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The potential of nematophagous fungi to control the free-living stages of nematode parasites of sheep: feeding and block studies with Duddingtonia flagrans
Veterinary Parasitology 102 (2001) 321–330
The potential of nematophagous fungi to control the free-living stages of nematode parasites of sheep: feeding and block studies with Duddingtonia flagrans P.J. Waller a,∗ , M.R. Knox a , M. Faedo b,1 a
Pastoral Research Laboratory, CSIRO Livestock Industries, Armidale, NSW 2350, Australia b McMaster Laboratory, CSIRO Livestock Industries, Blacktown, NSW 2148, Australia
Received 8 December 2000; received in revised form 30 July 2001; accepted 7 August 2001
Abstract A series of feeding trials was conducted with penned sheep harboring Trichostrongylus colubriformis infections. They were offered barley grains supporting the growth of the nematophagous fungus Duddingtonia flagrans. It was shown that as little as 5 g of grain/sheep per day was sufficient to virtually eliminate larval numbers from faecal culture. This effect persisted for the time that the fungal grains were fed, and for up to 2 days following cessation of feeding this material. Macerated fungal grains were also incorporated into a range of feed block formulations. In all these, D. flagrans was found to survive the manufacturing process and resulted in significant reductions in larval numbers in faecal cultures set up during the feeding period to sheep. This was observed even for sheep that showed only modest and irregular block consumption. These studies demonstrate that supplementary feeding or block administration offer potential deployment options for D. flagrans as a means of biological control of nematode parasites of livestock. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Sheep-nematoda; Biological control; Nematophagous fungi; Duddingtonia flagrans; Feed blocks
1. Introduction In the pursuit of a suitable candidate organism for the biological control of the free-living stages of nematode parasites of livestock, research activities have now focused on the ∗ Corresponding author. Present address: SWEPAR, National Veterinary Institute, SE-751 89, Uppsala, Sweden. Tel.: +46-18-67-4127; fax: +46-18-30-9162. E-mail address: [email protected] (P.J. Waller). 1 Present address: DCEP, The Royal Veterinary and Agricultural University, 13 Bulowsvej, DK 1870 Frederiksberg C, Denmark.
0304-4017/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 1 ) 0 0 5 4 2 - 8
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nematophagous microfungus, Duddingtonia flagrans (for reviews, see Waller and Faedo, 1996; Larsen et al., 1997). This is because of the superior ability of this fungus to survive passage through the gastrointestinal tract of livestock. This ability is believed to be an essential pre-requisite for the practical deployment of such organisms in non-chemotherapeutic nematode parasite control options. D. flagrans produces thick walled chlamydospores in abundance and it is in this resting stage that the fungus can survive the harsh environmental conditions (anaerobic, enzymatic and thermal) of gut passage in livestock (Larsen et al., 1992, 1998; Grønvold et al., 1993). It is this harsh environment to which fungi that produce thin-walled conidia appear to succumb during gastrointestinal transit (Waller et al., 1994; Faedo et al., 1997). Various cereal grains provide an ideal substrate for the growth and production of substantial quantities of spores of nematode destroying fungi. Initial studies feeding this material to sheep in France resulted in the presence of fungi in faeces and reductions in the number of infective larvae recovered from faecal culture (Gruner et al., 1985; Peloille, 1991). However, these observations were based on feeding up to 0.5 kg of grain to each animal and the focus of attention was on members of the Arthrobotrys genera, which are more predisposed to the production of thin-walled conidia rather than chlamydospores. In further studies by Danish workers (Larsen et al., 1992; Grønvold et al., 1993), calves were fed 200 g of barley grains on which Arthrobotrys spp. or D. flagrans, had been cultivated. These experiments resulted in significant reductions in infective larval numbers in faecal cultures and in pasture samples where the latter fungal species was used. This work set the benchmark for the amount of fungal grains to be fed daily to test the concept of biological control of nematode parasites using D. flagrans in several livestock species (cattle: Grønvold et al., 1993; horses: Larsen et al., 1996; pigs: Nansen et al., 1996, and sheep: Githega et al., 1997). The success of these trials demonstrated, in principle, that biological control can be achieved using D. flagrans, against mixed parasite infections commonly found in a variety of livestock species. Clearly the prospect for biological control of nematode parasites is good. However, even in the most recent studies from Denmark, the large amounts of grain (supporting the growth of D. flagrans) used are impractical — even for the most intensive grazing livestock enterprise. The purpose of this work was to determine the least amount of fungal grain material needed to be ingested by sheep to result in substantial reductions in the number of infective larvae that develop from faecal cultures. In addition, this fungal grain material was macerated and incorporated into a range of supplementary feed block formulations to determine whether this form of administration could also be possible. 2. Materials and methods 2.1. Grain feeding trials Young (6–9-month-old) Merino wether sheep, harboring infections of Trichostrongylus colubriformis, were used in these experiments. These sheep were maintained in individual pen accommodation and provided with a standard ration of 600 g of chaff mix (lucerne, wheat and oats) per day. For 3–4 days prior to the commencement of each feeding trial, faecal samples were collected daily from each sheep for estimates of nematode faecal egg
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count and larval and fungal development, as described by Faedo et al. (1997). Briefly, faecal cultures for larval development were prepared using 5 g faeces, vermicullite and water and incubated for 10 days at 25◦ C before recovery and enumeration of total larvae. Percent development was then calculated by dividing the number of larvae harvested by the number of nematode eggs as determined by faecal egg count. For fungal presence, three replicate 5 g sub-samples of faeces from the remaining pooled and thoroughly mixed faeces from lambs in each treatment, were applied to water agar Petri dishes and incubated at 25◦ C for 10 days. After this time a quantitative assessment of the presence of fungal colonies was determined microscopically, using the following classifications: (䊏) ( ) ( ) ( )
no D. flagrans growth on colonies; D. flagrans growth on 1 of 3 cultures — generally few, scattered colonies; D. flagrans growth on 2 of 3 cultures — generally many colonies; D. flagrans growth on 3 of 3 culture plates — profuse growth (colonies overlapping and covering whole plate).
