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The effect of diet fed to lambs on subsequent development of Trichostrongylus colubriformis larvae in vitro and on pasture
Veterinary Parasitology 105 (2002) 269–283
The effect of diet fed to lambs on subsequent development of Trichostrongylus colubriformis larvae in vitro and on pasture J.H. Niezen∗ , G.C. Waghorn, T. Graham, J.L. Carter, D.M. Leathwick AgResearch Grasslands, P.O. Box 11008, Palmerston North, New Zealand Received 20 August 2001; received in revised form 23 January 2002; accepted 6 February 2002
Abstract Contrasting herbage diets were fed to lambs to evaluate their effect on subsequent development of Trichostrongylus colubriformis larvae in faeces and on pasture. The diets had either no condensed tannin (CT), lucerne (Medicago sativa cv. Otaio), white clover (Trifolium repens cv. Tahora), or had moderate to high concentrations of CT, sulla (Hedysarum coronarium cv. Grassland Aokau), Lotus corniculatus (cv. Grasslands Goldie), L. pedunculatus (cv. Grassland Maku), Dorycnium pentophyllum, and Dorycnium rectum. Trials were carried out in summer (warm) and in autumn (cool and moist). In summer, egg viability was evaluated in vitro with egg hatch and larval development assays. In both seasons faeces were placed on pasture to compare recovery of eggs and larvae from faeces and larvae from herbage on the high and low fertility farmlets on the AgResearch Ballantrae Hill Country Research Station. D. rectum and D. pentophyllum diets decreased (P < 0.01) egg hatching and larval development in laboratory assays relative to other diets. In summer, the number of larvae recovered from faeces placed on pasture was far greater (P < 0.001) if the lambs had been fed lucerne than any other diet, whereas recovery was always lowest from faeces of sheep fed D. rectum and D. pentophyllum. Although dietary differences were lower in autumn than in summer, larval recoveries were lower (P < 0.05) from faeces of lambs fed D. rectum and L. corniculatus than from white clover, lucerne and sulla diets. This study indicates that the diet of the host can have a significant impact on egg hatching and the subsequent development of T. colubriformis larvae in the laboratory and in the field. In particular, D. rectum consistently reduced T. colubriformis development. Effects measured in vitro generally under-estimated effects measured under field conditions. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Sheep-nematoda; Trichostrongylus colubriformis; Larval development; Condensed tannins; Feeding and nutrition ∗ Corresponding author. Present address: International Livestock Research Institute (ILRI), C/o IITA, PMB 5320, Oyo Rd., Ibadan, Nigeria. Tel.: +234-2-241-2626; fax: +234-2-241-2221. E-mail address: [email protected] (J.H. Niezen).
0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 2 ) 0 0 0 2 5 - 0
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1. Introduction With increasing levels of anthelmintic resistance in New Zealand (McKenna et al., 1995) alternative methods of gastrointestinal parasite control must be explored. One possibility is to change the composition of the diet of the grazing ruminant by grazing different pasture species. This can increase the plane of nutrition of the host which, in turn, can increase immunity and reduce gastrointestinal parasite establishment (Abbott and Holmes, 1990; Roberts and Adams, 1990; Coop et al., 1995; van Houtert et al., 1995) as well as reduce the pathology of parasite infection (Bown et al., 1986). Changing the pastures grazed by ruminants can also introduce into the diet plant secondary compounds which may be detrimental to gastrointestinal parasites. Previous studies have shown that feeding forages which contain condensed tannins (CTs) can lower gastrointestinal parasite establishment in lambs (Niezen et al., 1995, 1998c) and can reduce established gastrointestinal parasite burdens (Niezen et al., 1998b), although the mechanisms which cause these effects remain unclear. It has also been shown that CT are toxic to Trichostrongylus colubriformis and Dictyocaulus viviparus larvae in in vitro assays (Molan et al., 2000a,b). Because CTs are not absorbed from the gastrointestinal tract of ruminants (Terrill et al., 1994) and thus remain in the faeces of animals fed forages containing CT, the possibility remains that they may directly impair gastrointestinal parasite larval development within faeces. This study investigated this possibility by comparing a range of dietary species, particularly those containing CT on the development of T. colubriformis larvae in egg hatch and larval development assays (LDAs) and on pasture plots.
2. Animals and forages 2.1. Summer Forty Romney ram lambs, 4–5 months of age (average liveweight 28.8 kg) were orally treated with ivermectin (Ivomec® Merial) at twice the manufacturer’s recommended dose rate and grazed on pasture which had been grazed only by cattle for the previous year. Five days after drenching, each lamb received a single dose of 20,000 T. colubriformis third stage larvae (L3 ). Three weeks later the lambs were housed indoors in individual crates and restrictively randomised, based on faecal nematode egg count (FEC), to one of the six dietary treatments with six lambs per treatment. The four lambs with the lowest FEC were not used in the trial. The six treatments comprised ad libitum feeding of either, Lotus corniculatus (cv. Grasslands Goldie), L. pedunculatus (cv. Grasslands Maku), Dorycnium pentophyllum, Dorycnium rectum, white clover (Trifolium repens cv. Grasslands Tahora), or lucerne (Medicago sativa cv. Otaio). Each forage was fed for 7 days. Daily dry matter intakes (DMIs) were measured over the final 4 days when faeces were also collected. Dry matter (DM) concentrations in feeds (duplicates) and refusals were determined by drying at 95 ◦ C for 24 h and samples of feeds were bulked over the final 4 days for chemical analyses. All forages were cut daily from vegetative, monospecific stands which had not been grazed by sheep for the previous year. Lucerne was lush and pre-flowering and the white
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clover contained small proportions of dead leaf because it was relatively mature when harvested. Some flowers were present in the Lotus spp. but the stems were palatable and eaten by sheep. D. rectum is a herbaceous shrub and 30–45 cm spikes were given to sheep which selected leaf from stem. D. pentophyllum was mature and flowering and the lambs consumed all parts of the plant. Parasite development was assessed by egg hatch assay (EHA), LDA and larval development on pasture over 128 days commencing mid-January (see below). 2.2. Autumn Thirty Romney ram lambs, 7–8 months of age, were treated as described above. Diets fed were L. corniculatus, lucerne, D. rectum, sulla (Hedysarum coronarium cv. Grasslands Aokau) or white clover. All the forages were lush when fed and all lambs were offered 1000 g DM/day. Parasite development was assessed on pasture only. Faeces were deposited on field plots on 14 May and were collected until day 64. 3. Egg hatch and LDAs Four days after the lambs had been introduced to the diets, rectal faecal samples were collected and eggs extracted and placed in (EHAs) as described by Sutherland et al. (2000) and LDAs as described by Amarante et al. (1997). Egg hatch was determined by the number of eggs and larvae present in each assay cell after 24 h incubation at 25 ◦ C. Larval development was measured by counting the number of eggs, first stage (L1 ), second stage (L2 ), third stage (L3 ) and dead larvae after 7 days incubation at 25 ◦ C. For each assay three replicates of each assay were undertaken for each animal with approximately 50 eggs added to each cell. 4. Larval development on pasture 4.1. Summer Faeces were collected in 40 × 30 cm2 trays placed under the grating floor at the rear of each sheep crate. These enabled approximately 70% of total faecal output to be collected without any urine contamination. Faeces were removed from the trays at 0800, 1100, 1600 and 2000 h each day and placed into individually labelled bags, which were left open to allow aeration and held at 4 ◦ C for no longer than 2 days. Equal amounts of faeces from each lamb were pooled within herbage type. Faecal DM content was determined. Faecal egg counts were determined using a modified McMaster technique. Faeces of sheep fed D. rectum and D. pentophyllum were always pelleted, those from Lotus spp. and white clover comprised a mixture of pellets and stools, but faeces from lambs fed lucerne were soft, and liquid in consistency. Pasture comparisons of larval development were carried out on two plots on each of the low and high fertility farmlets at the Ballantrae Hill Country Research Station located 30 km east of Palmerston North. The farmlets have either received a maintenance dressing
