M.R. Knox

Enhancing immunity to nematode parasites in single-bearing Merino ewes through nutrition and genetic selection

The effectiveness of protein supplementation and genetic selection to enhance the resistance of periparturient Merino ewes to infection from gastrointestinal parasites was tested in a replicated grazing experiment. One hundred and twenty ewes from lines selected for increased resistance (R) to Haemonchus contortus or at random (C) were subjected to one of the three supplement groups that provided 0 or 250 g per day cottonseed meal for 5 weeks prior to, or for 6 weeks after the start of lambing. Faecal egg counts (FEC) of R ewes were consistently lower than those of C ewes but both groups exhibited a periparturient rise in FEC. Supplementation during the pre-partum period reduced FEC and increased ewe body weight gain. The benefits of pre-partum supplementation in reducing FEC continued to be apparent up to 10 weeks after supplementation ceased. There was a strong suggestion that the benefits to parasite resistance from protein supplementation were greatest in C ewes. Wool growth rates (15%) and birth weights (5%) were greater for C ewes but differences between the lines for lamb body weight had disappeared by day 97. The greatest benefit to resistance from protein supplementation was observed when ewes were experiencing a loss of maternal body weight. Conversely, no benefits to resistance were observed when ewes had moderate (78-107 g per day) rates of maternal weight gain. These results suggest that increased resistance as a result of protein supplementation is dependent on the prevailing supply and demand for scarce nutrients such as metabolisable protein (MP). Both genetic selection and protein nutrition are effective strategies to enhance host resistance to nematode infection during the periparturient period.

Nutritional enhancement of parasite control in small ruminant production systems in developing countries of south-east Asia and the Pacific

Nutritional insufficiency and gastrointestinal nematode parasitism are major constraints to small ruminant production in south-east Asia and the Pacific Islands. Research on the effects of low cost supplements which supply nitrogen and essential minerals on the ability of small ruminants to resist infection is summarised. In controlled pen studies in young Merino sheep offered a low quality roughage diet of oaten chaff and essential minerals, supplementation with urea reduced the effects of parasitic infection by increasing weight gain and wool production and reducing faecal egg output and parasite burden. In Fiji, field studies have shown that supplementation with urea-molasses blocks can result in increased live-weights of lambs at weaning, increased reproduction rates in maiden ewes and reduction in faecal egg output in grazing sheep. Additional benefits were derived from the inclusion of anthelmintic in the blocks in similar groups of sheep particularly during periods of greater susceptibility to parasites. Pen studies with young goats have shown that urea supplements alone gave no production benefits, but when accompanied by 100 g/d of cotton seed meal beneficial responses were observed. It is expected that parasite control in the small ruminant production systems of developing countries in south-east Asia and the Pacific Islands will benefit from the introduction of low cost nitrogen supplements along with anthelmintic therapy delivered strategically by molasses blocks.

The potential of nematophagous fungi to control the free-living stages of nematode parasites of sheep: feeding and block studies with Duddingtonia flagrans.

A series of feeding trials was conducted with penned sheep harboring Trichostrongylus colubriformis infections. They were offered barley grains supporting the growth of the nematophagous fungus Duddingtonia flagrans. It was shown that as little as 5g of grain/sheep per day was sufficient to virtually eliminate larval numbers from faecal culture. This effect persisted for the time that the fungal grains were fed, and for up to 2 days following cessation of feeding this material. Macerated fungal grains were also incorporated into a range of feed block formulations. In all these, D. flagrans was found to survive the manufacturing process and resulted in significant reductions in larval numbers in faecal cultures set up during the feeding period to sheep. This was observed even for sheep that showed only modest and irregular block consumption. These studies demonstrate that supplementary feeding or block administration offer potential deployment options for D. flagrans as a means of biological control of nematode parasites of livestock.

Regulation of the resistance to nematode parasites of single- and twin-bearing Merino ewes through nutrition and genetic selection

Periparturient Merino ewes obtained from lines of sheep that had been selected either for increased resistance to Haemonchus contortus (R) or at random (C) were supplemented, while grazing at pasture, with either nil or 250 g/day cottonseed meal (CSM) for the 6 weeks prior to or the 6 weeks after the start of parturition. Ewes from both supplement groups had lower (mean 66% reduction) faecal egg counts (FECs) during the postpartum period and this coincided with a period of maternal body weight loss. Factors which increased the rate of maternal body weight loss, such as pregnancy and lactation status, also increased FEC. Evidence is presented that the magnitude of the periparturient rise (PPR) in FEC in grazing ewes will be greatest during periods of maternal weight loss and at these times supplementation to increase metabolisable protein (MP) supply will be most effective in increasing resistance to nematode parasites. The resistance of R ewes to nematode parasites was greater than that of C ewes throughout the experiment and was sufficiently low such that anthelmintic treatment in a commercial environment may not have been required. Irrespective of actual FEC, ewes from all treatment combinations exhibited a PPR in FEC. Reduced FEC of R ewes resulted in reduced apparent pasture larval contamination after 18 weeks of continuous grazing but supplementation was ineffective in this regard. It is suggested that integrated parasite management (IPM) programs for periparturient ewes should make use of both protein supplementation and genetic selection to increase worm resistance and reduce dependency on anthelmintics for worm control.

