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The potential of nematophagus fungi to control the free-living stages of nematode parasites of sheep: in vitro and in vivo studies
Veterinary Parasitology, 51 (1994) 289-299 0304-4017/94/$07.00 © 1994 - Elsevier Science B.V. All fights reserved
289
The potential of nematophagous fungi to control the free-living stages of nematode parasites of sheep: in vitro and in vivo studies P.J. Waller*, M. Larsen, M. Faedo, D.R. Hennessy CSIRO Division of Animal Health, McMaster Laboratory, Private Bag No. 1, Glebe, N.S.W. 2037, Australia (Accepted 16 March 1993)
Abstract
Following in vitro screening investigations on approximately 100 nematophagous fungi reported previously, eight species were selected for further investigation. Fungal elements (mycelium and conidia) were subjected to in vitro stress selection designed to simulate rumen and abomasal conditions. From these studies, three species, namely, Arthrobotrys oligospora, Arthrobotrys oviformis and Geniculifera eudermata, were selected for in vivo survival studies in sheep surgically fitted with abomasal and ileal cannulae. Doses of fungal conidia were administered orally or via the abomasal cannulae and samples of digesta were taken from the abomasum, the terminal ileum and faeces. The viability of the three fungal species at these sites was demonstrated. The abundance of fungi throughout the gut was dose-dependent but in all cases only very small volumes of fungal suspension containingunprotected conidia were used. These results demonstrate that a practical means of orally administering nematophagous fungi to control free-living stages of nematodes in faeces may become an achievable objective. Key words: Arthrobotrys spp.; Geniculifera eudermata; Fungi; Sheep-Nematoda; Control methodsNematoda
Introduction
An essential requirement for any nematophagous fungi to be exploited as biological control agents of nematode parasites of ruminant livestock, is the ability to colonise and to assume the nematode capturing mode in faeces. A number of studies have identified fungi with these attributes, but they have been restricted to only a few fungal species (for review see Waller and Larsen, 1992). Accordingly, a screening investigation involving approximately 100 fungi with known nematode destroying properties was undertaken, with the objective of identifying those species previously overlooked (Waller and Faedo, 1992 ). Experiments were carried out with sheep faecal cultures and several species of the genera Arthrobotrys, Geniculifera and Monacrosporium were shown to have the greatest activity. Reductions of infective larval num*Corresponding author. SSDI 0 3 0 4 - 4 0 1 7 ( 9 3 ) 005 05-S
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bers exceeding 80% compared with controls were consistently recorded when less than 250 conidia g-1 faeces were tested in laboratory culturing experiments. The next more demanding requirement is that fungal elements must survive gut passage in ruminants, so that oral administration can be pursued as a practical means of deploying these organisms as biological control agents of nematode parasites. The purpose of this investigation, therefore, was first to determine optimal culture media for the production of resting spores, or conidia. Secondly, these stages were tested, both in vitro and in vivo, for their capacity to survive passage through the gastrointestinal tract of sheep. Materials and methods
Fungi Following earlier screening investigations (Waller and Faedo, 1992), eight species of nematophagous fungi with superior larval trapping ability in faecal cultures were selected for further investigation. These consisted of four species of Arthrobotrys (A. javanica, A. oligospora, A. oviformis, A. polycephala ) , two species of Geniculifera (G. bogoriensis, G. eudermata) and two species of
Monacrosporum ( M. rutgeriense, M. thaumasium ). Media Fungal isolates were subcultured on the following media in 9 cm diameter petri dishes and were cultured for 7 days at 25 ° C, when estimates of growth and sporulation were made: ATCC 196, yeast malt extract agar (Difco 0770); MA, 2% malt agar; Blood, sheep blood agar (7% defibrinated sheep blood in nutrient agar); CMA, 0.6% corn meal agar; Water, 2% water agar; Faecal, sheep faecal agar (50 g macerated faeces, filtered through a 75/tm sieve, filtrate adjusted to 500 ml, added to 2% agar and then autoclaved; Faecal and worms, sheep faecal agar, to which a concentrated suspension of infective Haemonchus contortus larvae were added prior to autoclaving.