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of fertiliser annually (low fertility) or a high allowance of fertiliser annually (high fertility) for the past 25 years (Lambert et al., 1986). Each plot was divided into one hundred and ninety-two 60×60 cm2 sub-plots mowed to a height of 5 cm 2 days prior to faecal deposition. There was a 40 cm margin between sub-plots and 45 g of faeces were deposited in the centre of each sub-plot on 18 January (day 0). Faeces were collected from sub-plots on days 1, 2, 4, 8, 16, 32, 64 and 128. On days 4, 8, 16, 32, 64, and 128 herbage was cut to ground level in an area of 0.1 m2 around the site of faecal deposition to extract and count larvae. Faeces recovered from the plots were used for FEC and T. colubriformis larvae were extracted from faeces and herbage using a modified Baermann technique and counted in 10% (2 × 5%) aliquots. 4.2. Autumn The autumn contamination followed a similar protocol to the summer but as only five herbage treatments were evaluated, each plot was divided into 160, 60 × 60 cm2 sub-plots. Faeces were deposited on the 160 plots on 14 May (day 0) and faeces collected on days 1, 2, 4, 8, 16, 32, and 64. Herbage was cut to ground level and collected on days 8, 16, 32, and 64. 5. Chemical and statistical analysis Feed samples were freeze dried and ground through a 1 mm screen prior to analysis by near infrared reflectance spectroscopy (NIRS) for main components and butanol HCl sequential extraction (Terrill et al., 1992) to determine concentrations of CTs. Dietary treatment effects on the EHA, LDA and FEC, DMI, faecal DM and larval culture comparisons were evaluated on the basis of least squared means using Genstat (version 5.42) with animal as the experimental unit. In summer, DMI, FEC and faecal DM and in autumn faecal DM were compared by Kruskal–Wallis one-way ANOVA. In autumn FEC and larval cultures were loge transformed to normalise data. Herbage DM was determined on each of 4 days with two replicates for each feed sample per day. The EHA and FEC were compared by analysis of variance while the LDA used multivariate analyses. For studies on pasture, individual plots were used as the experimental units. Dietary comparisons for larval development were made within seasons but not between seasons. In both summer and autumn the proportion of eggs remaining in faeces and L3 larvae on herbage were loge (x + 1) transformed, while L3 larvae recovered from faeces were loge (x + 0.1) transformed to normalise data. Where ANOVAs produced a significant F-value means were compared using least significant differences calculated for P < 0.05, <0.01 and <0.001. Where data had been transformed means were compared on the transformed scale. 6. Results 6.1. Diet analyses Chemical composition of diets fed in both summer and autumn are shown in Table 1. Diets varied substantially in chemical composition with crude protein (CP) content of the DM ranging from 13.1 to 29.9% and fibre concentrations from 24 to 48% of the DM. This
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Table 1 Chemical composition (% of the DM), estimates of OMD (%) and metabolisable energy (ME) content (MJ/kg DM) of L. corniculatus, L. pedunculatus, D. pentophyllum, D. rectum, white clover, lucerne and sulla diets fed to lambs in summer and autumn experimentsa CP
Lipid Ash
ADF NDF CHO OMD ME
CT concentration Free
Protein bound
Fibre bound
Total
Summer L. corniculatus L. pedunculatus D. pentophyllum D. rectum White clover Lucerne
21.9 17.5 13.1 14.4 25.6 29.9
4.1 3.6 3.8 3.6 3.3 3.6
7.1 7.0 6.4 9.4 11.3 10.5
25.7 34.0 33.6 30.7 21.6 22.7
31.6 41.0 47.5 39.8 28.5 29.8
6.9 4.8 2.8 7.2 11.1 8.6
72.6 66.1 64.7 70.2 77.7 72.0
11.6 9.8 9.6 10.5 11.6 10.7
3.6 7.7 8.2 14.2 NAb NA
1.6 4.2 2.3 2.8 NA NA
0.3 0.3 0.4 0.4 NA NA
5.5 12.2 10.9 17.4 NA NA
Autumn L. corniculatus Lucerne D. rectum Sulla White clover
22.6 25.1 15.8 16.6 25.5
3.8 3.5 3.7 2.0 3.5
8.1 9.7 6.5 10.9 11.5
20.3 25.8 18.8 24.4 20.0
26.9 33.6 23.8 24.4 24.1
12.9 9.7 22.5 18.7 14.5
71.5 69.1 78.0 77.9 81.6
10.7 10.3 11.6 11.6 12.2
0.9 NA 8.2 2.0 NA
0.6 NA 0.3 0.9 NA
0.1 NA 0.2 0.1 NA
1.6 NA 8.7 3.0 NA
a b
CP: crude protein; ADF: acid detergent fibre; NDF: neutral detergent fibre; CHO: soluble carbohydrate. Not analysed as these forages do not contain CT.
range in composition was associated with CT concentrations as high as 17.4% of the DM in D. rectum in summer, whilst lucerne and white clover do not contain CT. D. rectum had the highest CT and lowest CP content. Predicted organic matter digestibilities (OMDs) ranged from 65 to 78% (Table 1). The principal differences between diets fed in summer and autumn were the lower CT concentrations in autumn and higher quality of D. rectum on offer. 6.2. Intakes and faecal characteristics 6.2.1. Summer When offered ad libitum, DMI was lower (P < 0.01) in lambs fed D. pentophyllum and D. rectum than lambs fed the other diets (Table 2). Faecal DM was higher (P < 0.001) in lambs fed D. pentophyllum and D. rectum than in lambs fed L. pedunculatus and white clover, which was higher (P < 0.05) than in lambs fed L. corniculatus and lucerne. Lamb FEC was lower (P < 0.05) for the L. pedunculatus diet than all other diets except D. pentophyllum, which was intermediate (Table 2). Greater numbers (P < 0.05) of larvae were recovered from cultured faeces of lambs fed white clover than L. corniculatus. All other diets did not differ. 6.2.2. Autumn The older lambs used in autumn consumed virtually all material on offer with sufficient D. rectum given to ensure that 1 kg of leafy DM was consumed daily. Lamb FEC did not differ (Table 3) between diets. Faecal DM was lowest for white clover and highest for L. corniculatus (P < 0.001), Lucerne, sulla and D. rectum were intermediate and did not differ
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Table 2 Lamb DM intake (g DM/day), herbage DM (%), faecal egg count, faecal DM and larval development (larvae recovered from culturing 45 g fresh faeces at 25 ◦ C for 7 days) from lambs fed either L. corniculatus, L. pedunculatus, D. pentophyllum, D. rectum, white clover or lucerne diets in summer Herbage
DMI (g DM/day)
Herbage DM (%)
Faecal egg count (eggs/g faeces)
Faecal DM (%)
Larval recovery from culture
L. corniculatus L. pedunculatus D. pentophyllum D. rectum White clover Lucerne
1000 965 784 669 1091 1100
15.0 15.0 24.3 15.2 16.3 17.8
2467 1142 1467 2025 2058 2175
22.7 26.7 35.2 35.0 27.3 22.7
4725 6783 9917 8033 10350 5850
Pooled S.E.M. Herbage effect (P)
46.1 <0.001
1.26 <0.001
287.7 <0.05
0.52 <0.001
140.5 <0.04
from one another. The number of larvae recovered from cultures was lower (P < 0.05) for white clover than for the other diets which did not differ. 6.3. Egg hatch and LDAs There was a significant (P < 0.05) diet effect on the proportion of T. colubriformis eggs which remained unhatched after 24 h in the EHA (Fig. 1). More eggs remained in the D. rectum treatment (36%) than in most other herbage treatments, which averaged 15%, except D. pentophyllum which was intermediate on 22%. There were also significant (P < 0.001) diet effects on larval development in the LDA (Fig. 2). When lambs were fed L. pedunculatus, lucerne and white clover diets the greatest proportion of larvae recovered after 1 week were third stage larvae (L3 ) with small proportions of first and second stage larvae (L1 and L2 ). The L. corniculatus diet resulted in over 60% of the eggs remaining unhatched while D. rectum and D. pentophyllum diets resulted in similar numbers of eggs, L1 –L3 larval stages. Table 3 Lamb faecal egg count (eggs/g faeces), faecal DM and larval development (L3 recovered from culturing 45 g fresh faeces at 25 ◦ C for 7 days) from lambs fed either L. corniculatus, lucerne, D. rectum, sulla or white clover in autumn. For faecal egg count and larval culture data was loge transformed prior to analyses. Arithmetic means are shown in parentheses Herbage
Faecal egg count
L. corniculatus Lucerne D. rectum Sulla White clover
6.81 (1285) 6.67 (964) 6.70 (1000) 6.65 (957) 6.63 (885)
28.1 25.6 24.8 25.1 21.5
7.33 (1825) 7.42 (1995) 8.07 (3820) 7.68 (2340) 6.99 (1235)
Pooled S.E.M. Herbage effect (P)
0.267 NSa
0.356 <0.001
0.246 0.05
a
Not significant (P > 0.05).
