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Trapping efficacy of Duddingtonia flagrans against Haemonchus contortus at temperatures existing at lambing in Australia
Veterinary Parasitology 146 (2007) 83–89 www.elsevier.com/locate/vetpar
Trapping efficacy of Duddingtonia flagrans against Haemonchus contortus at temperatures existing at lambing in Australia L.P. Kahn *, T.M. Norman, S.W. Walkden-Brown, A. Crampton 1, L.J. O’Connor Centre for Animal Health and Welfare, School of Rural Science and Agriculture, University of New England, Armidale, NSW 2351, Australia Received 7 August 2006; received in revised form 15 January 2007; accepted 7 February 2007
Abstract The aim of this study was to determine the trapping efficacy of Duddingtonia flagrans against Haemonchus contortus at the temperature ranges experienced around lambing in the major sheep producing regions of Australia. Faeces were collected from Merino wethers, maintained in an animal house and which had received either D. flagrans chlamydospores for a 6-day period (DF) or not (NIL). Faeces were incubated at one of four daily temperature regimens which were composed of hourly steps to provide 6– 19 8C, 9–25 8C, 14–34 8C and 14–39 8C to mimic normal diurnal air temperature variation. Enumeration of the number of preinfective and infective larvae that had migrated from or remained in faecal pellets was used to calculate percentage recovery and trapping efficacy of D. flagrans. Recovery of H. contortus larvae of both stages was significantly lower in DF faeces but the magnitude of the effect was considerably greater for infective larvae. Mean recovery of infective larvae from NIL and DF faeces was 10.6 and 0.4%, respectively, indicating a mean trapping efficacy of 96.4%. The lowest trapping efficacy (80.7%) was observed at 6–19 8C but total recovery of infective larvae, from DF faeces, was greatest at the two highest temperature regimens, although still less than 0.9%. The results of this study indicate that typical Australian lambing temperatures should not be a barrier to the use of D. flagrans as an effective biocontrol of H. contortus in Australia. # 2007 Elsevier B.V. All rights reserved. Keywords: Duddingtonia flagrans; Haemonchus contortus; Sheep; Temperature
1. Introduction Parasitism from gastrointestinal nematodes is the major health cost to the Australian sheep industry (McLeod, 1995) and in regions of eastern Australia with summer-dominant rainfall, sporadic but significant losses of sheep from haemonchosis compound this cost. Underlying the significance of gastrointestinal parasitism has been the increasing severity of resistance
* Corresponding author. Tel.: +61 2 67732997; fax: +61 2 67733922. E-mail address: [email protected] (L.P. Kahn). 1 Present address: School of Biomedical Science, Charles Sturt University, Wagga Wagga, NSW 2650, Australia. 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.02.004
by gastrointestinal parasites to most anthelmintic classes currently available (Besier and Love, 2003). Control of Haemonchus contortus through the use of the persistent drug, closantel, is now limited to less then 30% of properties (Love et al., 1998). Recently, Bailey and Nielsen (2005) reported that 41% of properties that had administered moxidectin to sheep had positive H. contortus counts within the combined period of claimed persistence and prepatency. There are a number of non-chemotherapeutic strategies that can reduce the mortality and productive consequences of H. contortus infections including sheep-cattle alternation (Southcott and Barger, 1975), grazing management (Healey et al., 2004), genetic selection (Woolaston et al., 1990) and host nutrition
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(Kahn, 2003). Of these approaches, only sheep-cattle alternation and grazing management offer scope, in the short-term, for reducing the level of H. contortus larvae on pasture but industry adoption has been hampered by the consequent increased complexity of the grazing system. Predation of the free-living stages of gastrointestinal nematodes by various fungal genera with nematophagous activity has been demonstrated (Larsen et al., 1994; Hay et al., 1997). Of these, Duddingtonia flagrans has been reported to offer most promise (Larsen et al., 1998). Daily administration of D. flagrans chlamydospores to grazing sheep has been demonstrated to result in modest (Knox and Faedo, 2001) or large reductions in H. contortus burdens (Fontenot et al., 2003) and worm egg count (Chandrawathani et al., 2004). In contrast, Eysker and Bakker (in press) reported that while the provision of D. flagrans reduced burdens of H. contortus, it failed to reduce pasture infectivity to safe levels as assessed from the development of haemonchosis. The role that ambient temperature may play in accounting for some of this variation in claimed efficacy of D. flagrans has not been fully explored. Grønvold et al. (1996) demonstrated that the rate of growth and trap formation of D. flagrans increased with temperature above 5 8C; attained a maximum at a constant 30 8C and declined sharply at greater temperatures. However, Paraud et al. (2006) reported that the efficacy (as assessed from the reduction in the recovery of thirdstage larvae) of D. flagrans against H. contortus was greater at a constant 21 8C (efficacy = 100%) than at 28 8C (efficacy = 89%). In addition to the absolute effect of temperature, the effect of temperature fluctuation on trapping efficacy against Cooperia oncophora has been demonstrated to be temperaturedependent (Ferna´ndez et al., 1999). A better definition of the trapping efficacy of D. flagrans against H. contortus third-stage infective larvae under temperature regimens that reflect natural diurnal variation is still required. This information is important for devising the best use of D. flagrans in integrated parasite control programs, and particularly so for countries, such as Australia, where temperature varies considerably throughout the major sheep producing zones. Waller et al. (2004) suggest that the obvious time to deploy biocontrol is with the lambing ewe which is the major source of pasture contamination (O’Sullivan and Donald, 1970). For these reasons the aim of this study was to determine the trapping efficacy of D. flagrans against H. contortus at the temperature ranges experienced