In vitro assays In vitro stress selection experiments, performed according to the procedures described by Larsen et al. ( 1991 ) were used for both mycelial and conidial fractions of each isolate. To obtain cultures which had profuse mycelial growth, but with very sparse (or no) production of conidia, 7-day-old CMA
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cultures were used for each fungal isolate. For tests on conidia, older cultures (2-4 weeks old) grown on faecal agar were used. For both studies, fungal elements were harvested from cultures by adding 1 ml of dilute ( 1 : 4) rumen fluid to each petri dish and gently scraping the surface of the fungal colony with a microscope slide. Dislodged material was harvested with a pasteur pipette and transferred to two separate tubes each containing 5 ml of sheep ruminal fluid for each isolate. After 24 h incubation at 39°C in a shaking water bath, one tube from each isolate was used to set up three replicates of 0.5 ml of fungal suspension spread on tetracycline chloride-water agar (TCCWA) plates. The other tube was centrifuged (2500 rev min -1 for 10 min), the supernatant discarded, 5 ml of pepsin-HC1 solution added, thoroughly mixed and incubated for a further 4 h to simulate conditions in the abomasum. Following this procedure, three additional TCC-WA plates were set up by the above method. To each of these plates approximately 5000 third-stage H. contortus were added and incubated for 7 days at 25°C. The plates were then observed for larval entrapment and fungal growth, characteristic of each species. A second study was carried out to compare the survival of second generation conidia with conidia from the parent strains, using the procedures described above. In vivo passage experiments
Sheep, surgically modified by the procedures described by Harmon and Seid (1982) were used in these studies. The sheep had abomasal and ileal cannulae inserted and were kept in metabolism cages where they were fed 600 g lucerne/wheaten chaff per day for the duration of the experiments. Conidia harvested from fungal isolates grown on faecal agar were quantified using a haemocytometer for both of the following experiments. Oral administration experiments The total number of conidia administered for each fungal species was divided into four equal doses suspended in 1 ml of water and administered hourly in a gelatin capsule via stomach tube to individual sheep. Samples were taken from the abomasal cannulae at hourly intervals, commencing at the time of administration of the first capsule and continued for 12 h. Two final samples were made after a further 12 h and 24 h had elapsed. Three replicate 5 ml abomasal subsamples were mixed thoroughly with vermiculite to achieve a well aerated, moist culture and separately placed in sealed plastic petri dishes (9 cm diameter). These were incubated for 14 days at 25°C. Approximately 15 000 infective H. contortus larvae were added to each culture at the time of establishment and again after 7 days incubation, as a source of prey for any fungal elements that survived gut passage. After incubation
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for 2 weeks, each culture was visually inspected for the presence of nematophagous fungal activity. The total number of larvae was then recovered by placing the cultures on small wire mesh screens and immersing them in a chamber filled with warm water for 12 h. Faeces were also collected from each sheep commencing 12 h after the first fungal dose and subsequently at 24, 31, 48, 57 and 72 h. Three replicate 5 g faecal samples were also set up for each of these bulk collections in sealed petri dishes. Incubation procedures, addition of larvae, inspection, and finally larval recovery, were carded out as described above.
Abomasal administration experiments Similar to the oral dosing experiment, the total conidial dose for each fungal species to be tested was divided into four equal doses. These were administered every 30 rain into the abomasal cannulae using a hypodermic syringe with a fine rubber tube attachment. Samples were taken from the ileal cannulae at approximately 2 h intervals for the first 8 h, then hourly for the following 6 h and finally the two last samples were taken 2 h apart. Faecal samples were collected at the time of the first fungal administration and then 9, 16, 19, 26, 29, 32 and 35 h later. Triplicate cultures for both ileal and faecal samples were set up, examined and tested with larvae according to the procedures outlined for the oral dosing experiments. Results Growth and sporulation of all fungal isolates varied with the test media (see Table 1 ). Not unexpectedly, the various nutrient media provided a better substrate for mycelial growth than water agar, but the best medium for inducing conidia production was sheep faecal agar. There was no added benefit from including nematode larvae in this medium with regard to further stimulating growth or spore formation. Therefore, for all subsequent experiments, sheep faecal agar was the medium used to produce fungal elements. Variations in growth and particularly conidia production were observed between species. The four Arthrobotrys species were good growers and sporulators, with G. eudermata showing good growth but rather sparse conidial production. The two species of Monacrosporium grew well but produced very few conidia whilst G. bogoriensis showed poor growth with almost no conidia production. Incubation for up to 4 weeks, in some instances, resulted in colonies producing more conidia than after 7 days. In vitro stress selection experiments showed only very limited survival of mycelium, in contrast to the good survival of conidia from most fungal species, particularly after the first phase of incubation in rumen fluid at 39 °C for 24 h (see Table 2 ). The additional 4 h incubation in pepsin-HC1 solution had a much more profound effect on viability. Conidia derived from cultures that
++++/+1 ++++/+ ++++/+ ++++/+ +++/+ +++/+ ++++/++++/-
ATCC196
Media
+++/++ +++/++ +++/++ +++/++ ++/+ ++/++ +++/+++/-
Malt +++/_ +++/++++/++ +++/+++/++++/++ +++/+++/-
Blood ++++/+ ++++/++ ++++/++ ++++/+ ++/+++/+++/++++/-
CMA ++/++/++/++/+++/++/++/++/-
Water ++++/+++ ++++/+++ ++++/+++ ++++/+++ ++/++++/+++ ++++/++++/+
Faecal
++++/+++ ++++/+++ ++++/+++ ++++/+++ ++/++++/+++ ++++/++++/+
Faecal/worms
~Mycelial growth/sporulation. Mycelial growth: + +, approximately 50% plate; + + +, approximately 75% plate; + + + +, 100% plate. Sporulation: - , no conidia; +, very few, patchy distribution; + +, sparse, even distribution; + + +, dense, even distribution.