The effects of urea supplementation on production and parasitological responses of sheep infected with Haemonchus contortus and Trichostrongylus colubriformis

Merino wether lambs were individually confined and fed a basal diet of oaten chaff containing essential minerals which was untreated or contained 3% urea. Within each dietary group animals were orally infected with either 200 H. contortus (H), 1000 T. colubriformis (T) or both species (H + T) thrice weekly or remained uninfected (C). Weight gain, wool production, and parasite burden were measured over a 19-week period. Sheep on the diet containing urea gained more weight, consumed more feed and grew more wool of higher fibre diameter than their counterparts given no urea. On both diets uninfected sheep consumed more feed than infected sheep and the sheep given no urea and infected with both H and T worm species consumed the least feed. Parasitised sheep gained less weight than uninfected control sheep. Sheep with urea in their diet had lower faecal egg counts when infected with H alone or with H and T but there was no effect of urea on egg count of sheep infected with T alone. In contrast, T numbers after slaughter were reduced in sheep fed diets containing urea whereas H numbers were not affected by diet. It was concluded that supplementation with urea can increase resilience to parasitism thereby improving production and also enhance resistance mechanisms against worms in young sheep on low quality roughage diets. These responses can be partly attributed to stimulation of feed intake, presumably due to enhanced ruminal digestion, but also to elevated rumen NH3-N levels which would be expected to have increased rumen microbial protein synthesis and availability to the intestines.

Phylogenetic characterization of β-tubulins and development of pyrosequencing assays for benzimidazole resistance in cattle nematodes

Control of helminth infections is a major task in livestock production to prevent health constraints and economic losses. However, resistance to established anthelmintic substances already impedes effective anthelmintic treatment in many regions worldwide. Thus, there is an obvious need for sensitive and reliable methods to assess the resistance status of at least the most important nematode populations. Several single nucleotide polymorphisms (SNPs) in the β-tubulin isotype 1 gene of various nematodes correlate with resistance to benzimidazoles (BZ), a major anthelmintic class. Here we describe the full-length β-tubulin isotype 1 and 2 and α-tubulin coding sequences of the cattle nematode Ostertagia ostertagi. Additionally, the Cooperia oncophora α-tubulin coding sequence was identified. Phylogenetic maximum-likelihood analysis revealed that both isotype 1 and 2 are orthologs to the Caenorhabditis elegans ben-1 gene which is also associated with BZ resistance upon mutation. In contrast, a Trichuris trichiura cDNA, postulated to be β-tubulin isotype 1 involved in BZ resistance in this human parasite, turned out to be closely related to C. elegans β-tubulins tbb-4 and mec-7 and would therefore represent the first non-ben-1-like β-tubulin to be under selection through treatment with BZs. A pyrosequencing assay was established to detect BZ resistance associated SNPs in β-tubulin isotype 1 codons 167, 198 and 200 of C. oncophora and O. ostertagi. PCR-fragments representing either of the two alleles were combined in defined ratios to evaluate the pyrosequencing assay. The correlation between the given and the measured allele frequencies of the respective SNPs was very high. Subsequently laboratory isolates and field populations with known resistance status were analyzed. With the exception of codon 167 in Cooperia, increases of resistance associated alleles were detected for all codons in at least one of the phenotypically resistant population. Pyrosequencing provides a fast, inexpensive and sensitive alternative to conventional resistance detection methods.

Influence of diet type on the kinetic disposition of fenbendazole in cattle and buffalo.

The plasma concentration profiles of fenbendazole (FBZ), FBZ-sulphoxide and FBZ-sulphone were measured following intraruminal administration of FBZ at 7.5 mg kg-1 body weight in cattle and buffalo offered 3 different diets: 100% dry mature sorghum hay, 100% green Pennisetum spp. and a 50:50 mix of these 2 diets. Changing the diet from dry to green fodder resulted in significantly lower systemic availability of FBZ and its metabolites in both species. Buffalo had a lower systemic availability of the drug than cattle on the dry diet and the difference between species increased when the diet included green fodder. It is suggested that decreased transit time of digesta on the green fodder reduced systemic concentrations by reducing the time available for gastrointestinal absorption of the drug.

Effects of diet and species on the pharmacokinetics of fenbendazole in cattle

Knox, M.R. and Steel, J.W., 1997. Effects of diet and species on the pharmacokinetics of fenbendazole in cattle. Veterinary Research Communications, 21 (1), 37-43.The plasma concentration profiles of fenbendazole (FBZ), FBZ-sulphoxide (OFZ) and FBZ-sulphone were measured following intraruminal administration of FBZ at 7.5 mg/kg bodyweight in Bos taurus and B. indicus cattle offered three different diets: 100% wheaten chaff, 100% lucerne, and a 50:50 mix of these two diets. No differences between the species were apparent except for a longer time to peak plasma concentration for OFZ in the B. taurus steers fed 100% wheaten chaff. Cattle fed wheaten chaff alone gave greater areas under the concentration-time curve and longer persistence for all metabolites than when the same cattle were fed the other diets. It is concluded that the reduced rate of passage of digesta on lower-quality fibrous diets allows greater time for absorption of FBZ and its metabolites from the gut, thereby increasing systemic availability.