around lambing in the major sheep producing regions of Australia. 2. Materials and methods 2.1. Animals and conditions Four Merino wethers (6 months of age) that had been born and grazed on pasture were used in the experiment. The mean (S.D.) live weight when animals entered the animal house was 16.4 2.77 kg. Animals were treated to remove existing gastrointestinal nematode infections (abamectin 0.2 mg/kg live weight; albendazole oxide 3.4 mg/kg live weight in combination with levamisole hydrochloride 7.0 mg/kg live weight and naphthalophos 30.0 mg/kg live weight) upon entering the animal house. Animals were maintained in individual pens with continuous access to water and fed once daily a ration of 700 g/day containing 0.25 oaten chaff (Avena sativa) and 0.75 lucerne chaff (Medicago sativa). 2.2. Experimental design and infection details The effectiveness of anthelmentic treatment was confirmed 10 days later when all animals had negative faecal worm egg count. Three days later (day 1), all animals received an oral dose of 350 Haemonchus contortus L3 (Kirby strain; CSIRO Livestock Industries, Armidale) per kg live weight followed by 500 L3 on days 3 and 5 and 200 L3 on 3 days each week thereafter. Oral infections ceased on day 24 when mean worm egg count was 21,255 epg. Animals were then allocated at random to biocontrol treatment, such that two animals (DF) received a daily intake of 500,000 Duddingtonia flagrans chlamydospores per kg live weight (spore density of 1 107 /g material; Chr Hansen, Denmark) and the remaining two animals (NIL) received no biocontrol. The D. flagrans chlamydospores were stored at 4 8C and each day the required mass of material for each animal was removed and thoroughly mixed with 100 g of the daily ration and 10 g of water. From days 27 to 32 and 40 to 45 inclusive, all animals were offered the 100 g ration D. flagrans chlamydospores and after consumption were offered the remaining 600 g of ration, of which there were no refusals. 2.3. Animal sampling Faecal collection bags were fitted to all animals at 16:00 h on day 32 and removed at 9:00 h on day 33. This process was repeated on day 45.
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2.4. Faecal culture Faeces were removed from collection bags and thoroughly mixed within biocontrol treatment. Two 30 g faecal subsamples were taken from both treatments, each blended, diluted with 150 ml water and mixed to form a homogenous slurry. Within each subsample, 5 0.140 ml aliquots were removed through a 600 mm sieve and each combined with 0.560 ml saturated salt (s.g. = 1.2 g/ml) in a ruled glass chamber (Universal). Worm egg count was calculated from 1 egg = 60 epg. A further faecal subsample (approx. 10 g) was taken to determine faecal dry matter content by drying at 105 8C for 4 days as faecal dry matter is known to influence H. contortus development (Rossanigo and Gruner, 1995) From the remaining faeces, 5 g intact subsamples (i.e. intact faecal pellets) were taken and placed in culture containers. The culture containers were made of an 800 mm length of PVC pipe (50 mm diameter) with a layer of nylon mesh (200 mm weave) located midway along the length. Culture containers were inserted into plastic vessels (65 mm diameter 95 mm height) filled with deionised water to the height of the mesh. Faecal subsamples were placed in the middle of the mesh layer and culture containers placed in either of two programmable incubators (MIR 253, Sanyo Electric Biomedical Co., Ltd.) to constitute a factorial design with two levels of biocontrol, two periods of incubation, two shelf positions within the cabinet (upper and lower) and two replicates (N = 16 per incubator). Incubators were programmed to cycle through one of four daily temperature regimens, with hourly steps, as detailed in Fig. 1. An error in the programming of temperature steps resulted in the temperature regimens being based on a 20 h and not a 24 h cycle. The temperature regimens used for faecal samples obtained from the first collection (i.e. day 32) were 6–19 8C and
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14–34 8C and from the second collection (i.e. day 45) were 9–25 8C and 14–39 8C. Incubation at cool and warm temperature regimens was conducted at both collections to minimise the confounding effect of time and collection period. The recorded mean values of the four temperature regimens (lowest to highest) were 12.2, 17.1, 23.3 and 27.1 8C, respectively. These temperatures regimens were chosen as being representative of lambing times in the major sheep producing regions of Australia. The level of water in the culture containers was checked on a daily basis, and adjusted if required, to ensure the level was equivalent to the nylon mesh. Within each incubator, temperature was recorded halfhourly (Tinytag, Gemini Data Loggers) and evaporation rate recorded weekly. Evaporation rate (mm/day) was calculated from loss of water (ml) in six containers (63 mm diameter, containing 150 ml water). Half of the culture containers were removed from the incubators after 7 days for enumeration of larvae and the remainder after 14 days. Faecal material was removed from the culture containers, weighed, deionised water added to achieve a six-fold dilution and mixed to form homogenous slurry. Duplicate aliquots (60 mL) were removed through a 600 mm sieve, each combined with 180 mL water and four drops of Lugol’s iodine on a ruled glass slide. The number of preinfective and infective larvae was enumerated at 40 magnification. Faecal dry matter content was estimated by dividing starting faecal dry mass by faecal fresh mass at harvest and assuming no losses of faeces into the culture container. Water from the culture containers (150 mL) was removed and stored for 18 h at room temperature to allow sedimentation of larvae. Supernatant was removed, the remaining water (40 mL) weighed, mixed and duplicate aliquots (200 mL) taken for enumeration of the number of preinfective and infective larvae at 40 magnification. The number of preinfective larvae that emerged from the faecal pellet and were collected in water may be an artifact of the culture procedure, which provided contact between the pellet and water. 2.5. Statistical analysis
Fig. 1. Temperature (8C) profiles over a typical 40 h period within each temperature regimen recorded from the incubators.