A.javanica A. oligospora A. oviformis A. polycephala G. bogoriensis G. eudermata M. rutgeriense M. thaumasium
Species
Table 1 Growth and sporulation of nematophagous fungi after 7 days incubation at 25 °C on various culture media
bJ ~D
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Table 2 Survival capability of nematophagous fungi following in vitro exposure to simulate the gut environment of ruminants
Species
Rumen fluid Mycelium
A. j a v a n i c a A. oligospora A. oligospora selected A. o v i f o r m i s A. o v i f o r m i s selected A. p o l y c e p h a l a G. b o g o r i e n s i s G. e u d e r m a t a G. e u d e r m a t a selected M. rutgeriense M. thaumasium M . t h a u m a s i u m selected
Rumen fluid + pepsin/HC1 Conidia
+ + -
Mycelium
Expt. 1
Expt. 2
+ +
+ +
Conidia Expt. 1
Expt. 2
-
+ +
-
+ +
+ +
+
-
-
-
+ + +
+ + + + +
+ +
. +
+
+ + +
-
+ + + .
. + +
+
. +
+ + + . -
.
. + +
. + + +
+
+ -
+ + +
+ +
+ + +
+ + +
Proportion of plates in which fungal growth recorded: - , no growth; + , one-third; + + , two-thirds; + + + , three-thirds. Table 3 Total number of conidia administered to sheep in the in vivo survival studies
Species
A. oligospora A. o v i f o r m i s G. e u d e r m a t a
Route of administration OraP
Abomasal 2
1.2 X 10 6
1.2 × 10 6
2.4× 106 2.4× 10 5
1.1 X 10 7 2.5 × l0 s
1Oral dose divided into four equal doses, 1 h apart. 2Abomasal dose divided into four equal doses, 30 rain apart.
developed from conidia surviving the first in vitro experiment (i.e. the second generation) were tested in a further study with conidia derived from the parent cultures. Conidia from the selected lines showed no greater propensity to survive the simulated rumen or abomasal conditions than those from the parent cultures. The fungal species that showed the greatest ability to survive these conditions were A. oligospora, A. oviformis, G. eudermata and M. thaumasium. All these species, except M. thaumasium, which was a very poor producer of conidia, were used for the subsequent in vivo investigations. The total numbers of conidia administered to separate sheep for both the oral and abomasal administration experiments are shown in Table 3. The results of the oral dosing experiment are summarised in Fig. 1.
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Fig. 1. Presence of fungal growth and number ofH. contortus infective larvae in 14 day cultures of abomasal samples following oral administration of fungal conidia: ( A ) A. oligospora; ( B ) A. oviformis; (C) G. euderrnata. N u m b e r of asterisks represents number of cultures with fungal growth; * *, larval numbers.
Fungal growth of all three species was observed for abomasal samples taken from sheep during the first 12 h after oral dosing. The three replicate culture plates were all positive for both A. oligospora and A. oviformis 1 h after dosing. One out of three plates showed A. oligospora growth in each of the hourly samples for the following 3 h. All plates were again positive 5 h after administration. Fungal growth was also observed in two-thirds and one-third of the plates, 7 h and 8 h after dosing, respectively. Apart from the first hour after dosing, the period when A. oviformis was most frequently recorded was 4-8 h
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pJ. Walleret al. I VeterinaryParasitology51 (1994)289-299
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Fig. 2. Presence of fungal growth and number ofH. contortus infective larvae in 14 day cultures of ileal and faecal samples following abomasal administration of fungal conidia: ( A ) A. oligospora; (B) A. oviformis; (C) G. eudermata. Ileal samples: number of asterisks represents number of cultures with fungal growth; *--*, larval numbers. Faecal samples: number of stars represents number of cultures with fungal growth; ~ *, larval numbers.
after dosing. Geniculifera eudermata tended to appear later and in greater abundance than the two Arthrobotrys species. The numbers of infective larvae recovered from cultures were, by and large, inversely related to the abundance of fungal growth. Fungal growth in faecal samples was only recorded on one occasion for one species, namely A. oligospora, at 24 h after oral dosing when two of the three cultures were positive.