Faecal DM (%)
Larval recovery from culture
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Fig. 1. Percentage of T. colubriformis eggs recovered from faeces of lambs fed either L. corniculatus, L. pedunculatus, lucerne, D. pentophyllum, D. rectum, or white clover which remained unhatched when cultured at 25 ◦ C for 24 h. Herbage effect, P < 0.05; error bars represent pooled S.E.M.
6.4. Larval development on pasture 6.4.1. Summer Most of the faeces placed on pasture were recovered until at least 64 days after deposition but little remained by day 128 (Fig. 3). The amount of faeces remaining was higher (P < 0.001) for L. corniculatus than for the other diets, this being particularly noticeable
Fig. 2. Percentage of T. colubriformis eggs, first (L1 ), second (L2 ), third stage (L3 ) and dead larvae in the LDA. Eggs were extracted from faeces of lambs fed L. corniculatus, L. pedunculatus, lucerne, D. pentophyllum, D. rectum, or white clover. Herbage effect, P < 0.001.
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Fig. 3. Recovery of faeces, expressed as percentage of DM deposited on day 0, in summer. Faeces were obtained from lambs fed L. corniculatus, L. pedunculatus, D. pentophyllum, D. rectum, white clover or lucerne. Error bars represent pooled S.E.M.
from days 16 to 64. By day 128 there were no herbage effects (P > 0.05) on the amount of faeces remaining, with 18–40% of initial faecal DM mass recovered. One day after depositing faeces on pasture, 6–31% of eggs remained in faeces, but by day 4 virtually no eggs remained (Table 4). One and 2 days after faecal deposition, there were fewer eggs remaining (P < 0.05) in the D. pentophyllum, L. pedunculatus and D. rectum faeces compared to faeces from lambs fed lucerne, white clover and L. corniculatus. The number of L3 larvae recovered from faeces was far greater (P < 0.001) from lambs fed lucerne than from any other diet (Table 4). Maximum larval numbers were recovered 32 days after faecal deposition followed by a slow decline to day 128. In contrast to lucerne, the numbers of larvae recovered from D. rectum and D. pentophyllum remained low throughout the trial with the other diets intermediate. The pattern between diets was similar when larval numbers are expressed as a proportion of eggs deposited (data not shown). The relative dietary effects on the numbers of L3 recovered from herbage were similar to numbers recovered from faeces (Table 4) in that the greatest numbers of L3 were recovered from plots contaminated with faeces from sheep fed lucerne and the lowest numbers from those contaminated with faeces from sheep fed D. pentophyllum and D. rectum. The lucerne diet resulted in an earlier recovery of L3 from herbage following faecal deposition (P < 0.001) (days 16 and 32; Table 4) compared with faeces from lambs fed the other diets. Pasture growth was similar for the herbage treatments and density of L3 on herbage (L3 /g DM) followed a similar pattern to L3 numbers (data not shown). 6.4.2. Autumn The rate of faecal DM disappearance from pasture was greater in autumn than in summer with significant (P < 0.01) differences attributable to diet (Fig. 4). Disappearance was
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Fig. 4. Recovery of faeces, expressed as percentage of DM deposited on day 0, in autumn. Faeces were obtained from lambs fed L. corniculatus, D. rectum, white clover, lucerne or sulla. Error bars represent pooled S.E.M.
slowest from lambs fed D. rectum (Fig. 4) so that by day 64 markedly greater faecal mass (P < 0.05) remained from plots which had faeces from lambs fed D. rectum than from any other treatment. Differences between dietary treatments were significant (P < 0.05) by day 8 and continued until the end of the trial. Few eggs remained in faeces by day 8 (Table 5), except for faeces from lambs fed white clover which developed more slowly from day 4 until day 32. There was a rapid rise in L3 recovered from faeces to day 4 after which recovery was at a lower level to day 32. Dietary effects on L3 larval recovery from faeces were significant (P < 0.05) on two occasions. On day 2 more (P < 0.05) L3 were recovered from faeces of lambs fed sulla, lucerne and L. corniculatus than D. rectum, and white clover. On day 32, more L3 were recovered from faeces of lambs fed sulla and white clover, with lowest recoveries from faeces of lambs fed L. corniculatus and D. rectum. The pattern between diets was similar when larval numbers are expressed as a proportion of eggs deposited (data not shown). No dietary treatment differences were evident in numbers of L3 recovered from herbage until day 32 when highest (P < 0.05) recoveries were obtained from plots with faeces from lucerne and L. corniculatus diets (Table 5). By day 64 greatest (P < 0.05) numbers of L3 were recovered from herbage which had faeces collected from lambs fed white clover, lucerne and sulla and lowest (P < 0.05) numbers from L. corniculatus and D. rectum diets. As with the summer contamination, herbage growth did not differ due to type of faeces deposited, so that larval density on herbage (L3 /g DM) closely reflected L3 numbers (data not shown). However, larval density in the autumn was markedly lower than in the summer because herbage growth was greater in autumn and larval development was lower.