The number of H. contortus preinfective and infective larvae enumerated after incubation was expressed as a percentage of the number of H. contortus eggs in the original faecal deposition. Data were analysed by fitting a General Linear Model (SAS Institute, 2003) followed by analysis of variance. The
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fixed effects fitted in the model were biocontrol treatment, temperature regimen, length of incubation (day 7 or 14), shelf, replicate and all two-way interactions. Effects, which failed to reach statistical significance, were removed from the analysis. The effect of length of incubation (day) and the interaction between day and temperature regimen will be reported elsewhere as these data were not central to testing the effect of ambient temperature on trapping efficacy of D. flagrans against H. contortus. Data that were not normally distributed (i.e. Shapiro–Wilks P 0.05) were subjected to transformation (cube-root) prior to analysis. The effectiveness of the transformation was confirmed on the transformed data and on the model residuals by reference to the Shapiro–Wilks statistic. Back transformed least squares mean 68% confidence intervals (c.i.) are presented in the results. These confidence intervals give a close approximation to standard errors, which cannot be backtransformed as they are asymmetric about the mean.
and 14–34 8C and 24.6% (NIL) and 26.6% (DF) for 9– 25 8C and 14–39 8C. 3.2. Recovery from faeces Significantly lower (P < 0.001) recovery of infective but not preinfective larvae was obtained from DF faecal material. Recovery of infective larvae from NIL and DF faeces was 1.57% and 0.11%, respectively. Recovery of H. contortus preinfective larvae remaining in faecal material was lowest (P < 0.001) at the two highest temperature regimens. The interaction between the effects of temperature and biocontrol was significant for recovery of preinfective larvae (P < 0.05) (Table 1). The interaction arose primarily because maximum recovery of preinfective larvae from NIL faeces was achieved at 6–19 8C and declined as temperature increased. In contrast, recovery of preinfective larvae from DF faeces did not differ between the two lowest temperature regimens but rose as maximum temperature increased from 34 to 39 8C.
3. Results 3.3. Recovery from water 3.1. Culture and faecal conditions Daily evaporation rates for the four temperature regimens (i.e. 6–19 8C, 9–25 8C, 14–34 8C and 14– 39 8C) were 0.38, 0.72, 1.17 and 1.77 mm/day, respectively. Faecal dry matter (%) and WEC at the start of the first and second incubation periods were 33.5% and 16,398 epg (NIL) and 32.6% and 20,856 epg (DF) (temperatures 6–19 8C and 14–34 8C) and 39.0% and 25,296 epg (NIL) and 42.2% and 24,792 epg (DF) (temperatures 9–25 8C and 14–39 8C). Mean faecal dry matter (%) at the end of the first and second incubation periods was 21.9% (NIL) and 20.5% (DF) for 6–19 8C
Recovery of H. contortus preinfective (P < 0.01) and infective (P < 0.001) stages that emerged from faeces was greatly reduced in the presence of D. flagrans. Averaged across temperature regimens, recovery of preinfective larvae was 10.03% and 6.74% and infective larvae 6.02% and 0.07% for NIL and DF, respectively. Recovery of preinfective larvae that had emerged from faecal material was affected (P < 0.001) by temperature regimen and was greatest at 9–25 8C (21.77%) and least at the two highest (mean of 2.94%) temperatures. In contrast, recovery of infective larvae was greatest (P < 0.001) at the two highest
Table 1 Recovery (%) of Haemonchus contortus preinfective and infective larvae remaining in faecal material obtained from sheep fed Duddingtonia flagrans chlamydospores (DF) or no biocontrol (NIL)a Biocontrol
Temperature
Preinfective larvae Lower c.i.
Infective larvae
lsmean (%) a
Upper c.i.
Lower c.i.
lsmean (%)
Upper c.i.
NIL
6–19 9–25 14–34 14–39
3.69 2.37 1.22 1.01
22.5 13.0b 5.0 c 3.8 cd
4.15 2.70 1.46 1.23
0.48 1.12 0.52 0.47
1.0 3.5 1.2 1.0
0.71 1.41 0.73 0.67
DF
6–19 9–25 14–34 14–39
2.55 3.13 0.54 1.30
14.5ab 19.6ab 1.5 d 5.5 c
2.89 3.51 0.70 1.55
0.06 0.04 0.06 0.18
0.0 0.0 0.0 0.3
0.15 0.11 0.14 0.31
Means within columns with different superscripts differ significantly (P < 0.05). a Values are averaged over length of incubation (days 7 and 14).
L.P. Kahn et al. / Veterinary Parasitology 146 (2007) 83–89
Fig. 2. Recovery (backtransformed mean 68% c.i.) of Haemonchus contortus preinfective and infective larval stages that had emerged from faeces collected from sheep fed Duddingtonia flagrans chlamydospores (DF) or that received no biocontrol (NIL) and incubated for 7 or 14 days. Values are averaged over temperature regimen.
temperatures. The effectiveness of D. flagrans to reduce recovery of preinfective and infective larval stages that had emerged from faeces differed (P < 0.06 preinfective; P < 0.001 infective) from day 7 to day 14 (Fig. 2). Recovery of preinfective stages was unaffected by D. flagrans at day 7 but was reduced by 50.1% at day 14. In contrast, D. flagrans reduced recovery of infective stages at both day 7 (97.2%) and day 14 (99.5%). 3.4. Total recovery Total recovery was calculated from the addition of recovery of H. contortus stages remaining in, and emerged from, faecal material. Total recovery of preinfective (P < 0.05) and infective stages (P < 0.001) was reduced from DF faeces (Fig. 3). Biocontrol with D. flagrans reduced recovery of preinfective larvae by 20.7% and infective larvae by 96.4%. The effectiveness of D. flagrans to alter recovery of H. contortus preinfective (P < 0.001) and infective (P < 0.001) larvae varied with temperature regimen
Fig. 3. Total recovery (backtransformed mean 68% c.i.) of Haemonchus contortus preinfective and infective larvae from faeces collected from sheep fed Duddingtonia flagrans chlamydospores (DF) or that received no biocontrol (NIL). Values are averaged over length of incubation (days 7 and 14) and temperature regimen.