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The results of the abomasal administration experiment are summarised in Fig. 2. In samples collected from the ileum, fungal growth was observed on only one occasion, 4 h after administration, for both A. oligospora and G. eudermata. In contrast, fungal growth of A. oviformis was observed in cultures at all sampling times, from 2 to 16 h after administration. In general, larval numbers reflected the patterns of fungal presence, showing greatest reductions at the times of highest fungal growth in cultures. Growth of both A. oligospora and G. eudermata was only recorded on one faecal sampling occasion, 9 h after abomasal administration, whereas A. oviformis was recorded on all faecal sampling occasions, except for the 16 h samples, commencing 9 h after administration. A substantial reduction in larval numbers occurred in the 9 h cultures when all samples exhibited profuse fun-
gal growth. Discussion
The in vitro procedures described by Larsen et al. ( 1991 ), which are designed to mimic environmental stresses of rumen and abomasal passage, proved to be a valuable screening procedure for the in vivo investigation. Fungi that survived in vitro stress selection also survived in vivo. However, the times imposed by the in vitro procedures clearly indicate that, at least for sheep, the duration of stress selection is too long. This may eliminate fungal species which may at least be able to survive through the anterior regions of the gastrointestinal tract. The protocols recommend a 24 h rumen fluid and 4 h pepsin-HC1 solution incubation period to simulate the environmental conditions and transit times through the rumen and abomasum, respectively. The in vivo work reported here using surgically modified sheep clearly shows that residence times in the rumen and abomasum are much shorter. In fact, conidia of all three species were recovered from samples collected from cannulae located in the pyloric region of the abomasum within 1 h after oral administration. The greatest flow concentration past this site appeared to be from 4 to 12 h after administration, which is substantially less than the 28 h of stress imposed in the in vitro protocols. This indicates that the very small size of conidia ofA. oligospora, A. oviformis and G. eudermata are present mainly in the fluid phase, rather than associated with particulate digesta. Consequently, the conidia experienced a much more rapid gut passage than was expected. Passage through the small and large intestine was also relatively rapid. Conidia which were administered immediately anterior of the duodenum could be detected in the ileum even at the time of the first collection 2 h later, and peak flow occurred 4-10 h after administration. Conidia were also present in faecal samples when collected 9 h after abomasal administration but, as expected, there was a protracted tail-off, with conidia being detected in faecal samples
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over the following 24 h. Taken together, the results of both in vivo experiments indicate that the total mean gut transit time of orally administered conidia in sheep is of the order of 24 h. Successful passage of conidia through the various gut compartments was determined directly by inspecting cultures for fungal growth, and indirectly by estimating the reduction of known numbers of infective larvae added to cultures. Neither procedure is entirely satisfactory owing to the difficulties associated with accurate visual assessment and because larval numbers often become concentrated in small droplets of water which escape fungal traps. Despite these limitations, there was relatively good correlation between both estimates; however, both procedures would underestimate the presence and predacity of fungi. Although A. oligospora was the only species found in faeces following oral dosing, evidence suggests that both A. oviformis and G. eudermata are also capable of entire gut passage. The presence of all three species was recorded at each different sampling site. Simply, detection of surviving conidia was related to the number of conidia in the inoculating dose. This was particularly evident in the abomasal dosing experiment where the total A. oviformis dose was approximately 10-fold and 100-fold greater than A. oligospora and G. eudermata, respectively. Consequently, A. oviformis was found in the majority of faecal samples collected during the course of the experiment. Although previous experiments have also demonstrated successful ruminant gut passage of predacious fungi (Gruner et al., 1985; Larsen et al., 1992), the methodology used by these workers did not sufficiently define the survival of discrete fungal elements. Rather, both of these trials involved feeding animals with fungal material which had been grown on grains. Not only were quite prodigious amounts of fungal material administered, e.g. infected grains amounting to 1.7% of lamb liveweight (Gruner et al., 1985 ), but fungal survival was estimated by recovering intact grains from faeces within which fungi could well be protected against the gut environment (Larsen et al., 1992). In our investigation, unprotected conidia were administered in very small volumes of water and the results indicate that a proportion of these minute, thinwalled structures are capable of survival through the various gut compartments. This represents a significant advance towards the ultimate objective of developing a practical means of biological control of nematode parasites. Neither massive amounts of fungal material, nor some means of protection from the gut environment appear to be necessary. Further investigations on dose-response and sustained delivery to determine threshold dose levels of conidia in prototype delivery systems of these and other fungal species are being undertaken.
Acknowledgements The authors wish to thank Natalie Miller and Tom Parker for their expert technical assistance.
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References Gruner, L., Peloille, M., Sauv6, C. and Cortet, J., 1985. Parasitologie animale. Survie et conservation de ractivit6 pr6datrice vis-A-vis de n6matodes trichostrongylides apr6s ingestion par des ovins de trois hyphomyc6tes pr6dateurs. C. R. Acad. Sci., III, 300: 525-528. Harmon, J.W. and Seid, W., 1982. Surgical approaches to studying gastrointestinal physiology. In: D.A. Titchen (Editor), Techniques in the Life Sciences, P2 Digestive Physiology, p. 204, Elsevier, Shannon Ireland, pp. 1-20. Larsen, M., Wolstrup, J., Henriksen, S.A., Dackman, C., Gronvold, J. and Nansen, P., 1991. In vitro stress selection of nematophagous fungi for biocontrol of parasitic nematodes in ruminants. J. Helminthol., 65:193-200. Larsen, M., Wolstrup, J., Henriksen, S.A., Gronvold, J. and Nansen, P., 1992. In vivo passage through calves of nematophagous fungi selected for biocontrol of parasitic nematodes. J. Helminthol., 66: 137-141. Waller, P.J. and Faedo, M., 1992. The potential of nematophagous fungi to control the freeliving stages of nematode parasites of sheep: screening studies. Vet. Parasitol., 49: 285-297. Waller, P.J. and Larsen, M., 1992. Biological control of nematode parasites of livestock. Int. J. Parasitol., 23: 539-546.