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7. Discussion This study provides the first data showing that host diet can affect subsequent larval development both in vitro and on pasture. This was particularly evident during the warm summer conditions which are associated with greatest larval development on pasture in the Manawatu region of New Zealand (Niezen, 1996; Niezen et al., 1998a). While mechanisms which caused the dietary differences in the number of L3 recovered are not known, the short timeframe between initiation of feeding and faecal collection was deliberate, to minimise changes in the animal (possibly immunological) and focus on direct dietary effects. Thus, we believe that the observed differences in egg hatching and larval development are a direct consequence of dietary differences and most likely reflect the effects of CT in faeces. As CT are not absorbed in the digestive tract (Terrill et al., 1994) they become concentrated in the faeces. For instance, if herbage has a CT concentration of 5% and the herbage has 66% digestibility, then the CT concentration in the faeces will be 15%. Currently, the form or function of CT in faeces is unknown but it is likely to bind to protein and other substrates. It has been shown that CT extracted from herbage can inhibit the migration of T. colubriformis L3 and the L1 and L3 larvae of D. viviparous in in vitro assays (Molan et al., 2000a) indicating that CT can be detrimental to both egg hatching and larval development. The EHA and LDA results indicate that both D. rectum and D. pentophyllum have ovicidal and larval development inhibitory activity. This is supported by the fact that they also substantially reduced larval contamination of pasture, with low numbers of larvae recovered from faeces and surrounding herbage from lambs fed these two forages. Further, the reduction in larval development (relative to lucerne) observed on pasture with these two diets was far greater than measured in vitro. Similarly, while L. pedunculatus and white clover demonstrated similar activity to lucerne in the LDA and EHA these diets resulted in lower recovery of larvae (relative to lucerne) from faeces placed on pasture in the summer. The difference between in vitro and pasture results suggests that the EHA and LDA are conservative estimators of diet related differences on larval development on pasture. This might be explained by the time the eggs and larvae are in contact with CT in the faeces. In the EHA and LDA, the eggs were extracted from the faeces, and hence removed from further contact with CT, soon after removal from the rectum (12–24 h after the eggs came into contact with CT). In contrast, on pasture, eggs and developing larvae would remain in contact with CT for days if not weeks. Recently, Molan et al. (2000a) demonstrated that CT extracted from four common legumes were more deleterious to D. viviparous larvae when exposure was increased from 2 to 48 h. In contrast to the in vitro assays, results from larval cultures bore no relationship to diet related differences in numbers of larvae recovered from faeces placed on pasture suggesting that it is a poor estimator of diet related differences in larval development. The reason why larval cultures gave results in variance to in vitro and pasture results is unknown, and is the subject of current studies. The herbage effects between the EHA and LDA were not consistent for L. corniculatus in particular. In the EHA, 15% of the eggs did not hatch while in the LDA 60% of the eggs did not develop. As the eggs for the assays came from a common source on the same day the reason for the difference is hard to explain. The difference between the assays is in the type of media used. It is possible that the media in the LDA interacted with
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compounds on the eggs extracted from L. corniculatus faeces, thereby reducing egg hatch in the LDA. There were less marked herbage effects on larval development on pasture during autumn than summer, in part due to lower numbers of L3 recovered from lucerne in autumn compared with summer. This may reflect the use of older lambs which could be expected to reduce larval development (Jørgensen et al., 1998). The cooler autumn climatic conditions may also slow larval development and biological control agents may be more active (Hay et al., 2002). The autumn trial was not extended out to 128 days as previous studies have shown that by this time insufficient faeces would remain on pasture (Niezen et al., 1998a). Despite large dietary differences in numbers of L3 recovered from faeces in summer, corresponding smaller differences were observed on larval numbers recovered from herbage. For example, the ratio of L3 recovered from herbage on day 64 relative to the number of L3 recovered from faeces on day 32, were higher from L. corniculatus and L. pedunculatus treatments, than lucerne, suggesting that the diet affected the translation from faeces to herbage. The composition of faeces could alter the ability of larvae to migrate from it, but this remains unstudied. In this trial, L3 were recovered in faeces 1 day after faeces were deposited on pasture. This would be unlikely in a grazing situation where animals defecate directly on to pasture and highlights important differences between the experiments reported in this paper and actual grazing conditions. While every attempt was made to minimise larval development it would still have occurred during the collection, weighing and transportation of faeces to the trial site, and during collection and weighing of recovered faeces. Faeces were also placed on Baermann funnels for 24 h further allowing larval development to occur. The combination of these processes probably allowed some L3 to develop from samples collected on days 1 and 2. While handling the faeces may have shifted the temporal pattern of larval development relative to grazing conditions all treatments were subject to identical conditions and the observed dietary differences are still valid. The decline in faecal DM mass recovered from pasture was only slightly slower in the autumn than in the summer, and less than in previous reports from similar locations (Niezen, 1996; Niezen et al., 1998a). The lack of seasonal differences observed in this study may be a consequence of feeding fresh forages compared to pelleted lucerne (Niezen et al., 1998a). The drying and fine grinding of herbage for pelleting appears to affect faecal decomposition or disintegration in some cases (Niezen, unpublished data). Further work in this area is warranted. The incomplete egg hatch in faeces from lambs fed white clover in autumn may be associated with the high moisture content of, and hence lack of oxygen within (Table 3). Although egg viability was not measured, their appearance in the McMaster slides suggested that they remained intact and may have hatched at a later time. Moist faeces could preserve eggs in some instances and act as a reservoir of gastrointestinal parasites eggs for release at a later time as has been observed with O. ostertagi in cattle faeces (Young et al., 1980). This study has shown that the host diet can have a marked effect of the subsequent development of T. colubriformis larvae. The reasons for the dietary difference have yet to be ascertained, but CT in the diet are suspected to have an important role. Judicious selection of diets could have a major role in the reduction of larval development in grazing ruminants.
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Acknowledgements The authors are grateful to Rebecca Edwards and Shona Brock who assisted with larval counting. Scott and Luke Waghorn fed and looked after the lambs. John Napier was most co-operative and readily provided all the fencing materials. Bob Fletcher assisted with the trial design and Fred Potter did the statistical analyses. References Abbott, E.M., Holmes, P.H., 1990. Influence of dietary protein on the immune responsiveness of sheep to Haemonchus contortus. Res. Vet. Sci. 48, 103–107. Amarante, A.F.T., Pomroy, W.E., Charleston, W.A.G., Leathwick, D.M., Tornero, M.T.T., 1997. Evaluation of a larval development assay for the detection of anthelmintic resistance in Ostertagia circumcincta. Int. J. Parasitol. 27, 305–311. Bown, M.D., Poppi, D.P., Sykes, A.R., 1986. The effect of post-ruminal infusion of protein or energy on the pathology of Trichostrongylus colubriformis infection and body composition in lambs. Proc. NZ Soc. Anim. Prod. 46, 27–30. Coop, R.L., Huntley, J.F., Smith, W.D., 1995. Effect of dietary protein supplementation on the development of immunity to Ostertagia circumcincta in growing lambs. Res. Vet. Sci. 59, 24–29. Hay, F.S., Niezen, J.H., Leathwick, D.M., Skipp, R.A., 2002. Nematophagus fungi in pasture: colonisation of sheep faeces and their potential for control of free-living stages of gastro-intestinal parasites of sheep. Aust. J. Exp. Agric. 42, 7–13. Jørgensen, L.T., Leathwick, D.M., Charleston, W.A.G., Godfrey, P.L., Vlassoff, A., Sutherland, I.A., 1998. Variation between hosts in the developmental success of the free-living stages of trichostrongyle infections of sheep. Int. J. Parasitol. 28, 1347–1352. Lambert, M.G., Clark, D.A., Grant, D.A., Costall, D.A., 1986. Influence of fertiliser and grazing management on North Island moist hill country. 2. Pasture botanical composition. NZ J. Agric. Res. 29, 1–10. McKenna, P.B., Allan, C.M., Taylor, M.J., Townsend, K.G., 1995. The prevalence of anthelmintic resistance in ovine case submissions to animal health laboratories in New Zealand in 1993. NZ Vet. J. 43, 96–98. Molan, A.L., Hoskin, S.O., Barry, T.N., McNabb, W.C., 2000a. Effect of the condensed tannins extracted from four forages on the viability of the larvae of deer lungworms and gastrointestinal nematodes. Vet. Rec. 147, 44–48. Molan, A.L., Waghorn, G.C., Min, B.R., McNabb, W.C., 2000b. The effects of condensed tannins from seven herbages on Trichostrongylus colubriformis larval migration in vitro. Fol. Parasitol. 47, 39–44. Niezen, J.H., 1996. The effect of herbage species on internal parasite dynamics in sheep. Ph.D. Thesis. Massey University, New Zealand. Niezen, J.H., Waghorn, T.S., Charleston, W.A.G., Waghorn, G.C., 1995. Growth and gastrointestinal nematode parasitism in lambs grazing either lucerne (Medicago sativa) or sulla (Hedysarum coronarium) which contains condensed tannins. J. Agric. Sci. 125, 281–289. Niezen, J.H., Miller, C.M., Robertson, H.A., Wilson, S.R., MacKay, A.D., 1998a. Effect of topographical aspect and farm system on the population dynamics of Trichostrongylus larvae on a hill country farm. Vet. Parasitol. 78, 37–48. Niezen, J.H., Robertson, H.A., Waghorn, G.C., Charleston, W.A.G., 1998b. Production, faecal egg counts and worm burdens of ewe lambs which grazed six contrasting forages. Vet. Parasitol. 80, 15–27. Niezen, J.H., Waghorn, G.C., Charleston, W.A.G., 1998c. The effects of feeding ryegrass (Lolium perenne) or Lotus pedunculatus on Ostertagia circumcincta and Trichostrongylus colubriformis establishment and fecundity and lamb growth rate. Vet. Parasitol. 78, 13–21. Roberts, J.A., Adams, D.B., 1990. The effect of nutrition on the development of resistance to Haemonchus contortus in sheep. Aust. Vet. J. 67, 89–91. Sutherland, I.A., Brown, A.E., Leathwick, D.M., 2000. Selection for drug-resistant nematodes during and following extended exposure to anthelmintic. Parasitology 121, 217–226.