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Fig. 4. Total recovery (backtransformed mean 68% c.i.) of Haemonchus contortus preinfective and infective larvae from faeces collected from sheep fed Duddingtonia flagrans chlamydospores (DF) or that received no biocontrol (NIL) and incubated at a range of temperature regimens. Values are averaged over length of incubation (days 7 and 14). Means within larval stage with different superscripts differ significantly (P < 0.0001).
(Fig. 4). For example, total recovery of preinfective larvae from DF faeces (when compared to NIL faeces) was lower at 6–19 8C and 14–34 8C, unaffected at 14– 39 8C and greater at 9–25 8C. With infective larvae, total recovery in DF faeces at the four temperature regimens (coldest–hottest) was reduced by 80.7%, 99.1%, 97.9% and 94.5% relative to that recovered from faeces collected from sheep that did not receive biocontrol. While total recovery of infective larvae from NIL faeces increased until a maximum temperature of 34 8C no such increase was observed from DF faeces. 4. Discussion Provision of D. flagrans chlamydospores to Merino wethers led to a significant reduction in the faecal recovery of H. contortus infective larvae at temperature regimens reflective of those that may exist at lambing in the main sheep producing regions of Australia. That maximum temperature in the exposed faecal pellet is typically greater than that recorded in air (O’Connor et al., 2006) may mean that the maximum temperatures used in this study underestimate those experienced in faecal pellets deposited in the field. Mean recovery of infective larvae was reduced by D. flagrans by 96.4% but the efficacy of D. flagrans was least at the lowest temperature regimen of 6–19 8C. Nevertheless, absolute recovery of H. contortus infective larvae did not exceed 1% in the DF treatment and was greatest at the highest temperature regimen. Grønvold et al. (1996) reported maximum trap induction by D. flagrans occurred at a constant 30 8C but declined to almost negligible levels at 35 and 40 8C. In contrast to this, Paraud et al. (2006) suggested peak
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trapping efficacy occurs at lower temperatures as efficacy of D. flagrans against H. contortus larvae declined from 100% at a constant 21 8C to 89% at 28 8C. The highest temperature regimen in the present study exposed D. flagrans to temperatures that exceeded 30 8C for 8 h in a 20 h cycle, during which 35 8C was exceeded for 5 h. Recovery of infective larvae from NIL but not DF faeces declined at this highest temperature regimen highlighting the small but not statistically significant drop in D. flagrans trapping efficacy from 97.9 to 94.5%. High trapping efficacy of D. flagrans against H. contortus infective larvae at these temperature regimens may indicate that the trapping efficacy of D. flagrans may be dependent on the duration of exposure to high temperatures rather than temperature alone. There is support (Ferna´ndez et al., 1999) that a variable temperature regimen will improve trapping efficacy of D. flagrans at mean temperatures below 15 8C probably because the fluctuating regimen included periods of elevated temperature which was more conducive to fungal growth and trap formation (Grønvold et al., 1996). However, Ferna´ndez et al. (1999) also reported that a variable temperature regimen resulted in reduced D. flagrans efficacy at a mean temperature of 20 8C, which included 5 h at 35 8C. The sensitivity of D. flagrans trapping efficacy to temperature that is reported from in-vitro experiments is at odds with reports from the field. For example, Fontenot et al. (2003) investigated the effect of biocontrol with D. flagrans in ewes grazing pasture during the period June–October in subtropical Baton Rouge, Louisiana, USA (latitude = approx. 308 N). Mean daily minimum and maximum temperatures for this period are 19.7–32.7 8C producing a mean value of 26.7 8C; mean monthly rainfall is 139 mm (US Dept of Commerce, 2006). Tracer lambs which subsequently grazed the nil and D. flagrans biocontrol plots recorded worm burdens of 21,350 and 600, respectively indicating high trapping efficacy at this temperature regimen and consistent with the conclusions drawn from this study. The results of this study indicate that typical Australian lambing temperatures, with diurnal variation, should not be a barrier to the use of D. flagrans as an effective biocontrol of H. contortus. Acknowledgements We acknowledge Chr Hansen for the supply of D. flagrans chlamydospores and the experimental assistance provided by Dr. C. Scrivener, University of New
England. This study was funded by Australian woolgrowers and the Australian Government through Australian Wool Innovation’s ‘Integrated Parasite Management – sheep’ project.