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The potential of nematophagous fungi to control the free-living stages of nematode parasites of sheep: in vitro and in vivo studies P.J. Waller*, M. Larsen, M. Faedo, D.R. Hennessy CSIRO Division of Animal Health, McMaster Laboratory, Private Bag No. 1, Glebe, N.S.W. 2037, Australia (Accepted 16 March 1993)
Abstract
Following in vitro screening investigations on approximately 100 nematophagous fungi reported previously, eight species were selected for further investigation. Fungal elements (mycelium and conidia) were subjected to in vitro stress selection designed to simulate rumen and abomasal conditions. From these studies, three species, namely, Arthrobotrys oligospora, Arthrobotrys oviformis and Geniculifera eudermata, were selected for in vivo survival studies in sheep surgically fitted with abomasal and ileal cannulae. Doses of fungal conidia were administered orally or via the abomasal cannulae and samples of digesta were taken from the abomasum, the terminal ileum and faeces. The viability of the three fungal species at these sites was demonstrated. The abundance of fungi throughout the gut was dose-dependent but in all cases only very small volumes of fungal suspension containingunprotected conidia were used. These results demonstrate that a practical means of orally administering nematophagous fungi to control free-living stages of nematodes in faeces may become an achievable objective. Key words: Arthrobotrys spp.; Geniculifera eudermata; Fungi; Sheep-Nematoda; Control methodsNematoda
Introduction
An essential requirement for any nematophagous fungi to be exploited as biological control agents of nematode parasites of ruminant livestock, is the ability to colonise and to assume the nematode capturing mode in faeces. A number of studies have identified fungi with these attributes, but they have been restricted to only a few fungal species (for review see Waller and Larsen, 1992). Accordingly, a screening investigation involving approximately 100 fungi with known nematode destroying properties was undertaken, with the objective of identifying those species previously overlooked (Waller and Faedo, 1992 ). Experiments were carried out with sheep faecal cultures and several species of the genera Arthrobotrys, Geniculifera and Monacrosporium were shown to have the greatest activity. Reductions of infective larval num*Corresponding author. SSDI 0 3 0 4 - 4 0 1 7 ( 9 3 ) 005 05-S
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bers exceeding 80% compared with controls were consistently recorded when less than 250 conidia g-1 faeces were tested in laboratory culturing experiments. The next more demanding requirement is that fungal elements must survive gut passage in ruminants, so that oral administration can be pursued as a practical means of deploying these organisms as biological control agents of nematode parasites. The purpose of this investigation, therefore, was first to determine optimal culture media for the production of resting spores, or conidia. Secondly, these stages were tested, both in vitro and in vivo, for their capacity to survive passage through the gastrointestinal tract of sheep. Materials and methods
Fungi Following earlier screening investigations (Waller and Faedo, 1992), eight species of nematophagous fungi with superior larval trapping ability in faecal cultures were selected for further investigation. These consisted of four species of Arthrobotrys (A. javanica, A. oligospora, A. oviformis, A. polycephala ) , two species of Geniculifera (G. bogoriensis, G. eudermata) and two species of
Monacrosporum ( M. rutgeriense, M. thaumasium ). Media Fungal isolates were subcultured on the following media in 9 cm diameter petri dishes and were cultured for 7 days at 25 ° C, when estimates of growth and sporulation were made: ATCC 196, yeast malt extract agar (Difco 0770); MA, 2% malt agar; Blood, sheep blood agar (7% defibrinated sheep blood in nutrient agar); CMA, 0.6% corn meal agar; Water, 2% water agar; Faecal, sheep faecal agar (50 g macerated faeces, filtered through a 75/tm sieve, filtrate adjusted to 500 ml, added to 2% agar and then autoclaved; Faecal and worms, sheep faecal agar, to which a concentrated suspension of infective Haemonchus contortus larvae were added prior to autoclaving.