During each of the feeding trials, faecal samples were taken twice per day (09.00 and 17.00 h) for the same procedures. At the conclusion of the feeding period, collection of faecal samples for nematode egg count, larval and fungal culture reverted to once per day for a further 2 days in the first trial and for 5 days in the second trial. For each feeding trial, lambs had quantities of fungal grains incorporated into a small amount of their daily ration, which was offered each morning. After this material was consumed the remainder of the ration was provided. A total of 4–5-week-old cultures of the Walcha strain of D. flagrans (Larsen et al., 1994) growing on previously sterilized, moistened barley grains were used in each trial. Estimates of chlamydospore numbers per gram of grains were made by taking at random, five, 1 g sub-samples of grains and homogenizing in 10 ml of water and then conducting at least 10 counts of spores using a haemocytometer. For the first trial, two lambs were offered 10 g of fungal grains (containing 3 × 106 chlamydospores) each day for 9 days and another two lambs were given 10 g of normal barley. For the second trial, four groups of two sheep were used which were fed each day for 5 days the following: Controls Group A Group B Group C
15 g clean barley 5 g D. flagrans barley (4 × 106 spores per day) 10 g D. flagrans barley (9 × 106 spores per day) 15 g D. flagrans barley (13 × 106 spores per day)
2.2. Block trials Barley grains used in culturing D. flagrans for 4–5 weeks were air dried, milled and then chlamydospore estimates were made by the procedures outlined above. This material was then incorporated into a range of feed block formulations as detailed in Table 1. Prior to the commencement of the fungal block treatments, parasitized sheep (T. colubriformis) were maintained on a diet of chopped oaten hay and allowed ad libitum access to a fungi-free urea molasses block (UMB) to habituate them to block consumption. Faecal samples were taken each day for nematode egg counts and larval and fungal development. For each trial,
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Table 1 Block ingredients (%) used in experimental formulations with D. flagrans Ingredient
Formulation UMB
A
B
C
D
Molasses NaCl Urea Mineralsa Wheat bran Fungi barleyb CaO Ag. lime (CaCO3 ) H3 PO4 Bentonite Triple superphosphate Compression
45.5 5.0 10.0 1.0 26.5 Nil 3.5 4.0 4.5 Nil Nil Nil
45.5 5.0 10.0 1.0 9.0 17.5 3.5 4.0 4.5 Nil Nil Nil
30.0 8.0 15.0 Nil Nil 5.0 Nil 10.0 5.0 3.0 Nil Yesc
43.0 5.0 10.0 1.0 21.0 5.0 2.0 5.0 3.0 Nil 1.0 Nil
43.0 5.0 10.0 1.0 29.0 5.0 7.0 Nil Nil Nil Nil Nil
a
Mineral Premix — Pfizer 422 (Pfizer Agricare, NSW). Barley grains on which D. flagrans had been cultured. These were dried and milled before inclusion in the blocks. c Compression using a hydraulic ram press delivering approximately 15 t over a 320 cm2 plate. b
sheep were provided with UMB for a minimum of 2 weeks, followed by fungal block for 5 days and then 9 days of UMB block. In the first trial, where block A was used, four sheep were each given 100 g of block in a reduced portion of their daily ration and then provided with the balance once the initial allocation was consumed. This was followed by a series of trials using 12 individually penned sheep to which blocks A–D were offered for ad libitum consumption. Blocks were weighed each day to estimate the amount of block (and fungal chlamydospores) consumed. Following each trial, the remaining blocks were retained in the laboratory to estimate the shelf-life of chlamydospores incorporated into these blocks, with storage of blocks at both ambient and refrigerated temperatures. Estimates of shelf-life were conducted by mixing 20 g of block material into the daily ration of groups of four parasitized sheep and feeding for 7 days. 2.3. Statistical procedures For the block trials the numbers of larvae produced from faecal cultures were estimated daily and expressed as a percentage developing from available eggs as estimated by faecal egg counts. Mean percentage estimates after exposure to the fungi (days 6–10) were compared to those prior to exposure (days 2–4) by means of the paired sample t-test. 3. Results 3.1. Feeding trials Faecal egg counts for the first trial were 4087 ± 1211 eggs/g faeces (mean ± S.D.). Larval cultures showed that the feeding of 10 g per day of D. flagrans barley (3 × 106
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Fig. 1. Mean percentage development of nematode eggs to infective larvae in lambs fed 10 g of D. flagrans barley grains for 9 days: (䊊—䊊) percent larval development; (䊏) no D. flagrans growth; ( ) D. flagrans growth on 2 of 3 cultures; ( ) D. flagrans growth on 3 of 3 cultures.