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Rossanigo, C.E., Gruner, L., 1995. Moisture and temperature requirements in faeces for the development of free-living stages of gastrointestinal nematodes of sheep, cattle and deer. J. Helminthol. 69, 357–362. SAS Institute, 2003. Version 9.1 3. SAS Institute Inc., Cary, NC, USA. Southcott, W.H., Barger, I.A., 1975. Control of nematode parasites by grazing management—II. Decontamination of sheep and cattle pastures by varying periods of grazing with the alternate host. Int. J. Parasitol. 5, 45–48. US Dept of Commerce 2006. National Oceanic and Atmospheric Administration. http://www.ncdc.noaa.gov. Waller, P.J., Schwan, O., Ljungstrom, B.-L., Rydzik, A., Yeates, G.W., 2004. Evaluation of biological control of sheep parasites using Duddingtonia flagrans under commercial farming conditions on the island of Gotland Sweden. Vet. Parasitol. 126, 299–315. Woolaston, R.R., Barger, I.A., Piper, L.R., 1990. Response to helminth infection of sheep selected for resistance to Haemonchus contortus. Int. J. Parasitol. 20, 1015–1018.
Trapping efficacy of Duddingtonia flagrans against Haemonchus contortus at temperatures existing at lambing in Australia L.P. Kahn *, T.M. Norman, S.W. Walkden-Brown, A. Crampton 1, L.J. O’Connor Centre for Animal Health and Welfare, School of Rural Science and Agriculture, University of New England, Armidale, NSW 2351, Australia Received 7 August 2006; received in revised form 15 January 2007; accepted 7 February 2007
Abstract The aim of this study was to determine the trapping efficacy of Duddingtonia flagrans against Haemonchus contortus at the temperature ranges experienced around lambing in the major sheep producing regions of Australia. Faeces were collected from Merino wethers, maintained in an animal house and which had received either D. flagrans chlamydospores for a 6-day period (DF) or not (NIL). Faeces were incubated at one of four daily temperature regimens which were composed of hourly steps to provide 6– 19 8C, 9–25 8C, 14–34 8C and 14–39 8C to mimic normal diurnal air temperature variation. Enumeration of the number of preinfective and infective larvae that had migrated from or remained in faecal pellets was used to calculate percentage recovery and trapping efficacy of D. flagrans. Recovery of H. contortus larvae of both stages was significantly lower in DF faeces but the magnitude of the effect was considerably greater for infective larvae. Mean recovery of infective larvae from NIL and DF faeces was 10.6 and 0.4%, respectively, indicating a mean trapping efficacy of 96.4%. The lowest trapping efficacy (80.7%) was observed at 6–19 8C but total recovery of infective larvae, from DF faeces, was greatest at the two highest temperature regimens, although still less than 0.9%. The results of this study indicate that typical Australian lambing temperatures should not be a barrier to the use of D. flagrans as an effective biocontrol of H. contortus in Australia. # 2007 Elsevier B.V. All rights reserved. Keywords: Duddingtonia flagrans; Haemonchus contortus; Sheep; Temperature
1. Introduction Parasitism from gastrointestinal nematodes is the major health cost to the Australian sheep industry (McLeod, 1995) and in regions of eastern Australia with summer-dominant rainfall, sporadic but significant losses of sheep from haemonchosis compound this cost. Underlying the significance of gastrointestinal parasitism has been the increasing severity of resistance
* Corresponding author. Tel.: +61 2 67732997; fax: +61 2 67733922. E-mail address: [email protected] (L.P. Kahn). 1 Present address: School of Biomedical Science, Charles Sturt University, Wagga Wagga, NSW 2650, Australia. 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.02.004
by gastrointestinal parasites to most anthelmintic classes currently available (Besier and Love, 2003). Control of Haemonchus contortus through the use of the persistent drug, closantel, is now limited to less then 30% of properties (Love et al., 1998). Recently, Bailey and Nielsen (2005) reported that 41% of properties that had administered moxidectin to sheep had positive H. contortus counts within the combined period of claimed persistence and prepatency. There are a number of non-chemotherapeutic strategies that can reduce the mortality and productive consequences of H. contortus infections including sheep-cattle alternation (Southcott and Barger, 1975), grazing management (Healey et al., 2004), genetic selection (Woolaston et al., 1990) and host nutrition
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(Kahn, 2003). Of these approaches, only sheep-cattle alternation and grazing management offer scope, in the short-term, for reducing the level of H. contortus larvae on pasture but industry adoption has been hampered by the consequent increased complexity of the grazing system. Predation of the free-living stages of gastrointestinal nematodes by various fungal genera with nematophagous activity has been demonstrated (Larsen et al., 1994; Hay et al., 1997). Of these, Duddingtonia flagrans has been reported to offer most promise (Larsen et al., 1998). Daily administration of D. flagrans chlamydospores to grazing sheep has been demonstrated to result in modest (Knox and Faedo, 2001) or large reductions in H. contortus burdens (Fontenot et al., 2003) and worm egg count (Chandrawathani et al., 2004). In contrast, Eysker and Bakker (in press) reported that while the provision of D. flagrans reduced burdens of H. contortus, it failed to reduce pasture infectivity to safe levels as assessed from the development of haemonchosis. The role that ambient temperature may play in accounting for some of this variation in claimed efficacy of D. flagrans has not been fully explored. Grønvold et al. (1996) demonstrated that the rate of growth and trap formation of D. flagrans increased with temperature above 5 8C; attained a maximum at a constant 30 8C and declined sharply at greater temperatures. However, Paraud et al. (2006) reported that the efficacy (as assessed from the reduction in the recovery of thirdstage larvae) of D. flagrans against H. contortus was greater at a constant 21 8C (efficacy = 100%) than at 28 8C (efficacy = 89%). In addition to the absolute effect of temperature, the effect of temperature fluctuation on trapping efficacy against Cooperia oncophora has been demonstrated to be temperaturedependent (Ferna´ndez et al., 1999). A better definition of the trapping efficacy of D. flagrans against H. contortus third-stage infective larvae under temperature regimens that reflect natural diurnal variation is still required. This information is important for devising the best use of D. flagrans in integrated parasite control programs, and particularly so for countries, such as Australia, where temperature varies considerably throughout the major sheep producing zones. Waller et al. (2004) suggest that the obvious time to deploy biocontrol is with the lambing ewe which is the major source of pasture contamination (O’Sullivan and Donald, 1970). For these reasons the aim of this study was to determine the trapping efficacy of D. flagrans against H. contortus at the temperature ranges experienced