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Terrill, T.H., Rowan, A.M., Douglas, G.B., Barry, T.N., 1992. Determination of extractable and bound condensed tannin concentrations in forage plants, protein concentrate meals and cereal grains. J. Sci. Food Agric. 58, 321–329. Terrill, T.H., Waghorn, G.C., Woolley, D.J., McNabb, W.C., Barry, T.N., 1994. Assay and digestion of 14 C-labelled condensed tannins in the gastrointestinal tract of sheep. Br. J. Nutr. 72, 467–477. van Houtert, M.J.F., Barger, I.A., Steel, J.W., Windon, R.G., Emery, D.L., 1995. Effects of dietary protein on responses of young sheep to infection with Trichostrongylus colubriformis. Vet. Parasitol. 56, 163–180. Young, R.R., Nicholson, R.M., Tweedie, R.L., Schuh, H.J., 1980. Quantitative modelling and prediction of development times of the free-living stages of Ostertagia ostertagi under controlled and field conditions. Parasitology 81, 493–505.
The effect of diet fed to lambs on subsequent development of Trichostrongylus colubriformis larvae in vitro and on pasture J.H. Niezen∗ , G.C. Waghorn, T. Graham, J.L. Carter, D.M. Leathwick AgResearch Grasslands, P.O. Box 11008, Palmerston North, New Zealand Received 20 August 2001; received in revised form 23 January 2002; accepted 6 February 2002
Abstract Contrasting herbage diets were fed to lambs to evaluate their effect on subsequent development of Trichostrongylus colubriformis larvae in faeces and on pasture. The diets had either no condensed tannin (CT), lucerne (Medicago sativa cv. Otaio), white clover (Trifolium repens cv. Tahora), or had moderate to high concentrations of CT, sulla (Hedysarum coronarium cv. Grassland Aokau), Lotus corniculatus (cv. Grasslands Goldie), L. pedunculatus (cv. Grassland Maku), Dorycnium pentophyllum, and Dorycnium rectum. Trials were carried out in summer (warm) and in autumn (cool and moist). In summer, egg viability was evaluated in vitro with egg hatch and larval development assays. In both seasons faeces were placed on pasture to compare recovery of eggs and larvae from faeces and larvae from herbage on the high and low fertility farmlets on the AgResearch Ballantrae Hill Country Research Station. D. rectum and D. pentophyllum diets decreased (P < 0.01) egg hatching and larval development in laboratory assays relative to other diets. In summer, the number of larvae recovered from faeces placed on pasture was far greater (P < 0.001) if the lambs had been fed lucerne than any other diet, whereas recovery was always lowest from faeces of sheep fed D. rectum and D. pentophyllum. Although dietary differences were lower in autumn than in summer, larval recoveries were lower (P < 0.05) from faeces of lambs fed D. rectum and L. corniculatus than from white clover, lucerne and sulla diets. This study indicates that the diet of the host can have a significant impact on egg hatching and the subsequent development of T. colubriformis larvae in the laboratory and in the field. In particular, D. rectum consistently reduced T. colubriformis development. Effects measured in vitro generally under-estimated effects measured under field conditions. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Sheep-nematoda; Trichostrongylus colubriformis; Larval development; Condensed tannins; Feeding and nutrition ∗ Corresponding author. Present address: International Livestock Research Institute (ILRI), C/o IITA, PMB 5320, Oyo Rd., Ibadan, Nigeria. Tel.: +234-2-241-2626; fax: +234-2-241-2221. E-mail address: [email protected] (J.H. Niezen).
0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 2 ) 0 0 0 2 5 - 0
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1. Introduction With increasing levels of anthelmintic resistance in New Zealand (McKenna et al., 1995) alternative methods of gastrointestinal parasite control must be explored. One possibility is to change the composition of the diet of the grazing ruminant by grazing different pasture species. This can increase the plane of nutrition of the host which, in turn, can increase immunity and reduce gastrointestinal parasite establishment (Abbott and Holmes, 1990; Roberts and Adams, 1990; Coop et al., 1995; van Houtert et al., 1995) as well as reduce the pathology of parasite infection (Bown et al., 1986). Changing the pastures grazed by ruminants can also introduce into the diet plant secondary compounds which may be detrimental to gastrointestinal parasites. Previous studies have shown that feeding forages which contain condensed tannins (CTs) can lower gastrointestinal parasite establishment in lambs (Niezen et al., 1995, 1998c) and can reduce established gastrointestinal parasite burdens (Niezen et al., 1998b), although the mechanisms which cause these effects remain unclear. It has also been shown that CT are toxic to Trichostrongylus colubriformis and Dictyocaulus viviparus larvae in in vitro assays (Molan et al., 2000a,b). Because CTs are not absorbed from the gastrointestinal tract of ruminants (Terrill et al., 1994) and thus remain in the faeces of animals fed forages containing CT, the possibility remains that they may directly impair gastrointestinal parasite larval development within faeces. This study investigated this possibility by comparing a range of dietary species, particularly those containing CT on the development of T. colubriformis larvae in egg hatch and larval development assays (LDAs) and on pasture plots.