chlamydospores per day) resulted in a virtual elimination of infective larvae that successfully developed from nematode eggs that were present in faeces (see Fig. 1). At the same time, a profuse growth of the fungus was recorded in all agar plate faecal cultures. Faecal egg counts for the second trial were 4293 ± 1779 eggs/g faeces (mean ± S.D.). As shown in Fig. 2, the larvae elimination observed in the first trial was confirmed in the second trial where the same result was recorded for feeding 5, 10 and 15 g of D. flagrans barley grains (4 × 106 , 9 × 106 and 13 × 106 chlamydospores per day, respectively). It should be noted that the chlamydospore concentration of the latter batch of grains was more than two-fold greater than in the first trial. In all cases, growth of D. flagrans was recorded on the majority of agar plate cultures from sheep fed D. flagrans throughout the feeding and was still evident during the 5 days post-fungal feeding period. Growth of small colonies of nematode-trapping fungi were found on fungal culture plates from faeces of the control sheep on a few occasions during the trial. This was attributed to adventitious contamination occurring during the plating phase in the laboratory, as there was no coincident depression in larval numbers in the faecal cultures. 3.2. Block trials The details of the various block compositions and formulations are given in Table 1 The first trial, where sheep were fed 100 g of block A, resulted in a total elimination of infective larvae from faecal culture, concurrent with observed growth of D. flagrans in the majority of agar plate faecal cultures. With the ad libitum intake trials, considerable variation occurred between sheep for each block formulation as well as between formulations. Some formulations (B and D) resulted in very hard blocks and as a consequence negligible, or very little, block was consumed and results are not presented here. However, for all formulations the presence of D. flagrans together with larval reductions were recorded in faecal samples during the period in which the fungal blocks were on offer. Fig. 3 provides a summary of
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Fig. 3. Mean percentage (±S.D.) development of eggs to infective larvae in lambs offered formulation A fungi block ad libitum for 5 days.
the larval recovery data for 12 sheep with faecal egg counts of 3477 ± 1737 (mean ± S.D.) offered block A formulation ad libitum for 5 days. The percentage of larvae produced from available eggs before fungal block offer (37.1 ± 12.1%; mean for days 2–4 ± S.D.) was significantly higher (P < 0.0005) than that after fungal block offer (7.7 ± 7.6%; mean for days 6–10 ± S.D.). Larvae numbers, as a percentage of available eggs, remained low for 3 days after the fungal blocks were withdrawn and then returned to the pre-fungal block levels (see Fig. 3). Estimates of shelf-life showed that D. flagrans spores incorporated into compressed blocks (block B), which had negligible water content, survived well for at least 18 weeks, particularly when stored at refrigerated temperatures (∼4◦ C) (see Fig. 4). The viability of spores in blocks that had been made by processes involving some moisture (blocks A, C and D), declined to negligible levels after 12 weeks storage at room temperature. We surmise
Fig. 4. Mean percentage development of eggs to infective larvae in lambs fed 20 g of compressed block after storage at 5◦ C for 18 weeks, compared with development of eggs from sheep fed normal block: (䉱—䉱) percent larval development — normal blocks; (䊏—䊏) percent larval development — compressed blocks.
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that this may be attributed to the induction of germination of the spores by moisture and, as a consequence, a reduction in their capacity to survive gut passage through the sheep.
4. Discussion Our studies showed that in pen fed, parasitized sheep, the quantities of fungal grains required to virtually eliminate larval numbers from faecal culture are much less than what would be expected from the studies by the Danish workers (Larsen et al., 1992; Grønvold et al., 1993; Nansen et al., 1996). As little as 5 g/sheep per day of barley grain, supporting the growth of D. flagrans, incorporated into the daily ration of sheep achieved this result. In Australia, grain feeding of sheep is becoming a more common management procedure, particularly at times of expected nutritional and/or environmental stress of young or pregnant stock. In the winter and non-seasonal rainfall zones, which encompass more than 75% of the sheep raising regions in Australia, this period usually occurs in autumn/early winter. This coincides with the period in which pasture contamination with nematode eggs results in the seasonal peaks of larval availability in the following late winter/spring (Anderson et al., 1978). Not only are weather conditions favorable for the development and survival of the free-living stages of nematode parasites, but they are also favorable for the germination and colonization of dung by nematophagous fungi (Faedo et al., 1998). It could be expected that feeding sheep fungal grains, rather than the normal grains, during autumn/early winter, would result in a substantial reduction in the subsequent peaks in larval numbers. Further, unpublished studies in our laboratories have shown that D. flagrans will grow on a variety of grain substrates (e.g. maize, wheat, sorghum, lupins and unmilled rice) and often these are more popular with sheep farmers, because of price and availability, to be fed to sheep. Therefore, the feeding of fungal chlamydospores produced on cereal substrates is not restricted to barley grain. Our studies on prototype block formulations have, for the first time, demonstrated the practical feasibility of deploying fungal spores by these means. Block administration, developed mainly for nutritional supplementation and utilized to a lesser extent for anthelmintic administration, has the disadvantage of voluntary, therefore, variable, intake when offered to grazing livestock. A concern has been raised that optimal reduction in larval development, which is achieved when all animals consume the required daily dose of spores, may only rarely, and never consistently, occur. On a flock basis this should not be a problem, because the objective of this form of fungal spore delivery is to reduce the seasonal peaks in larval numbers and not eliminate them. Under pen conditions, we have demonstrated that profound reduction in larval numbers in faeces can be achieved even in animals showing irregular and modest consumption of blocks on offer. With relatively fewer infective larvae on pasture, productivity losses would possibly be eliminated, whilst providing the necessary antigenic stimulation to provoke naturally acquired immunity in grazing sheep. Block administration is now becoming recognized as a practical means of providing low-cost nutrient supplementation to livestock in the developing world by utilizing previously unwanted plant by-products (Knox, 1996). The types of block formulations used in our study were chosen to represent those currently employed in the manufacture of blocks by simple, cheap procedures in the developing world (Sansoucy, 1995). Plant by-product
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(second-quality grains, rice hulls and sugar cane pulp) can also support the growth of D. flagrans. In many instances, block manufacturing operations are relatively small, producing only small batches of blocks for immediate use by farmers. Therefore, reasonable shelf-life of spores in these blocks is not an important consideration. Block administration has possibly greater potential for success in the tropical/sub-tropical regions of the world where tethered husbandry and night housing with stall feeding are common management practices. Consumption is likely to be greater, both in terms of quantity and number of animals, than in permanent grazing systems. The other great incentive for farmers in the tropics/sub-tropics to adopt alternative, non-chemotherapeutic means of parasite control is the alarming increase in the prevalence of multiple anthelmintic resistance in these regions of the world (Waller, 1997).