around lambing in the major sheep producing regions of Australia. 2. Materials and methods 2.1. Animals and conditions Four Merino wethers (6 months of age) that had been born and grazed on pasture were used in the experiment. The mean (S.D.) live weight when animals entered the animal house was 16.4 2.77 kg. Animals were treated to remove existing gastrointestinal nematode infections (abamectin 0.2 mg/kg live weight; albendazole oxide 3.4 mg/kg live weight in combination with levamisole hydrochloride 7.0 mg/kg live weight and naphthalophos 30.0 mg/kg live weight) upon entering the animal house. Animals were maintained in individual pens with continuous access to water and fed once daily a ration of 700 g/day containing 0.25 oaten chaff (Avena sativa) and 0.75 lucerne chaff (Medicago sativa). 2.2. Experimental design and infection details The effectiveness of anthelmentic treatment was confirmed 10 days later when all animals had negative faecal worm egg count. Three days later (day 1), all animals received an oral dose of 350 Haemonchus contortus L3 (Kirby strain; CSIRO Livestock Industries, Armidale) per kg live weight followed by 500 L3 on days 3 and 5 and 200 L3 on 3 days each week thereafter. Oral infections ceased on day 24 when mean worm egg count was 21,255 epg. Animals were then allocated at random to biocontrol treatment, such that two animals (DF) received a daily intake of 500,000 Duddingtonia flagrans chlamydospores per kg live weight (spore density of 1 107 /g material; Chr Hansen, Denmark) and the remaining two animals (NIL) received no biocontrol. The D. flagrans chlamydospores were stored at 4 8C and each day the required mass of material for each animal was removed and thoroughly mixed with 100 g of the daily ration and 10 g of water. From days 27 to 32 and 40 to 45 inclusive, all animals were offered the 100 g ration D. flagrans chlamydospores and after consumption were offered the remaining 600 g of ration, of which there were no refusals. 2.3. Animal sampling Faecal collection bags were fitted to all animals at 16:00 h on day 32 and removed at 9:00 h on day 33. This process was repeated on day 45.
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2.4. Faecal culture Faeces were removed from collection bags and thoroughly mixed within biocontrol treatment. Two 30 g faecal subsamples were taken from both treatments, each blended, diluted with 150 ml water and mixed to form a homogenous slurry. Within each subsample, 5 0.140 ml aliquots were removed through a 600 mm sieve and each combined with 0.560 ml saturated salt (s.g. = 1.2 g/ml) in a ruled glass chamber (Universal). Worm egg count was calculated from 1 egg = 60 epg. A further faecal subsample (approx. 10 g) was taken to determine faecal dry matter content by drying at 105 8C for 4 days as faecal dry matter is known to influence H. contortus development (Rossanigo and Gruner, 1995) From the remaining faeces, 5 g intact subsamples (i.e. intact faecal pellets) were taken and placed in culture containers. The culture containers were made of an 800 mm length of PVC pipe (50 mm diameter) with a layer of nylon mesh (200 mm weave) located midway along the length. Culture containers were inserted into plastic vessels (65 mm diameter 95 mm height) filled with deionised water to the height of the mesh. Faecal subsamples were placed in the middle of the mesh layer and culture containers placed in either of two programmable incubators (MIR 253, Sanyo Electric Biomedical Co., Ltd.) to constitute a factorial design with two levels of biocontrol, two periods of incubation, two shelf positions within the cabinet (upper and lower) and two replicates (N = 16 per incubator). Incubators were programmed to cycle through one of four daily temperature regimens, with hourly steps, as detailed in Fig. 1. An error in the programming of temperature steps resulted in the temperature regimens being based on a 20 h and not a 24 h cycle. The temperature regimens used for faecal samples obtained from the first collection (i.e. day 32) were 6–19 8C and
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14–34 8C and from the second collection (i.e. day 45) were 9–25 8C and 14–39 8C. Incubation at cool and warm temperature regimens was conducted at both collections to minimise the confounding effect of time and collection period. The recorded mean values of the four temperature regimens (lowest to highest) were 12.2, 17.1, 23.3 and 27.1 8C, respectively. These temperatures regimens were chosen as being representative of lambing times in the major sheep producing regions of Australia. The level of water in the culture containers was checked on a daily basis, and adjusted if required, to ensure the level was equivalent to the nylon mesh. Within each incubator, temperature was recorded halfhourly (Tinytag, Gemini Data Loggers) and evaporation rate recorded weekly. Evaporation rate (mm/day) was calculated from loss of water (ml) in six containers (63 mm diameter, containing 150 ml water). Half of the culture containers were removed from the incubators after 7 days for enumeration of larvae and the remainder after 14 days. Faecal material was removed from the culture containers, weighed, deionised water added to achieve a six-fold dilution and mixed to form homogenous slurry. Duplicate aliquots (60 mL) were removed through a 600 mm sieve, each combined with 180 mL water and four drops of Lugol’s iodine on a ruled glass slide. The number of preinfective and infective larvae was enumerated at 40 magnification. Faecal dry matter content was estimated by dividing starting faecal dry mass by faecal fresh mass at harvest and assuming no losses of faeces into the culture container. Water from the culture containers (150 mL) was removed and stored for 18 h at room temperature to allow sedimentation of larvae. Supernatant was removed, the remaining water (40 mL) weighed, mixed and duplicate aliquots (200 mL) taken for enumeration of the number of preinfective and infective larvae at 40 magnification. The number of preinfective larvae that emerged from the faecal pellet and were collected in water may be an artifact of the culture procedure, which provided contact between the pellet and water. 2.5. Statistical analysis
Fig. 1. Temperature (8C) profiles over a typical 40 h period within each temperature regimen recorded from the incubators.