In vitro assays In vitro stress selection experiments, performed according to the procedures described by Larsen et al. ( 1991 ) were used for both mycelial and conidial fractions of each isolate. To obtain cultures which had profuse mycelial growth, but with very sparse (or no) production of conidia, 7-day-old CMA
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cultures were used for each fungal isolate. For tests on conidia, older cultures (2-4 weeks old) grown on faecal agar were used. For both studies, fungal elements were harvested from cultures by adding 1 ml of dilute ( 1 : 4) rumen fluid to each petri dish and gently scraping the surface of the fungal colony with a microscope slide. Dislodged material was harvested with a pasteur pipette and transferred to two separate tubes each containing 5 ml of sheep ruminal fluid for each isolate. After 24 h incubation at 39°C in a shaking water bath, one tube from each isolate was used to set up three replicates of 0.5 ml of fungal suspension spread on tetracycline chloride-water agar (TCCWA) plates. The other tube was centrifuged (2500 rev min -1 for 10 min), the supernatant discarded, 5 ml of pepsin-HC1 solution added, thoroughly mixed and incubated for a further 4 h to simulate conditions in the abomasum. Following this procedure, three additional TCC-WA plates were set up by the above method. To each of these plates approximately 5000 third-stage H. contortus were added and incubated for 7 days at 25°C. The plates were then observed for larval entrapment and fungal growth, characteristic of each species. A second study was carried out to compare the survival of second generation conidia with conidia from the parent strains, using the procedures described above. In vivo passage experiments
Sheep, surgically modified by the procedures described by Harmon and Seid (1982) were used in these studies. The sheep had abomasal and ileal cannulae inserted and were kept in metabolism cages where they were fed 600 g lucerne/wheaten chaff per day for the duration of the experiments. Conidia harvested from fungal isolates grown on faecal agar were quantified using a haemocytometer for both of the following experiments. Oral administration experiments The total number of conidia administered for each fungal species was divided into four equal doses suspended in 1 ml of water and administered hourly in a gelatin capsule via stomach tube to individual sheep. Samples were taken from the abomasal cannulae at hourly intervals, commencing at the time of administration of the first capsule and continued for 12 h. Two final samples were made after a further 12 h and 24 h had elapsed. Three replicate 5 ml abomasal subsamples were mixed thoroughly with vermiculite to achieve a well aerated, moist culture and separately placed in sealed plastic petri dishes (9 cm diameter). These were incubated for 14 days at 25°C. Approximately 15 000 infective H. contortus larvae were added to each culture at the time of establishment and again after 7 days incubation, as a source of prey for any fungal elements that survived gut passage. After incubation
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for 2 weeks, each culture was visually inspected for the presence of nematophagous fungal activity. The total number of larvae was then recovered by placing the cultures on small wire mesh screens and immersing them in a chamber filled with warm water for 12 h. Faeces were also collected from each sheep commencing 12 h after the first fungal dose and subsequently at 24, 31, 48, 57 and 72 h. Three replicate 5 g faecal samples were also set up for each of these bulk collections in sealed petri dishes. Incubation procedures, addition of larvae, inspection, and finally larval recovery, were carded out as described above.
Abomasal administration experiments Similar to the oral dosing experiment, the total conidial dose for each fungal species to be tested was divided into four equal doses. These were administered every 30 rain into the abomasal cannulae using a hypodermic syringe with a fine rubber tube attachment. Samples were taken from the ileal cannulae at approximately 2 h intervals for the first 8 h, then hourly for the following 6 h and finally the two last samples were taken 2 h apart. Faecal samples were collected at the time of the first fungal administration and then 9, 16, 19, 26, 29, 32 and 35 h later. Triplicate cultures for both ileal and faecal samples were set up, examined and tested with larvae according to the procedures outlined for the oral dosing experiments. Results Growth and sporulation of all fungal isolates varied with the test media (see Table 1 ). Not unexpectedly, the various nutrient media provided a better substrate for mycelial growth than water agar, but the best medium for inducing conidia production was sheep faecal agar. There was no added benefit from including nematode larvae in this medium with regard to further stimulating growth or spore formation. Therefore, for all subsequent experiments, sheep faecal agar was the medium used to produce fungal elements. Variations in growth and particularly conidia production were observed between species. The four Arthrobotrys species were good growers and sporulators, with G. eudermata showing good growth but rather sparse conidial production. The two species of Monacrosporium grew well but produced very few conidia whilst G. bogoriensis showed poor growth with almost no conidia production. Incubation for up to 4 weeks, in some instances, resulted in colonies producing more conidia than after 7 days. In vitro stress selection experiments showed only very limited survival of mycelium, in contrast to the good survival of conidia from most fungal species, particularly after the first phase of incubation in rumen fluid at 39 °C for 24 h (see Table 2 ). The additional 4 h incubation in pepsin-HC1 solution had a much more profound effect on viability. Conidia derived from cultures that
++++/+1 ++++/+ ++++/+ ++++/+ +++/+ +++/+ ++++/++++/-
ATCC196
Media
+++/++ +++/++ +++/++ +++/++ ++/+ ++/++ +++/+++/-
Malt +++/_ +++/++++/++ +++/+++/++++/++ +++/+++/-
Blood ++++/+ ++++/++ ++++/++ ++++/+ ++/+++/+++/++++/-
CMA ++/++/++/++/+++/++/++/++/-
Water ++++/+++ ++++/+++ ++++/+++ ++++/+++ ++/++++/+++ ++++/++++/+
Faecal
++++/+++ ++++/+++ ++++/+++ ++++/+++ ++/++++/+++ ++++/++++/+
Faecal/worms
~Mycelial growth/sporulation. Mycelial growth: + +, approximately 50% plate; + + +, approximately 75% plate; + + + +, 100% plate. Sporulation: - , no conidia; +, very few, patchy distribution; + +, sparse, even distribution; + + +, dense, even distribution.