Acknowledgements We wish to acknowledge the skilled assistance of Lester Anderson, Ben Foster and Tom Parker in the course of these studies. References Anderson, N., Dash, K.M., Donald, A.D., Southcott, W.H., Waller, P.J., 1978. Epidemiology and control of nematode infections. In: Donald, A.D., Southcott, W.H., Dineen, J.K. (Eds.), The Epidemiology and Control of Gastrointestinal Parasites of Sheep in Australia. Academic Press, Australia, pp. 23–51. Faedo, M., Waller, P.J., Larsen, M., 1997. The potential of nematophagous fungi to control the free-living stages of nematode parasites of sheep: comparison between Australian isolates of Arthrobotrys spp. and Duddingtonia flagrans. Vet. Parasitol. 72, 149–155. Faedo, M., Barnes, E.H., Dobson, R.J., Waller, P.J., 1998. The potential of nematophagous fungi to control the free-living stages of nematode parasites of sheep: pasture plot study with Duddingtonia flagrans. Vet. Parasitol. 76, 129–135. Githega, S.M., Thamsborg, S.M., Larsen, M., Kyvsgaard, N.C., Nansen, P., 1997. The preventive effect of the fungus Duddingtonia flagrans on trichostrongyle infections of lambs on pasture. Int. J. Parasitol. 27, 931–939. Grønvold, J., Wolstrup, J., Larsen, M., Henriksen, S.A., Nansen, P., 1993. Biological control of Ostertagia ostertagi by feeding selected nematode-trapping fungi to calves. J. Helminthol. 67, 31–36. Gruner, L., Peloille, M., Sauv´e, C., Cortet, J., 1985. Parasit`ogie animale. Survie et conservation de l’activit´e pr`edatrice vis-`a-vis de n´ematodes trichostrongylides apr`es ingestion par des ovins de trois hypomyc`es predateurs, Vol. 300. Compte Rendu de l’Acad´emie des Sciences Paris III, pp. 525–528. Knox, M.R., 1996. Integrated control programs using medicated blocks. In: Le Jambre, L.F., Knox, M.R. (Eds.), Proceedings of the ACIAR on Sustainable Parasite Control in Small Ruminants (Proceedings No. 74). Canberra, Australia, pp. 141–145. Larsen, M., Wolstrup, J., Henriksen, S.A., Grønvold, J., Nansen, P., 1992. In vivo passage through calves of nematophagous fungi selected for biocontrol of parasitic nematodes. J. Helminthol. 66, 137–141. Larsen, M., Faedo, M., Waller, P.J., 1994. The potential of nematophagous fungi to control the free-living stages of nematode parasites of sheep: survey for the presence of fungi in fresh faeces of grazing livestock in Australia. Vet. Parasitol. 53, 275–281. Larsen, M., Nansen, P., Grondahl, C., Thamsborg, S.M., Grønvold, J., Wolstrup, J., Henriksen, S.A., Monrad, J., 1996. The capacity of the fungus Duddingtonia flagrans to prevent strongyle infections in foals on pasture. Vet. Parasitol. 113, 1–6. Larsen, M., Nansen, P., Grønvold, J., Wolstrup, J., Henriksen, S.A., 1997. Biological control of gastro-intestinal nematodes-facts, future or fiction. Vet. Parasitol. 72, 479–492.