The number of H. contortus preinfective and infective larvae enumerated after incubation was expressed as a percentage of the number of H. contortus eggs in the original faecal deposition. Data were analysed by fitting a General Linear Model (SAS Institute, 2003) followed by analysis of variance. The
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fixed effects fitted in the model were biocontrol treatment, temperature regimen, length of incubation (day 7 or 14), shelf, replicate and all two-way interactions. Effects, which failed to reach statistical significance, were removed from the analysis. The effect of length of incubation (day) and the interaction between day and temperature regimen will be reported elsewhere as these data were not central to testing the effect of ambient temperature on trapping efficacy of D. flagrans against H. contortus. Data that were not normally distributed (i.e. Shapiro–Wilks P 0.05) were subjected to transformation (cube-root) prior to analysis. The effectiveness of the transformation was confirmed on the transformed data and on the model residuals by reference to the Shapiro–Wilks statistic. Back transformed least squares mean 68% confidence intervals (c.i.) are presented in the results. These confidence intervals give a close approximation to standard errors, which cannot be backtransformed as they are asymmetric about the mean.
and 14–34 8C and 24.6% (NIL) and 26.6% (DF) for 9– 25 8C and 14–39 8C. 3.2. Recovery from faeces Significantly lower (P < 0.001) recovery of infective but not preinfective larvae was obtained from DF faecal material. Recovery of infective larvae from NIL and DF faeces was 1.57% and 0.11%, respectively. Recovery of H. contortus preinfective larvae remaining in faecal material was lowest (P < 0.001) at the two highest temperature regimens. The interaction between the effects of temperature and biocontrol was significant for recovery of preinfective larvae (P < 0.05) (Table 1). The interaction arose primarily because maximum recovery of preinfective larvae from NIL faeces was achieved at 6–19 8C and declined as temperature increased. In contrast, recovery of preinfective larvae from DF faeces did not differ between the two lowest temperature regimens but rose as maximum temperature increased from 34 to 39 8C.
3. Results 3.3. Recovery from water 3.1. Culture and faecal conditions Daily evaporation rates for the four temperature regimens (i.e. 6–19 8C, 9–25 8C, 14–34 8C and 14– 39 8C) were 0.38, 0.72, 1.17 and 1.77 mm/day, respectively. Faecal dry matter (%) and WEC at the start of the first and second incubation periods were 33.5% and 16,398 epg (NIL) and 32.6% and 20,856 epg (DF) (temperatures 6–19 8C and 14–34 8C) and 39.0% and 25,296 epg (NIL) and 42.2% and 24,792 epg (DF) (temperatures 9–25 8C and 14–39 8C). Mean faecal dry matter (%) at the end of the first and second incubation periods was 21.9% (NIL) and 20.5% (DF) for 6–19 8C
Recovery of H. contortus preinfective (P < 0.01) and infective (P < 0.001) stages that emerged from faeces was greatly reduced in the presence of D. flagrans. Averaged across temperature regimens, recovery of preinfective larvae was 10.03% and 6.74% and infective larvae 6.02% and 0.07% for NIL and DF, respectively. Recovery of preinfective larvae that had emerged from faecal material was affected (P < 0.001) by temperature regimen and was greatest at 9–25 8C (21.77%) and least at the two highest (mean of 2.94%) temperatures. In contrast, recovery of infective larvae was greatest (P < 0.001) at the two highest
Table 1 Recovery (%) of Haemonchus contortus preinfective and infective larvae remaining in faecal material obtained from sheep fed Duddingtonia flagrans chlamydospores (DF) or no biocontrol (NIL)a Biocontrol
Temperature
Preinfective larvae Lower c.i.
Infective larvae
lsmean (%) a
Upper c.i.
Lower c.i.
lsmean (%)
Upper c.i.
NIL
6–19 9–25 14–34 14–39
3.69 2.37 1.22 1.01
22.5 13.0b 5.0 c 3.8 cd
4.15 2.70 1.46 1.23
0.48 1.12 0.52 0.47
1.0 3.5 1.2 1.0
0.71 1.41 0.73 0.67
DF
6–19 9–25 14–34 14–39
2.55 3.13 0.54 1.30
14.5ab 19.6ab 1.5 d 5.5 c
2.89 3.51 0.70 1.55
0.06 0.04 0.06 0.18
0.0 0.0 0.0 0.3
0.15 0.11 0.14 0.31
Means within columns with different superscripts differ significantly (P < 0.05). a Values are averaged over length of incubation (days 7 and 14).