Role of host and environment in mediating reduced gastrointestinal nematode infections in sheep due to intensive rotational grazing

We have previously reported marked reductions in faecal worm egg counts (WECs) and drenching frequency in sheep on an intensive rotational grazing system (IRG) in a cool temperate environment with summer-dominant rainfall. These experiments were designed to determine the role of the host and environmental factors in mediating this. The role of host factors was investigated by administering a fixed larval challenge in each of the 4 seasons of the year to groups of 20 young sheep on three different management systems, including IRG. This comprised a mixed larval challenge containing infective larvae of Haemonchus contortus and Trichostrongylus colubriformis 7 days after short-acting anthelmintic treatment. A range of measurements was then made up to day 35 post-challenge. The role of environmental factors was determined by assessing pasture infectivity in four seasons using faecal worm egg counts (WECs) and pooled faecal culture of worm-free tracer sheep. The management systems were high input (HI) with high fertiliser inputs high stocking rate and relatively long grazing periods; typical New England management system (TYP) with moderate fertiliser inputs and stocking rate and relatively long grazing periods and; Intensive rotational grazing (IRG) with moderate fertiliser inputs and stocking rate but very short (mean 5 days) grazing periods and long (mean 103 days) rest periods. IRG sheep had higher mean WEC at 28 and 35 days after fixed larval challenge than HI and TYP sheep in spring (IRG: 9500 ± 1000; HI: 4000 ± 1000; TYP: 7200 ± 1000 eggs/g, P<0.01) and summer (IRG: 8400 ± 750; HI: 5300 ± 800; TYP: 4400 ± 700 eggs/g; P<0.001) and also had lower live weights during these seasons. There was no difference in WEC after the autumn challenge (IRG: 5100 ± 450 HI: 4500 ± 450; TYP: 4200 ± 450 eggs/g; P ≈ 0.36) but IRG had lower WEC than TYP following the winter challenge (IRG: 2900 ± 400; HI: 2300 ± 400; TYP: 4300 ± 400 eggs/g, P<0.01). The tracer sheep (used to determine pasture infectivity) on IRG had significantly lower WECs during winter, spring and summer than those under the other management systems. Faecal culture and larval differentiation revealed that faeces from tracers on IRG contained significantly lower proportions of H. contortus and significantly higher proportions of Trichostrongylus spp. and Teladorsagia circumcincta than faeces from tracers on the HI and TYP treatments. Thus, when IRG was most efficacious for worm control, during spring and summer when short graze and long rest periods were maintained, sheep on this system exhibited greater susceptibility to larval challenge while tracer sheep indicated lower pasture infectivity. This demonstrates that the effects of IRG on WEC are mediated by reduced larval challenge rather than increased host resistance to infection.

Anthelmintic efficacy of five tropical native Australian plants against Haemonchus contortus and Trichostrongylus colubriformis in experimentally infected goats (Capra hircus)

The study of the anthelmintic properties of plants rich in plant secondary metabolites can provide ecologically sound methods for the treatment of parasites on grazing animals. The purpose of the present study was to evaluate the anthelmintic effect of five tropical native Australian plant species rich in plant secondary metabolites on adult Haemonchus contortus and Trichostrongylus colubriformis in experimentally infected goats. Thirty young, nematode-free goats were infected with 2500 H. contortus and 5000 T. colubriformis infective larvae thrice weekly for a week (day 1-7 of the experiment). On day 27 after first infection, the goats were allocated into six groups of five animals per group. From day 28 to day 35, fresh leaves from Acacia salicina, Acacia nilotica, Eucalyptus corymbia, Casuarina cunninghamiana and Eucalyptus drepanophylla were included in the goats diet. Five groups were offered leaves from one of these plant species and one group, the untreated control, received only the basal diet formulated with 20% Medicago sativa and 80% Avena sativa. Following plant material administration, the goats were monitored daily until day 40 and then slaughtered on day 41. Total faecal worm egg output, total production of larvae recovered from faecal cultures, total post-mortem worm burdens and the per capita fecundity of female worms were estimated. The toxicity of the plant species for the goats was measured by histopathological analyses of liver and kidney samples. Results showed that goats feeding on the plant material rich in plant secondary metabolites had significantly lower egg output compared to the control goats (P<0.05). A similar response was found for larval production in both H. contortus and T. colubriformis supporting that egg output was affected in both species. Although the total worm burdens were not affected by the plant material (P>0.05), the per capita fecundity was significantly reduced by E. corymbia, A. nilotica and A. salicina (P<0.05). No signs of toxicity were detected in the liver or kidney samples. It is concluded that goats can benefit from the short-term ingestion of plant secondary metabolites, which reduce the total faecal egg output and thus decrease the potential for re-infection from the pasture.