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Larsen, M., Faedo, M., Waller, P.J., Hennessy, D.R., 1998. The potential of nematophagous fungi to control the free-living stages of nematode parasites of sheep: studies with Duddingtonia flagrans. Vet. Parasitol. 76, 121–128. Nansen, P., Larsen, M., Roepstorff, A., Grønvold, J., Wolstrup, J., Henriksen, S.A., 1996. Control of Oesophagostomum dentatum and Hyostrongylus rubidus in outdoor-reared pigs through daily feeding with the microfungus Duddingtonia flagrans. Parasitol. Res. 82, 580–584. Peloille, M., 1991. Selection of Nematode-trapping Fungi for Use in Biological Control, Vol. 14. Bulletin SROP, International Organization for Biological Control of Noxious Animals and Plants, West Paleartic Regional Section, pp. 13–17. Sansoucy, R., 1995. New developments in the manufacture and utilization of multinutrient blocks. World Anim. Rev. 82, 78–83. Waller, P.J., 1997. A global perspective of anthelmintic resistance in nematode parasites of sheep — excluding Australia. In: Proceedings of the 4th International Sheep Veterinary Conference. Australian Sheep Veterinary Society, Indooroopilly, University of New England, Armidale, NSW, Australia, pp. 59–63. Waller, P.J., Faedo, M., 1996. The prospects for biological control of the free-living stages of nematode parasites of livestock. Int. J. Parasitol. 26, 915–925. Waller, P.J., Larsen, M., Faedo, M., Hennessy, D.R., 1994. The potential of nematophagous fungi to control the free-living stages of nematode parasites of sheep: in vitro and in vivo studies. Vet. Parasitol. 51, 289–299.
The potential of nematophagous fungi to control the free-living stages of nematode parasites of sheep: feeding and block studies with Duddingtonia flagrans P.J. Waller a,∗ , M.R. Knox a , M. Faedo b,1 a
Pastoral Research Laboratory, CSIRO Livestock Industries, Armidale, NSW 2350, Australia b McMaster Laboratory, CSIRO Livestock Industries, Blacktown, NSW 2148, Australia
Received 8 December 2000; received in revised form 30 July 2001; accepted 7 August 2001
Abstract A series of feeding trials was conducted with penned sheep harboring Trichostrongylus colubriformis infections. They were offered barley grains supporting the growth of the nematophagous fungus Duddingtonia flagrans. It was shown that as little as 5 g of grain/sheep per day was sufficient to virtually eliminate larval numbers from faecal culture. This effect persisted for the time that the fungal grains were fed, and for up to 2 days following cessation of feeding this material. Macerated fungal grains were also incorporated into a range of feed block formulations. In all these, D. flagrans was found to survive the manufacturing process and resulted in significant reductions in larval numbers in faecal cultures set up during the feeding period to sheep. This was observed even for sheep that showed only modest and irregular block consumption. These studies demonstrate that supplementary feeding or block administration offer potential deployment options for D. flagrans as a means of biological control of nematode parasites of livestock. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Sheep-nematoda; Biological control; Nematophagous fungi; Duddingtonia flagrans; Feed blocks
1. Introduction In the pursuit of a suitable candidate organism for the biological control of the free-living stages of nematode parasites of livestock, research activities have now focused on the ∗ Corresponding author. Present address: SWEPAR, National Veterinary Institute, SE-751 89, Uppsala, Sweden. Tel.: +46-18-67-4127; fax: +46-18-30-9162. E-mail address: [email protected] (P.J. Waller). 1 Present address: DCEP, The Royal Veterinary and Agricultural University, 13 Bulowsvej, DK 1870 Frederiksberg C, Denmark.
0304-4017/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 1 ) 0 0 5 4 2 - 8
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nematophagous microfungus, Duddingtonia flagrans (for reviews, see Waller and Faedo, 1996; Larsen et al., 1997). This is because of the superior ability of this fungus to survive passage through the gastrointestinal tract of livestock. This ability is believed to be an essential pre-requisite for the practical deployment of such organisms in non-chemotherapeutic nematode parasite control options. D. flagrans produces thick walled chlamydospores in abundance and it is in this resting stage that the fungus can survive the harsh environmental conditions (anaerobic, enzymatic and thermal) of gut passage in livestock (Larsen et al., 1992, 1998; Grønvold et al., 1993). It is this harsh environment to which fungi that produce thin-walled conidia appear to succumb during gastrointestinal transit (Waller et al., 1994; Faedo et al., 1997). Various cereal grains provide an ideal substrate for the growth and production of substantial quantities of spores of nematode destroying fungi. Initial studies feeding this material to sheep in France resulted in the presence of fungi in faeces and reductions in the number of infective larvae recovered from faecal culture (Gruner et al., 1985; Peloille, 1991). However, these observations were based on feeding up to 0.5 kg of grain to each animal and the focus of attention was on members of the Arthrobotrys genera, which are more predisposed to the production of thin-walled conidia rather than chlamydospores. In further studies by Danish workers (Larsen et al., 1992; Grønvold et al., 1993), calves were fed 200 g of barley grains on which Arthrobotrys spp. or D. flagrans, had been cultivated. These experiments resulted in significant reductions in infective larval numbers in faecal cultures and in pasture samples where the latter fungal species was used. This work set the benchmark for the amount of fungal grains to be fed daily to test the concept of biological control of nematode parasites using D. flagrans in several livestock species (cattle: Grønvold et al., 1993; horses: Larsen et al., 1996; pigs: Nansen et al., 1996, and sheep: Githega et al., 1997). The success of these trials demonstrated, in principle, that biological control can be achieved using D. flagrans, against mixed parasite infections commonly found in a variety of livestock species. Clearly the prospect for biological control of nematode parasites is good. However, even in the most recent studies from Denmark, the large amounts of grain (supporting the growth of D. flagrans) used are impractical — even for the most intensive grazing livestock enterprise. The purpose of this work was to determine the least amount of fungal grain material needed to be ingested by sheep to result in substantial reductions in the number of infective larvae that develop from faecal cultures. In addition, this fungal grain material was macerated and incorporated into a range of supplementary feed block formulations to determine whether this form of administration could also be possible. 2. Materials and methods 2.1. Grain feeding trials Young (6–9-month-old) Merino wether sheep, harboring infections of Trichostrongylus colubriformis, were used in these experiments. These sheep were maintained in individual pen accommodation and provided with a standard ration of 600 g of chaff mix (lucerne, wheat and oats) per day. For 3–4 days prior to the commencement of each feeding trial, faecal samples were collected daily from each sheep for estimates of nematode faecal egg
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count and larval and fungal development, as described by Faedo et al. (1997). Briefly, faecal cultures for larval development were prepared using 5 g faeces, vermicullite and water and incubated for 10 days at 25◦ C before recovery and enumeration of total larvae. Percent development was then calculated by dividing the number of larvae harvested by the number of nematode eggs as determined by faecal egg count. For fungal presence, three replicate 5 g sub-samples of faeces from the remaining pooled and thoroughly mixed faeces from lambs in each treatment, were applied to water agar Petri dishes and incubated at 25◦ C for 10 days. After this time a quantitative assessment of the presence of fungal colonies was determined microscopically, using the following classifications: (䊏) ( ) ( ) ( )
no D. flagrans growth on colonies; D. flagrans growth on 1 of 3 cultures — generally few, scattered colonies; D. flagrans growth on 2 of 3 cultures — generally many colonies; D. flagrans growth on 3 of 3 culture plates — profuse growth (colonies overlapping and covering whole plate).