2. Animals and forages 2.1. Summer Forty Romney ram lambs, 4–5 months of age (average liveweight 28.8 kg) were orally treated with ivermectin (Ivomec® Merial) at twice the manufacturer’s recommended dose rate and grazed on pasture which had been grazed only by cattle for the previous year. Five days after drenching, each lamb received a single dose of 20,000 T. colubriformis third stage larvae (L3 ). Three weeks later the lambs were housed indoors in individual crates and restrictively randomised, based on faecal nematode egg count (FEC), to one of the six dietary treatments with six lambs per treatment. The four lambs with the lowest FEC were not used in the trial. The six treatments comprised ad libitum feeding of either, Lotus corniculatus (cv. Grasslands Goldie), L. pedunculatus (cv. Grasslands Maku), Dorycnium pentophyllum, Dorycnium rectum, white clover (Trifolium repens cv. Grasslands Tahora), or lucerne (Medicago sativa cv. Otaio). Each forage was fed for 7 days. Daily dry matter intakes (DMIs) were measured over the final 4 days when faeces were also collected. Dry matter (DM) concentrations in feeds (duplicates) and refusals were determined by drying at 95 ◦ C for 24 h and samples of feeds were bulked over the final 4 days for chemical analyses. All forages were cut daily from vegetative, monospecific stands which had not been grazed by sheep for the previous year. Lucerne was lush and pre-flowering and the white
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clover contained small proportions of dead leaf because it was relatively mature when harvested. Some flowers were present in the Lotus spp. but the stems were palatable and eaten by sheep. D. rectum is a herbaceous shrub and 30–45 cm spikes were given to sheep which selected leaf from stem. D. pentophyllum was mature and flowering and the lambs consumed all parts of the plant. Parasite development was assessed by egg hatch assay (EHA), LDA and larval development on pasture over 128 days commencing mid-January (see below). 2.2. Autumn Thirty Romney ram lambs, 7–8 months of age, were treated as described above. Diets fed were L. corniculatus, lucerne, D. rectum, sulla (Hedysarum coronarium cv. Grasslands Aokau) or white clover. All the forages were lush when fed and all lambs were offered 1000 g DM/day. Parasite development was assessed on pasture only. Faeces were deposited on field plots on 14 May and were collected until day 64. 3. Egg hatch and LDAs Four days after the lambs had been introduced to the diets, rectal faecal samples were collected and eggs extracted and placed in (EHAs) as described by Sutherland et al. (2000) and LDAs as described by Amarante et al. (1997). Egg hatch was determined by the number of eggs and larvae present in each assay cell after 24 h incubation at 25 ◦ C. Larval development was measured by counting the number of eggs, first stage (L1 ), second stage (L2 ), third stage (L3 ) and dead larvae after 7 days incubation at 25 ◦ C. For each assay three replicates of each assay were undertaken for each animal with approximately 50 eggs added to each cell. 4. Larval development on pasture 4.1. Summer Faeces were collected in 40 × 30 cm2 trays placed under the grating floor at the rear of each sheep crate. These enabled approximately 70% of total faecal output to be collected without any urine contamination. Faeces were removed from the trays at 0800, 1100, 1600 and 2000 h each day and placed into individually labelled bags, which were left open to allow aeration and held at 4 ◦ C for no longer than 2 days. Equal amounts of faeces from each lamb were pooled within herbage type. Faecal DM content was determined. Faecal egg counts were determined using a modified McMaster technique. Faeces of sheep fed D. rectum and D. pentophyllum were always pelleted, those from Lotus spp. and white clover comprised a mixture of pellets and stools, but faeces from lambs fed lucerne were soft, and liquid in consistency. Pasture comparisons of larval development were carried out on two plots on each of the low and high fertility farmlets at the Ballantrae Hill Country Research Station located 30 km east of Palmerston North. The farmlets have either received a maintenance dressing
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of fertiliser annually (low fertility) or a high allowance of fertiliser annually (high fertility) for the past 25 years (Lambert et al., 1986). Each plot was divided into one hundred and ninety-two 60×60 cm2 sub-plots mowed to a height of 5 cm 2 days prior to faecal deposition. There was a 40 cm margin between sub-plots and 45 g of faeces were deposited in the centre of each sub-plot on 18 January (day 0). Faeces were collected from sub-plots on days 1, 2, 4, 8, 16, 32, 64 and 128. On days 4, 8, 16, 32, 64, and 128 herbage was cut to ground level in an area of 0.1 m2 around the site of faecal deposition to extract and count larvae. Faeces recovered from the plots were used for FEC and T. colubriformis larvae were extracted from faeces and herbage using a modified Baermann technique and counted in 10% (2 × 5%) aliquots. 4.2. Autumn The autumn contamination followed a similar protocol to the summer but as only five herbage treatments were evaluated, each plot was divided into 160, 60 × 60 cm2 sub-plots. Faeces were deposited on the 160 plots on 14 May (day 0) and faeces collected on days 1, 2, 4, 8, 16, 32, and 64. Herbage was cut to ground level and collected on days 8, 16, 32, and 64. 5. Chemical and statistical analysis Feed samples were freeze dried and ground through a 1 mm screen prior to analysis by near infrared reflectance spectroscopy (NIRS) for main components and butanol HCl sequential extraction (Terrill et al., 1992) to determine concentrations of CTs. Dietary treatment effects on the EHA, LDA and FEC, DMI, faecal DM and larval culture comparisons were evaluated on the basis of least squared means using Genstat (version 5.42) with animal as the experimental unit. In summer, DMI, FEC and faecal DM and in autumn faecal DM were compared by Kruskal–Wallis one-way ANOVA. In autumn FEC and larval cultures were loge transformed to normalise data. Herbage DM was determined on each of 4 days with two replicates for each feed sample per day. The EHA and FEC were compared by analysis of variance while the LDA used multivariate analyses. For studies on pasture, individual plots were used as the experimental units. Dietary comparisons for larval development were made within seasons but not between seasons. In both summer and autumn the proportion of eggs remaining in faeces and L3 larvae on herbage were loge (x + 1) transformed, while L3 larvae recovered from faeces were loge (x + 0.1) transformed to normalise data. Where ANOVAs produced a significant F-value means were compared using least significant differences calculated for P < 0.05, <0.01 and <0.001. Where data had been transformed means were compared on the transformed scale. 6. Results 6.1. Diet analyses Chemical composition of diets fed in both summer and autumn are shown in Table 1. Diets varied substantially in chemical composition with crude protein (CP) content of the DM ranging from 13.1 to 29.9% and fibre concentrations from 24 to 48% of the DM. This
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Table 1 Chemical composition (% of the DM), estimates of OMD (%) and metabolisable energy (ME) content (MJ/kg DM) of L. corniculatus, L. pedunculatus, D. pentophyllum, D. rectum, white clover, lucerne and sulla diets fed to lambs in summer and autumn experimentsa CP
Lipid Ash
ADF NDF CHO OMD ME
CT concentration Free
Protein bound
Fibre bound
Total
Summer L. corniculatus L. pedunculatus D. pentophyllum D. rectum White clover Lucerne
21.9 17.5 13.1 14.4 25.6 29.9
4.1 3.6 3.8 3.6 3.3 3.6
7.1 7.0 6.4 9.4 11.3 10.5
25.7 34.0 33.6 30.7 21.6 22.7
31.6 41.0 47.5 39.8 28.5 29.8
6.9 4.8 2.8 7.2 11.1 8.6
72.6 66.1 64.7 70.2 77.7 72.0
11.6 9.8 9.6 10.5 11.6 10.7
3.6 7.7 8.2 14.2 NAb NA
1.6 4.2 2.3 2.8 NA NA
0.3 0.3 0.4 0.4 NA NA
5.5 12.2 10.9 17.4 NA NA
Autumn L. corniculatus Lucerne D. rectum Sulla White clover
22.6 25.1 15.8 16.6 25.5
3.8 3.5 3.7 2.0 3.5
8.1 9.7 6.5 10.9 11.5
20.3 25.8 18.8 24.4 20.0
26.9 33.6 23.8 24.4 24.1
12.9 9.7 22.5 18.7 14.5
71.5 69.1 78.0 77.9 81.6
10.7 10.3 11.6 11.6 12.2
0.9 NA 8.2 2.0 NA
0.6 NA 0.3 0.9 NA
0.1 NA 0.2 0.1 NA
1.6 NA 8.7 3.0 NA
a b
CP: crude protein; ADF: acid detergent fibre; NDF: neutral detergent fibre; CHO: soluble carbohydrate. Not analysed as these forages do not contain CT.
