e., -2). b interpolated exponent (i.e., 0.5) = 10’2-5 Number of propagules per ml of original sample is the reciprocal = 102-5 propagules/g feces Appendix G. Protocol for collection and recovery of infective larvae on pasture. 258 259 PROTOCOL FOR COLLECTION AND RECOVERY OF INFECTIVE LARVAE ON PASTURE. I. Grass collection A. Collector walked in zigzag fashion, making 2 N's (see diagram). B. 100 stops were made to pluck grass. Blades were collected, not roots or soil. C. 4 collections per stop were made: 1. At feet 2. At left hand side 3. At right hand side 4. At arm's reach in front. D. Pasture sample was well mixed; 300-400 g was weighed and retained. The rest of the pasture sample was discarded. II. Washing grass sample A. Grass sample was placed in 10 1 bucket with 1-1.5 ml non-ionic detergent (Acationox). Rinsings from sample bag were added to the bucket. Grass sample was gently agitated and allowed to soak overnight or at least 6 h in 6 1 of water. B. Grass sample was carefully removed without disturbing the sediment and rinsed in two separate 5 1 buckets containing 2 1 of lukewarm water. C. Grass sample was wrung out, placed in pan to dry in oven at 195° C. 260 D. Water from the two rinsing buckets was run through a coarse mesh sieve to remove remaining blades of grass. Grass in sieve was rinsed with a spray of water from a squeeze bottle and combined with the rest of grass to dry. E. Washings were added to main sample in 10 1 bucket and allowed to sediment for 3 h. G. Washings were siphoned to 2 1, discarding the 8 1 of supernatant. H. Remaining 2 1 of sediment was poured into two 1 1 graduated cylinders. The bucket was rinsed well and the rinsings added to the cylinders. The columns of suspension were allowed to sediment 3 h. I. The supernatant was siphoned off, leaving 250 ml. The sediment and rinsings were poured into a 500 ml graduated pharmaceutical flask and allowed to sediment for at least 3 h. J. The supernatant was drawn off with Venturi pump leaving a small volume of water and the sediment. III. Recovery of larvae using Baermann apparatus A. A Baermann apparatus was set up with a conical pyrex tube attacked to a siliconized 200 mm filtering funnel with latex tubing. A 1/8" galvanized wire mesh and piece of cheesecloth, both 19 cm in diameter were placed on top of the funnel. The funnel was filled with warm water (50-55° C. B. The sediment and rinsings were poured onto a Whatman No. 1 18.5 cm coarse filter paper placed on absorbent paper toweling. The first aliquot of sediment was placed in the center of the filter paper and the following aliquots were poured around it. C. After most of the water had been absorbed by the underlying paper towels (the shiny look of the sediment had disappeared), the filter paper was gently inverted onto the cheesecloth supported by the wire mesh. 261 D. More water was added to funnel to prevent the paper and sediment from drying. Baermann was allowed to stand for 36 h. E. The conical tube was pulled from the Baermann apparatus and the supernatant was drawn off. F. Sediment was examined for infective larvae. LIST OF REFERENCES Anderson, N., J. Armour, W.F.H. Jarret, F.W. Jennings, J.S.D. Ritchie and G.M. Urquhart. 1967. Experimental infections of Ostertaaia ostertaai in calves; results of two regimens of multiple inoculations. 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