During each of the feeding trials, faecal samples were taken twice per day (09.00 and 17.00 h) for the same procedures. At the conclusion of the feeding period, collection of faecal samples for nematode egg count, larval and fungal culture reverted to once per day for a further 2 days in the first trial and for 5 days in the second trial. For each feeding trial, lambs had quantities of fungal grains incorporated into a small amount of their daily ration, which was offered each morning. After this material was consumed the remainder of the ration was provided. A total of 4–5-week-old cultures of the Walcha strain of D. flagrans (Larsen et al., 1994) growing on previously sterilized, moistened barley grains were used in each trial. Estimates of chlamydospore numbers per gram of grains were made by taking at random, five, 1 g sub-samples of grains and homogenizing in 10 ml of water and then conducting at least 10 counts of spores using a haemocytometer. For the first trial, two lambs were offered 10 g of fungal grains (containing 3 × 106 chlamydospores) each day for 9 days and another two lambs were given 10 g of normal barley. For the second trial, four groups of two sheep were used which were fed each day for 5 days the following: Controls Group A Group B Group C
15 g clean barley 5 g D. flagrans barley (4 × 106 spores per day) 10 g D. flagrans barley (9 × 106 spores per day) 15 g D. flagrans barley (13 × 106 spores per day)
2.2. Block trials Barley grains used in culturing D. flagrans for 4–5 weeks were air dried, milled and then chlamydospore estimates were made by the procedures outlined above. This material was then incorporated into a range of feed block formulations as detailed in Table 1. Prior to the commencement of the fungal block treatments, parasitized sheep (T. colubriformis) were maintained on a diet of chopped oaten hay and allowed ad libitum access to a fungi-free urea molasses block (UMB) to habituate them to block consumption. Faecal samples were taken each day for nematode egg counts and larval and fungal development. For each trial,
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Table 1 Block ingredients (%) used in experimental formulations with D. flagrans Ingredient
Formulation UMB
A
B
C
D
Molasses NaCl Urea Mineralsa Wheat bran Fungi barleyb CaO Ag. lime (CaCO3 ) H3 PO4 Bentonite Triple superphosphate Compression
45.5 5.0 10.0 1.0 26.5 Nil 3.5 4.0 4.5 Nil Nil Nil
45.5 5.0 10.0 1.0 9.0 17.5 3.5 4.0 4.5 Nil Nil Nil
30.0 8.0 15.0 Nil Nil 5.0 Nil 10.0 5.0 3.0 Nil Yesc
43.0 5.0 10.0 1.0 21.0 5.0 2.0 5.0 3.0 Nil 1.0 Nil
43.0 5.0 10.0 1.0 29.0 5.0 7.0 Nil Nil Nil Nil Nil
a
Mineral Premix — Pfizer 422 (Pfizer Agricare, NSW). Barley grains on which D. flagrans had been cultured. These were dried and milled before inclusion in the blocks. c Compression using a hydraulic ram press delivering approximately 15 t over a 320 cm2 plate. b
sheep were provided with UMB for a minimum of 2 weeks, followed by fungal block for 5 days and then 9 days of UMB block. In the first trial, where block A was used, four sheep were each given 100 g of block in a reduced portion of their daily ration and then provided with the balance once the initial allocation was consumed. This was followed by a series of trials using 12 individually penned sheep to which blocks A–D were offered for ad libitum consumption. Blocks were weighed each day to estimate the amount of block (and fungal chlamydospores) consumed. Following each trial, the remaining blocks were retained in the laboratory to estimate the shelf-life of chlamydospores incorporated into these blocks, with storage of blocks at both ambient and refrigerated temperatures. Estimates of shelf-life were conducted by mixing 20 g of block material into the daily ration of groups of four parasitized sheep and feeding for 7 days. 2.3. Statistical procedures For the block trials the numbers of larvae produced from faecal cultures were estimated daily and expressed as a percentage developing from available eggs as estimated by faecal egg counts. Mean percentage estimates after exposure to the fungi (days 6–10) were compared to those prior to exposure (days 2–4) by means of the paired sample t-test. 3. Results 3.1. Feeding trials Faecal egg counts for the first trial were 4087 ± 1211 eggs/g faeces (mean ± S.D.). Larval cultures showed that the feeding of 10 g per day of D. flagrans barley (3 × 106
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Fig. 1. Mean percentage development of nematode eggs to infective larvae in lambs fed 10 g of D. flagrans barley grains for 9 days: (䊊—䊊) percent larval development; (䊏) no D. flagrans growth; ( ) D. flagrans growth on 2 of 3 cultures; ( ) D. flagrans growth on 3 of 3 cultures.