range in composition was associated with CT concentrations as high as 17.4% of the DM in D. rectum in summer, whilst lucerne and white clover do not contain CT. D. rectum had the highest CT and lowest CP content. Predicted organic matter digestibilities (OMDs) ranged from 65 to 78% (Table 1). The principal differences between diets fed in summer and autumn were the lower CT concentrations in autumn and higher quality of D. rectum on offer. 6.2. Intakes and faecal characteristics 6.2.1. Summer When offered ad libitum, DMI was lower (P < 0.01) in lambs fed D. pentophyllum and D. rectum than lambs fed the other diets (Table 2). Faecal DM was higher (P < 0.001) in lambs fed D. pentophyllum and D. rectum than in lambs fed L. pedunculatus and white clover, which was higher (P < 0.05) than in lambs fed L. corniculatus and lucerne. Lamb FEC was lower (P < 0.05) for the L. pedunculatus diet than all other diets except D. pentophyllum, which was intermediate (Table 2). Greater numbers (P < 0.05) of larvae were recovered from cultured faeces of lambs fed white clover than L. corniculatus. All other diets did not differ. 6.2.2. Autumn The older lambs used in autumn consumed virtually all material on offer with sufficient D. rectum given to ensure that 1 kg of leafy DM was consumed daily. Lamb FEC did not differ (Table 3) between diets. Faecal DM was lowest for white clover and highest for L. corniculatus (P < 0.001), Lucerne, sulla and D. rectum were intermediate and did not differ
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Table 2 Lamb DM intake (g DM/day), herbage DM (%), faecal egg count, faecal DM and larval development (larvae recovered from culturing 45 g fresh faeces at 25 ◦ C for 7 days) from lambs fed either L. corniculatus, L. pedunculatus, D. pentophyllum, D. rectum, white clover or lucerne diets in summer Herbage
DMI (g DM/day)
Herbage DM (%)
Faecal egg count (eggs/g faeces)
Faecal DM (%)
Larval recovery from culture
L. corniculatus L. pedunculatus D. pentophyllum D. rectum White clover Lucerne
1000 965 784 669 1091 1100
15.0 15.0 24.3 15.2 16.3 17.8
2467 1142 1467 2025 2058 2175
22.7 26.7 35.2 35.0 27.3 22.7
4725 6783 9917 8033 10350 5850
Pooled S.E.M. Herbage effect (P)
46.1 <0.001
1.26 <0.001
287.7 <0.05
0.52 <0.001
140.5 <0.04
from one another. The number of larvae recovered from cultures was lower (P < 0.05) for white clover than for the other diets which did not differ. 6.3. Egg hatch and LDAs There was a significant (P < 0.05) diet effect on the proportion of T. colubriformis eggs which remained unhatched after 24 h in the EHA (Fig. 1). More eggs remained in the D. rectum treatment (36%) than in most other herbage treatments, which averaged 15%, except D. pentophyllum which was intermediate on 22%. There were also significant (P < 0.001) diet effects on larval development in the LDA (Fig. 2). When lambs were fed L. pedunculatus, lucerne and white clover diets the greatest proportion of larvae recovered after 1 week were third stage larvae (L3 ) with small proportions of first and second stage larvae (L1 and L2 ). The L. corniculatus diet resulted in over 60% of the eggs remaining unhatched while D. rectum and D. pentophyllum diets resulted in similar numbers of eggs, L1 –L3 larval stages. Table 3 Lamb faecal egg count (eggs/g faeces), faecal DM and larval development (L3 recovered from culturing 45 g fresh faeces at 25 ◦ C for 7 days) from lambs fed either L. corniculatus, lucerne, D. rectum, sulla or white clover in autumn. For faecal egg count and larval culture data was loge transformed prior to analyses. Arithmetic means are shown in parentheses Herbage
Faecal egg count
L. corniculatus Lucerne D. rectum Sulla White clover
6.81 (1285) 6.67 (964) 6.70 (1000) 6.65 (957) 6.63 (885)
28.1 25.6 24.8 25.1 21.5
7.33 (1825) 7.42 (1995) 8.07 (3820) 7.68 (2340) 6.99 (1235)
Pooled S.E.M. Herbage effect (P)
0.267 NSa
0.356 <0.001
0.246 0.05
a
Not significant (P > 0.05).
Faecal DM (%)
Larval recovery from culture
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Fig. 1. Percentage of T. colubriformis eggs recovered from faeces of lambs fed either L. corniculatus, L. pedunculatus, lucerne, D. pentophyllum, D. rectum, or white clover which remained unhatched when cultured at 25 ◦ C for 24 h. Herbage effect, P < 0.05; error bars represent pooled S.E.M.
6.4. Larval development on pasture 6.4.1. Summer Most of the faeces placed on pasture were recovered until at least 64 days after deposition but little remained by day 128 (Fig. 3). The amount of faeces remaining was higher (P < 0.001) for L. corniculatus than for the other diets, this being particularly noticeable
Fig. 2. Percentage of T. colubriformis eggs, first (L1 ), second (L2 ), third stage (L3 ) and dead larvae in the LDA. Eggs were extracted from faeces of lambs fed L. corniculatus, L. pedunculatus, lucerne, D. pentophyllum, D. rectum, or white clover. Herbage effect, P < 0.001.
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Fig. 3. Recovery of faeces, expressed as percentage of DM deposited on day 0, in summer. Faeces were obtained from lambs fed L. corniculatus, L. pedunculatus, D. pentophyllum, D. rectum, white clover or lucerne. Error bars represent pooled S.E.M.
from days 16 to 64. By day 128 there were no herbage effects (P > 0.05) on the amount of faeces remaining, with 18–40% of initial faecal DM mass recovered. One day after depositing faeces on pasture, 6–31% of eggs remained in faeces, but by day 4 virtually no eggs remained (Table 4). One and 2 days after faecal deposition, there were fewer eggs remaining (P < 0.05) in the D. pentophyllum, L. pedunculatus and D. rectum faeces compared to faeces from lambs fed lucerne, white clover and L. corniculatus. The number of L3 larvae recovered from faeces was far greater (P < 0.001) from lambs fed lucerne than from any other diet (Table 4). Maximum larval numbers were recovered 32 days after faecal deposition followed by a slow decline to day 128. In contrast to lucerne, the numbers of larvae recovered from D. rectum and D. pentophyllum remained low throughout the trial with the other diets intermediate. The pattern between diets was similar when larval numbers are expressed as a proportion of eggs deposited (data not shown). The relative dietary effects on the numbers of L3 recovered from herbage were similar to numbers recovered from faeces (Table 4) in that the greatest numbers of L3 were recovered from plots contaminated with faeces from sheep fed lucerne and the lowest numbers from those contaminated with faeces from sheep fed D. pentophyllum and D. rectum. The lucerne diet resulted in an earlier recovery of L3 from herbage following faecal deposition (P < 0.001) (days 16 and 32; Table 4) compared with faeces from lambs fed the other diets. Pasture growth was similar for the herbage treatments and density of L3 on herbage (L3 /g DM) followed a similar pattern to L3 numbers (data not shown). 6.4.2. Autumn The rate of faecal DM disappearance from pasture was greater in autumn than in summer with significant (P < 0.01) differences attributable to diet (Fig. 4). Disappearance was
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Fig. 4. Recovery of faeces, expressed as percentage of DM deposited on day 0, in autumn. Faeces were obtained from lambs fed L. corniculatus, D. rectum, white clover, lucerne or sulla. Error bars represent pooled S.E.M.