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Fig. 2. Recovery (backtransformed mean 68% c.i.) of Haemonchus contortus preinfective and infective larval stages that had emerged from faeces collected from sheep fed Duddingtonia flagrans chlamydospores (DF) or that received no biocontrol (NIL) and incubated for 7 or 14 days. Values are averaged over temperature regimen.
temperatures. The effectiveness of D. flagrans to reduce recovery of preinfective and infective larval stages that had emerged from faeces differed (P < 0.06 preinfective; P < 0.001 infective) from day 7 to day 14 (Fig. 2). Recovery of preinfective stages was unaffected by D. flagrans at day 7 but was reduced by 50.1% at day 14. In contrast, D. flagrans reduced recovery of infective stages at both day 7 (97.2%) and day 14 (99.5%). 3.4. Total recovery Total recovery was calculated from the addition of recovery of H. contortus stages remaining in, and emerged from, faecal material. Total recovery of preinfective (P < 0.05) and infective stages (P < 0.001) was reduced from DF faeces (Fig. 3). Biocontrol with D. flagrans reduced recovery of preinfective larvae by 20.7% and infective larvae by 96.4%. The effectiveness of D. flagrans to alter recovery of H. contortus preinfective (P < 0.001) and infective (P < 0.001) larvae varied with temperature regimen
Fig. 3. Total recovery (backtransformed mean 68% c.i.) of Haemonchus contortus preinfective and infective larvae from faeces collected from sheep fed Duddingtonia flagrans chlamydospores (DF) or that received no biocontrol (NIL). Values are averaged over length of incubation (days 7 and 14) and temperature regimen.
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Fig. 4. Total recovery (backtransformed mean 68% c.i.) of Haemonchus contortus preinfective and infective larvae from faeces collected from sheep fed Duddingtonia flagrans chlamydospores (DF) or that received no biocontrol (NIL) and incubated at a range of temperature regimens. Values are averaged over length of incubation (days 7 and 14). Means within larval stage with different superscripts differ significantly (P < 0.0001).
(Fig. 4). For example, total recovery of preinfective larvae from DF faeces (when compared to NIL faeces) was lower at 6–19 8C and 14–34 8C, unaffected at 14– 39 8C and greater at 9–25 8C. With infective larvae, total recovery in DF faeces at the four temperature regimens (coldest–hottest) was reduced by 80.7%, 99.1%, 97.9% and 94.5% relative to that recovered from faeces collected from sheep that did not receive biocontrol. While total recovery of infective larvae from NIL faeces increased until a maximum temperature of 34 8C no such increase was observed from DF faeces. 4. Discussion Provision of D. flagrans chlamydospores to Merino wethers led to a significant reduction in the faecal recovery of H. contortus infective larvae at temperature regimens reflective of those that may exist at lambing in the main sheep producing regions of Australia. That maximum temperature in the exposed faecal pellet is typically greater than that recorded in air (O’Connor et al., 2006) may mean that the maximum temperatures used in this study underestimate those experienced in faecal pellets deposited in the field. Mean recovery of infective larvae was reduced by D. flagrans by 96.4% but the efficacy of D. flagrans was least at the lowest temperature regimen of 6–19 8C. Nevertheless, absolute recovery of H. contortus infective larvae did not exceed 1% in the DF treatment and was greatest at the highest temperature regimen. Grønvold et al. (1996) reported maximum trap induction by D. flagrans occurred at a constant 30 8C but declined to almost negligible levels at 35 and 40 8C. In contrast to this, Paraud et al. (2006) suggested peak
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trapping efficacy occurs at lower temperatures as efficacy of D. flagrans against H. contortus larvae declined from 100% at a constant 21 8C to 89% at 28 8C. The highest temperature regimen in the present study exposed D. flagrans to temperatures that exceeded 30 8C for 8 h in a 20 h cycle, during which 35 8C was exceeded for 5 h. Recovery of infective larvae from NIL but not DF faeces declined at this highest temperature regimen highlighting the small but not statistically significant drop in D. flagrans trapping efficacy from 97.9 to 94.5%. High trapping efficacy of D. flagrans against H. contortus infective larvae at these temperature regimens may indicate that the trapping efficacy of D. flagrans may be dependent on the duration of exposure to high temperatures rather than temperature alone. There is support (Ferna´ndez et al., 1999) that a variable temperature regimen will improve trapping efficacy of D. flagrans at mean temperatures below 15 8C probably because the fluctuating regimen included periods of elevated temperature which was more conducive to fungal growth and trap formation (Grønvold et al., 1996). However, Ferna´ndez et al. (1999) also reported that a variable temperature regimen resulted in reduced D. flagrans efficacy at a mean temperature of 20 8C, which included 5 h at 35 8C. The sensitivity of D. flagrans trapping efficacy to temperature that is reported from in-vitro experiments is at odds with reports from the field. For example, Fontenot et al. (2003) investigated the effect of biocontrol with D. flagrans in ewes grazing pasture during the period June–October in subtropical Baton Rouge, Louisiana, USA (latitude = approx. 308 N). Mean daily minimum and maximum temperatures for this period are 19.7–32.7 8C producing a mean value of 26.7 8C; mean monthly rainfall is 139 mm (US Dept of Commerce, 2006). Tracer lambs which subsequently grazed the nil and D. flagrans biocontrol plots recorded worm burdens of 21,350 and 600, respectively indicating high trapping efficacy at this temperature regimen and consistent with the conclusions drawn from this study. The results of this study indicate that typical Australian lambing temperatures, with diurnal variation, should not be a barrier to the use of D. flagrans as an effective biocontrol of H. contortus. Acknowledgements We acknowledge Chr Hansen for the supply of D. flagrans chlamydospores and the experimental assistance provided by Dr. C. Scrivener, University of New
England. This study was funded by Australian woolgrowers and the Australian Government through Australian Wool Innovation’s ‘Integrated Parasite Management – sheep’ project.
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