chlamydospores per day) resulted in a virtual elimination of infective larvae that successfully developed from nematode eggs that were present in faeces (see Fig. 1). At the same time, a profuse growth of the fungus was recorded in all agar plate faecal cultures. Faecal egg counts for the second trial were 4293 ± 1779 eggs/g faeces (mean ± S.D.). As shown in Fig. 2, the larvae elimination observed in the first trial was confirmed in the second trial where the same result was recorded for feeding 5, 10 and 15 g of D. flagrans barley grains (4 × 106 , 9 × 106 and 13 × 106 chlamydospores per day, respectively). It should be noted that the chlamydospore concentration of the latter batch of grains was more than two-fold greater than in the first trial. In all cases, growth of D. flagrans was recorded on the majority of agar plate cultures from sheep fed D. flagrans throughout the feeding and was still evident during the 5 days post-fungal feeding period. Growth of small colonies of nematode-trapping fungi were found on fungal culture plates from faeces of the control sheep on a few occasions during the trial. This was attributed to adventitious contamination occurring during the plating phase in the laboratory, as there was no coincident depression in larval numbers in the faecal cultures. 3.2. Block trials The details of the various block compositions and formulations are given in Table 1 The first trial, where sheep were fed 100 g of block A, resulted in a total elimination of infective larvae from faecal culture, concurrent with observed growth of D. flagrans in the majority of agar plate faecal cultures. With the ad libitum intake trials, considerable variation occurred between sheep for each block formulation as well as between formulations. Some formulations (B and D) resulted in very hard blocks and as a consequence negligible, or very little, block was consumed and results are not presented here. However, for all formulations the presence of D. flagrans together with larval reductions were recorded in faecal samples during the period in which the fungal blocks were on offer. Fig. 3 provides a summary of
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Fig. 3. Mean percentage (±S.D.) development of eggs to infective larvae in lambs offered formulation A fungi block ad libitum for 5 days.
the larval recovery data for 12 sheep with faecal egg counts of 3477 ± 1737 (mean ± S.D.) offered block A formulation ad libitum for 5 days. The percentage of larvae produced from available eggs before fungal block offer (37.1 ± 12.1%; mean for days 2–4 ± S.D.) was significantly higher (P < 0.0005) than that after fungal block offer (7.7 ± 7.6%; mean for days 6–10 ± S.D.). Larvae numbers, as a percentage of available eggs, remained low for 3 days after the fungal blocks were withdrawn and then returned to the pre-fungal block levels (see Fig. 3). Estimates of shelf-life showed that D. flagrans spores incorporated into compressed blocks (block B), which had negligible water content, survived well for at least 18 weeks, particularly when stored at refrigerated temperatures (∼4◦ C) (see Fig. 4). The viability of spores in blocks that had been made by processes involving some moisture (blocks A, C and D), declined to negligible levels after 12 weeks storage at room temperature. We surmise
Fig. 4. Mean percentage development of eggs to infective larvae in lambs fed 20 g of compressed block after storage at 5◦ C for 18 weeks, compared with development of eggs from sheep fed normal block: (䉱—䉱) percent larval development — normal blocks; (䊏—䊏) percent larval development — compressed blocks.
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that this may be attributed to the induction of germination of the spores by moisture and, as a consequence, a reduction in their capacity to survive gut passage through the sheep.