slowest from lambs fed D. rectum (Fig. 4) so that by day 64 markedly greater faecal mass (P < 0.05) remained from plots which had faeces from lambs fed D. rectum than from any other treatment. Differences between dietary treatments were significant (P < 0.05) by day 8 and continued until the end of the trial. Few eggs remained in faeces by day 8 (Table 5), except for faeces from lambs fed white clover which developed more slowly from day 4 until day 32. There was a rapid rise in L3 recovered from faeces to day 4 after which recovery was at a lower level to day 32. Dietary effects on L3 larval recovery from faeces were significant (P < 0.05) on two occasions. On day 2 more (P < 0.05) L3 were recovered from faeces of lambs fed sulla, lucerne and L. corniculatus than D. rectum, and white clover. On day 32, more L3 were recovered from faeces of lambs fed sulla and white clover, with lowest recoveries from faeces of lambs fed L. corniculatus and D. rectum. The pattern between diets was similar when larval numbers are expressed as a proportion of eggs deposited (data not shown). No dietary treatment differences were evident in numbers of L3 recovered from herbage until day 32 when highest (P < 0.05) recoveries were obtained from plots with faeces from lucerne and L. corniculatus diets (Table 5). By day 64 greatest (P < 0.05) numbers of L3 were recovered from herbage which had faeces collected from lambs fed white clover, lucerne and sulla and lowest (P < 0.05) numbers from L. corniculatus and D. rectum diets. As with the summer contamination, herbage growth did not differ due to type of faeces deposited, so that larval density on herbage (L3 /g DM) closely reflected L3 numbers (data not shown). However, larval density in the autumn was markedly lower than in the summer because herbage growth was greater in autumn and larval development was lower.
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7. Discussion This study provides the first data showing that host diet can affect subsequent larval development both in vitro and on pasture. This was particularly evident during the warm summer conditions which are associated with greatest larval development on pasture in the Manawatu region of New Zealand (Niezen, 1996; Niezen et al., 1998a). While mechanisms which caused the dietary differences in the number of L3 recovered are not known, the short timeframe between initiation of feeding and faecal collection was deliberate, to minimise changes in the animal (possibly immunological) and focus on direct dietary effects. Thus, we believe that the observed differences in egg hatching and larval development are a direct consequence of dietary differences and most likely reflect the effects of CT in faeces. As CT are not absorbed in the digestive tract (Terrill et al., 1994) they become concentrated in the faeces. For instance, if herbage has a CT concentration of 5% and the herbage has 66% digestibility, then the CT concentration in the faeces will be 15%. Currently, the form or function of CT in faeces is unknown but it is likely to bind to protein and other substrates. It has been shown that CT extracted from herbage can inhibit the migration of T. colubriformis L3 and the L1 and L3 larvae of D. viviparous in in vitro assays (Molan et al., 2000a) indicating that CT can be detrimental to both egg hatching and larval development. The EHA and LDA results indicate that both D. rectum and D. pentophyllum have ovicidal and larval development inhibitory activity. This is supported by the fact that they also substantially reduced larval contamination of pasture, with low numbers of larvae recovered from faeces and surrounding herbage from lambs fed these two forages. Further, the reduction in larval development (relative to lucerne) observed on pasture with these two diets was far greater than measured in vitro. Similarly, while L. pedunculatus and white clover demonstrated similar activity to lucerne in the LDA and EHA these diets resulted in lower recovery of larvae (relative to lucerne) from faeces placed on pasture in the summer. The difference between in vitro and pasture results suggests that the EHA and LDA are conservative estimators of diet related differences on larval development on pasture. This might be explained by the time the eggs and larvae are in contact with CT in the faeces. In the EHA and LDA, the eggs were extracted from the faeces, and hence removed from further contact with CT, soon after removal from the rectum (12–24 h after the eggs came into contact with CT). In contrast, on pasture, eggs and developing larvae would remain in contact with CT for days if not weeks. Recently, Molan et al. (2000a) demonstrated that CT extracted from four common legumes were more deleterious to D. viviparous larvae when exposure was increased from 2 to 48 h. In contrast to the in vitro assays, results from larval cultures bore no relationship to diet related differences in numbers of larvae recovered from faeces placed on pasture suggesting that it is a poor estimator of diet related differences in larval development. The reason why larval cultures gave results in variance to in vitro and pasture results is unknown, and is the subject of current studies. The herbage effects between the EHA and LDA were not consistent for L. corniculatus in particular. In the EHA, 15% of the eggs did not hatch while in the LDA 60% of the eggs did not develop. As the eggs for the assays came from a common source on the same day the reason for the difference is hard to explain. The difference between the assays is in the type of media used. It is possible that the media in the LDA interacted with
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compounds on the eggs extracted from L. corniculatus faeces, thereby reducing egg hatch in the LDA. There were less marked herbage effects on larval development on pasture during autumn than summer, in part due to lower numbers of L3 recovered from lucerne in autumn compared with summer. This may reflect the use of older lambs which could be expected to reduce larval development (Jørgensen et al., 1998). The cooler autumn climatic conditions may also slow larval development and biological control agents may be more active (Hay et al., 2002). The autumn trial was not extended out to 128 days as previous studies have shown that by this time insufficient faeces would remain on pasture (Niezen et al., 1998a). Despite large dietary differences in numbers of L3 recovered from faeces in summer, corresponding smaller differences were observed on larval numbers recovered from herbage. For example, the ratio of L3 recovered from herbage on day 64 relative to the number of L3 recovered from faeces on day 32, were higher from L. corniculatus and L. pedunculatus treatments, than lucerne, suggesting that the diet affected the translation from faeces to herbage. The composition of faeces could alter the ability of larvae to migrate from it, but this remains unstudied. In this trial, L3 were recovered in faeces 1 day after faeces were deposited on pasture. This would be unlikely in a grazing situation where animals defecate directly on to pasture and highlights important differences between the experiments reported in this paper and actual grazing conditions. While every attempt was made to minimise larval development it would still have occurred during the collection, weighing and transportation of faeces to the trial site, and during collection and weighing of recovered faeces. Faeces were also placed on Baermann funnels for 24 h further allowing larval development to occur. The combination of these processes probably allowed some L3 to develop from samples collected on days 1 and 2. While handling the faeces may have shifted the temporal pattern of larval development relative to grazing conditions all treatments were subject to identical conditions and the observed dietary differences are still valid. The decline in faecal DM mass recovered from pasture was only slightly slower in the autumn than in the summer, and less than in previous reports from similar locations (Niezen, 1996; Niezen et al., 1998a). The lack of seasonal differences observed in this study may be a consequence of feeding fresh forages compared to pelleted lucerne (Niezen et al., 1998a). The drying and fine grinding of herbage for pelleting appears to affect faecal decomposition or disintegration in some cases (Niezen, unpublished data). Further work in this area is warranted. The incomplete egg hatch in faeces from lambs fed white clover in autumn may be associated with the high moisture content of, and hence lack of oxygen within (Table 3). Although egg viability was not measured, their appearance in the McMaster slides suggested that they remained intact and may have hatched at a later time. Moist faeces could preserve eggs in some instances and act as a reservoir of gastrointestinal parasites eggs for release at a later time as has been observed with O. ostertagi in cattle faeces (Young et al., 1980). This study has shown that the host diet can have a marked effect of the subsequent development of T. colubriformis larvae. The reasons for the dietary difference have yet to be ascertained, but CT in the diet are suspected to have an important role. Judicious selection of diets could have a major role in the reduction of larval development in grazing ruminants.
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