A.javanica A. oligospora A. oviformis A. polycephala G. bogoriensis G. eudermata M. rutgeriense M. thaumasium
Species
Table 1 Growth and sporulation of nematophagous fungi after 7 days incubation at 25 °C on various culture media
bJ ~D
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Table 2 Survival capability of nematophagous fungi following in vitro exposure to simulate the gut environment of ruminants
Species
Rumen fluid Mycelium
A. j a v a n i c a A. oligospora A. oligospora selected A. o v i f o r m i s A. o v i f o r m i s selected A. p o l y c e p h a l a G. b o g o r i e n s i s G. e u d e r m a t a G. e u d e r m a t a selected M. rutgeriense M. thaumasium M . t h a u m a s i u m selected
Rumen fluid + pepsin/HC1 Conidia
+ + -
Mycelium
Expt. 1
Expt. 2
+ +
+ +
Conidia Expt. 1
Expt. 2
-
+ +
-
+ +
+ +
+
-
-
-
+ + +
+ + + + +
+ +
. +
+
+ + +
-
+ + + .
. + +
+
. +
+ + + . -
.
. + +
. + + +
+
+ -
+ + +
+ +
+ + +
+ + +
Proportion of plates in which fungal growth recorded: - , no growth; + , one-third; + + , two-thirds; + + + , three-thirds. Table 3 Total number of conidia administered to sheep in the in vivo survival studies
Species
A. oligospora A. o v i f o r m i s G. e u d e r m a t a
Route of administration OraP
Abomasal 2
1.2 X 10 6
1.2 × 10 6
2.4× 106 2.4× 10 5
1.1 X 10 7 2.5 × l0 s
1Oral dose divided into four equal doses, 1 h apart. 2Abomasal dose divided into four equal doses, 30 rain apart.
developed from conidia surviving the first in vitro experiment (i.e. the second generation) were tested in a further study with conidia derived from the parent cultures. Conidia from the selected lines showed no greater propensity to survive the simulated rumen or abomasal conditions than those from the parent cultures. The fungal species that showed the greatest ability to survive these conditions were A. oligospora, A. oviformis, G. eudermata and M. thaumasium. All these species, except M. thaumasium, which was a very poor producer of conidia, were used for the subsequent in vivo investigations. The total numbers of conidia administered to separate sheep for both the oral and abomasal administration experiments are shown in Table 3. The results of the oral dosing experiment are summarised in Fig. 1.
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~1o 5
0 0
1
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3
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6
7
8
9
10
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12
13
7
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11
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13
7
8
9
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12
13
Hours 20
~ 5
1
2
3
4
5
6 Hours
~
15
x o 103
~ 5
2
3
4
5
6 Hours
Fig. 1. Presence of fungal growth and number ofH. contortus infective larvae in 14 day cultures of abomasal samples following oral administration of fungal conidia: ( A ) A. oligospora; ( B ) A. oviformis; (C) G. euderrnata. N u m b e r of asterisks represents number of cultures with fungal growth; * *, larval numbers.
Fungal growth of all three species was observed for abomasal samples taken from sheep during the first 12 h after oral dosing. The three replicate culture plates were all positive for both A. oligospora and A. oviformis 1 h after dosing. One out of three plates showed A. oligospora growth in each of the hourly samples for the following 3 h. All plates were again positive 5 h after administration. Fungal growth was also observed in two-thirds and one-third of the plates, 7 h and 8 h after dosing, respectively. Apart from the first hour after dosing, the period when A. oviformis was most frequently recorded was 4-8 h
296
pJ. Walleret al. I VeterinaryParasitology51 (1994)289-299
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Fig. 2. Presence of fungal growth and number ofH. contortus infective larvae in 14 day cultures of ileal and faecal samples following abomasal administration of fungal conidia: ( A ) A. oligospora; (B) A. oviformis; (C) G. eudermata. Ileal samples: number of asterisks represents number of cultures with fungal growth; *--*, larval numbers. Faecal samples: number of stars represents number of cultures with fungal growth; ~ *, larval numbers.
after dosing. Geniculifera eudermata tended to appear later and in greater abundance than the two Arthrobotrys species. The numbers of infective larvae recovered from cultures were, by and large, inversely related to the abundance of fungal growth. Fungal growth in faecal samples was only recorded on one occasion for one species, namely A. oligospora, at 24 h after oral dosing when two of the three cultures were positive.