4. Discussion Our studies showed that in pen fed, parasitized sheep, the quantities of fungal grains required to virtually eliminate larval numbers from faecal culture are much less than what would be expected from the studies by the Danish workers (Larsen et al., 1992; Grønvold et al., 1993; Nansen et al., 1996). As little as 5 g/sheep per day of barley grain, supporting the growth of D. flagrans, incorporated into the daily ration of sheep achieved this result. In Australia, grain feeding of sheep is becoming a more common management procedure, particularly at times of expected nutritional and/or environmental stress of young or pregnant stock. In the winter and non-seasonal rainfall zones, which encompass more than 75% of the sheep raising regions in Australia, this period usually occurs in autumn/early winter. This coincides with the period in which pasture contamination with nematode eggs results in the seasonal peaks of larval availability in the following late winter/spring (Anderson et al., 1978). Not only are weather conditions favorable for the development and survival of the free-living stages of nematode parasites, but they are also favorable for the germination and colonization of dung by nematophagous fungi (Faedo et al., 1998). It could be expected that feeding sheep fungal grains, rather than the normal grains, during autumn/early winter, would result in a substantial reduction in the subsequent peaks in larval numbers. Further, unpublished studies in our laboratories have shown that D. flagrans will grow on a variety of grain substrates (e.g. maize, wheat, sorghum, lupins and unmilled rice) and often these are more popular with sheep farmers, because of price and availability, to be fed to sheep. Therefore, the feeding of fungal chlamydospores produced on cereal substrates is not restricted to barley grain. Our studies on prototype block formulations have, for the first time, demonstrated the practical feasibility of deploying fungal spores by these means. Block administration, developed mainly for nutritional supplementation and utilized to a lesser extent for anthelmintic administration, has the disadvantage of voluntary, therefore, variable, intake when offered to grazing livestock. A concern has been raised that optimal reduction in larval development, which is achieved when all animals consume the required daily dose of spores, may only rarely, and never consistently, occur. On a flock basis this should not be a problem, because the objective of this form of fungal spore delivery is to reduce the seasonal peaks in larval numbers and not eliminate them. Under pen conditions, we have demonstrated that profound reduction in larval numbers in faeces can be achieved even in animals showing irregular and modest consumption of blocks on offer. With relatively fewer infective larvae on pasture, productivity losses would possibly be eliminated, whilst providing the necessary antigenic stimulation to provoke naturally acquired immunity in grazing sheep. Block administration is now becoming recognized as a practical means of providing low-cost nutrient supplementation to livestock in the developing world by utilizing previously unwanted plant by-products (Knox, 1996). The types of block formulations used in our study were chosen to represent those currently employed in the manufacture of blocks by simple, cheap procedures in the developing world (Sansoucy, 1995). Plant by-product
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(second-quality grains, rice hulls and sugar cane pulp) can also support the growth of D. flagrans. In many instances, block manufacturing operations are relatively small, producing only small batches of blocks for immediate use by farmers. Therefore, reasonable shelf-life of spores in these blocks is not an important consideration. Block administration has possibly greater potential for success in the tropical/sub-tropical regions of the world where tethered husbandry and night housing with stall feeding are common management practices. Consumption is likely to be greater, both in terms of quantity and number of animals, than in permanent grazing systems. The other great incentive for farmers in the tropics/sub-tropics to adopt alternative, non-chemotherapeutic means of parasite control is the alarming increase in the prevalence of multiple anthelmintic resistance in these regions of the world (Waller, 1997).
Acknowledgements We wish to acknowledge the skilled assistance of Lester Anderson, Ben Foster and Tom Parker in the course of these studies. References Anderson, N., Dash, K.M., Donald, A.D., Southcott, W.H., Waller, P.J., 1978. Epidemiology and control of nematode infections. In: Donald, A.D., Southcott, W.H., Dineen, J.K. (Eds.), The Epidemiology and Control of Gastrointestinal Parasites of Sheep in Australia. Academic Press, Australia, pp. 23–51. Faedo, M., Waller, P.J., Larsen, M., 1997. The potential of nematophagous fungi to control the free-living stages of nematode parasites of sheep: comparison between Australian isolates of Arthrobotrys spp. and Duddingtonia flagrans. Vet. Parasitol. 72, 149–155. Faedo, M., Barnes, E.H., Dobson, R.J., Waller, P.J., 1998. The potential of nematophagous fungi to control the free-living stages of nematode parasites of sheep: pasture plot study with Duddingtonia flagrans. Vet. Parasitol. 76, 129–135. Githega, S.M., Thamsborg, S.M., Larsen, M., Kyvsgaard, N.C., Nansen, P., 1997. The preventive effect of the fungus Duddingtonia flagrans on trichostrongyle infections of lambs on pasture. Int. J. Parasitol. 27, 931–939. Grønvold, J., Wolstrup, J., Larsen, M., Henriksen, S.A., Nansen, P., 1993. Biological control of Ostertagia ostertagi by feeding selected nematode-trapping fungi to calves. J. Helminthol. 67, 31–36. Gruner, L., Peloille, M., Sauv´e, C., Cortet, J., 1985. Parasit`ogie animale. Survie et conservation de l’activit´e pr`edatrice vis-`a-vis de n´ematodes trichostrongylides apr`es ingestion par des ovins de trois hypomyc`es predateurs, Vol. 300. Compte Rendu de l’Acad´emie des Sciences Paris III, pp. 525–528. Knox, M.R., 1996. Integrated control programs using medicated blocks. In: Le Jambre, L.F., Knox, M.R. (Eds.), Proceedings of the ACIAR on Sustainable Parasite Control in Small Ruminants (Proceedings No. 74). Canberra, Australia, pp. 141–145. Larsen, M., Wolstrup, J., Henriksen, S.A., Grønvold, J., Nansen, P., 1992. In vivo passage through calves of nematophagous fungi selected for biocontrol of parasitic nematodes. J. Helminthol. 66, 137–141. Larsen, M., Faedo, M., Waller, P.J., 1994. The potential of nematophagous fungi to control the free-living stages of nematode parasites of sheep: survey for the presence of fungi in fresh faeces of grazing livestock in Australia. Vet. Parasitol. 53, 275–281. Larsen, M., Nansen, P., Grondahl, C., Thamsborg, S.M., Grønvold, J., Wolstrup, J., Henriksen, S.A., Monrad, J., 1996. The capacity of the fungus Duddingtonia flagrans to prevent strongyle infections in foals on pasture. Vet. Parasitol. 113, 1–6. Larsen, M., Nansen, P., Grønvold, J., Wolstrup, J., Henriksen, S.A., 1997. Biological control of gastro-intestinal nematodes-facts, future or fiction. Vet. Parasitol. 72, 479–492.
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