P.£ Waller et al. / Veterinary Parasitology 51 (1994) 289-299
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The results of the abomasal administration experiment are summarised in Fig. 2. In samples collected from the ileum, fungal growth was observed on only one occasion, 4 h after administration, for both A. oligospora and G. eudermata. In contrast, fungal growth of A. oviformis was observed in cultures at all sampling times, from 2 to 16 h after administration. In general, larval numbers reflected the patterns of fungal presence, showing greatest reductions at the times of highest fungal growth in cultures. Growth of both A. oligospora and G. eudermata was only recorded on one faecal sampling occasion, 9 h after abomasal administration, whereas A. oviformis was recorded on all faecal sampling occasions, except for the 16 h samples, commencing 9 h after administration. A substantial reduction in larval numbers occurred in the 9 h cultures when all samples exhibited profuse fun-
gal growth. Discussion
The in vitro procedures described by Larsen et al. ( 1991 ), which are designed to mimic environmental stresses of rumen and abomasal passage, proved to be a valuable screening procedure for the in vivo investigation. Fungi that survived in vitro stress selection also survived in vivo. However, the times imposed by the in vitro procedures clearly indicate that, at least for sheep, the duration of stress selection is too long. This may eliminate fungal species which may at least be able to survive through the anterior regions of the gastrointestinal tract. The protocols recommend a 24 h rumen fluid and 4 h pepsin-HC1 solution incubation period to simulate the environmental conditions and transit times through the rumen and abomasum, respectively. The in vivo work reported here using surgically modified sheep clearly shows that residence times in the rumen and abomasum are much shorter. In fact, conidia of all three species were recovered from samples collected from cannulae located in the pyloric region of the abomasum within 1 h after oral administration. The greatest flow concentration past this site appeared to be from 4 to 12 h after administration, which is substantially less than the 28 h of stress imposed in the in vitro protocols. This indicates that the very small size of conidia ofA. oligospora, A. oviformis and G. eudermata are present mainly in the fluid phase, rather than associated with particulate digesta. Consequently, the conidia experienced a much more rapid gut passage than was expected. Passage through the small and large intestine was also relatively rapid. Conidia which were administered immediately anterior of the duodenum could be detected in the ileum even at the time of the first collection 2 h later, and peak flow occurred 4-10 h after administration. Conidia were also present in faecal samples when collected 9 h after abomasal administration but, as expected, there was a protracted tail-off, with conidia being detected in faecal samples
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over the following 24 h. Taken together, the results of both in vivo experiments indicate that the total mean gut transit time of orally administered conidia in sheep is of the order of 24 h. Successful passage of conidia through the various gut compartments was determined directly by inspecting cultures for fungal growth, and indirectly by estimating the reduction of known numbers of infective larvae added to cultures. Neither procedure is entirely satisfactory owing to the difficulties associated with accurate visual assessment and because larval numbers often become concentrated in small droplets of water which escape fungal traps. Despite these limitations, there was relatively good correlation between both estimates; however, both procedures would underestimate the presence and predacity of fungi. Although A. oligospora was the only species found in faeces following oral dosing, evidence suggests that both A. oviformis and G. eudermata are also capable of entire gut passage. The presence of all three species was recorded at each different sampling site. Simply, detection of surviving conidia was related to the number of conidia in the inoculating dose. This was particularly evident in the abomasal dosing experiment where the total A. oviformis dose was approximately 10-fold and 100-fold greater than A. oligospora and G. eudermata, respectively. Consequently, A. oviformis was found in the majority of faecal samples collected during the course of the experiment. Although previous experiments have also demonstrated successful ruminant gut passage of predacious fungi (Gruner et al., 1985; Larsen et al., 1992), the methodology used by these workers did not sufficiently define the survival of discrete fungal elements. Rather, both of these trials involved feeding animals with fungal material which had been grown on grains. Not only were quite prodigious amounts of fungal material administered, e.g. infected grains amounting to 1.7% of lamb liveweight (Gruner et al., 1985 ), but fungal survival was estimated by recovering intact grains from faeces within which fungi could well be protected against the gut environment (Larsen et al., 1992). In our investigation, unprotected conidia were administered in very small volumes of water and the results indicate that a proportion of these minute, thinwalled structures are capable of survival through the various gut compartments. This represents a significant advance towards the ultimate objective of developing a practical means of biological control of nematode parasites. Neither massive amounts of fungal material, nor some means of protection from the gut environment appear to be necessary. Further investigations on dose-response and sustained delivery to determine threshold dose levels of conidia in prototype delivery systems of these and other fungal species are being undertaken.
Acknowledgements The authors wish to thank Natalie Miller and Tom Parker for their expert technical assistance.
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References Gruner, L., Peloille, M., Sauv6, C. and Cortet, J., 1985. Parasitologie animale. Survie et conservation de ractivit6 pr6datrice vis-A-vis de n6matodes trichostrongylides apr6s ingestion par des ovins de trois hyphomyc6tes pr6dateurs. C. R. Acad. Sci., III, 300: 525-528. Harmon, J.W. and Seid, W., 1982. Surgical approaches to studying gastrointestinal physiology. In: D.A. Titchen (Editor), Techniques in the Life Sciences, P2 Digestive Physiology, p. 204, Elsevier, Shannon Ireland, pp. 1-20. Larsen, M., Wolstrup, J., Henriksen, S.A., Dackman, C., Gronvold, J. and Nansen, P., 1991. In vitro stress selection of nematophagous fungi for biocontrol of parasitic nematodes in ruminants. J. Helminthol., 65:193-200. Larsen, M., Wolstrup, J., Henriksen, S.A., Gronvold, J. and Nansen, P., 1992. In vivo passage through calves of nematophagous fungi selected for biocontrol of parasitic nematodes. J. Helminthol., 66: 137-141. Waller, P.J. and Faedo, M., 1992. The potential of nematophagous fungi to control the freeliving stages of nematode parasites of sheep: screening studies. Vet. Parasitol., 49: 285-297. Waller, P.J. and Larsen, M., 1992. Biological control of nematode parasites of livestock. Int. J. Parasitol., 23: 539-546.