Veterinary Science - Theses
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ItemEffect of feeding slowly fermentable grains as protection against heat stress in ruminants( 2017)The combination of high environment temperatures, the heat increment resulting from feed fermentation and digestion (HI), high metabolic rates and metabolic heat production (HP) make ruminants susceptible to heat stress (HS). The reduction in the ratio forage-to-concentrate in the diet during HS is a traditional nutrition management aimed at increasing the energy content of the diet to compensate for reductions in dry matter intake (DMI) frequently observed in heat stressed ruminants. Wheat is a rapidly fermentable grain commonly used as an energy source for ruminants in Australia. However, the rapid rate of rumen starch fermentation of wheat is associated with rumen acidosis and elevated body temperature in cattle. By contrast, slowly fermentable grains, like corn, are associated with better utilization of metabolisable energy (ME) and reduced HI. Chemical treatment of wheat grains with sodium hydroxide (NaOH) or Bioprotect (BP) may reduce the rate of starch fermentation, HI and metabolic HP. The effect of feeding cereal grains differing in rumen fermentability on physiological, metabolic and productive responses of ruminants under high thermal load has not previously been evaluated. Therefore, it was hypothesised that by feeding slowly fermentable grains, the HI would be reduced, thereby reducing the effects of HS in ruminants. The research presented herein firstly investigated the differences between rapidly (barley and wheat) and slowly fermentable grains (corn, 3 % NaOH- and BP-treated wheat) on gas production kinetic parameters, pH, and starch and dry matter (DM) digestibility using in vitro experiments. Then, using in vivo experiments the effect of feeding either slowly or rapidly fermentable grain diets at different feed intake on HS related changes on physiological, metabolic and digestibility variables of wethers and the physiological, metabolic and productive responses of lactating dairy cows fed grain-based diets differing in grain fermentability during summer were investigated. In vitro experiments demonstrated the slower fermentability and higher pH during incubation of corn, BP- and 3 % NaOH-treated wheat compared with untreated wheat and barley. In vivo experiments demonstrated that HS induces changes in physiological variables, blood-acid base balance, and energy utilization in ruminants; that elevated DMI increases the thermal load of wethers under HS; that feeding wethers either a corn- or 3 % NaOH-treated wheat based diet, may increase the glucose available for intestinal absorption, can reduce the HI and ameliorates the physiological variables negatively affected by HS compared with untreated wheat; that the reduced total apparent starch digestibility of corn diets explains the improved responses observed in corn-diet fed wethers under HS condition; and that feeding 3 % NaOH-treated wheat improves apparent starch, DM digestibility, and DMI of wheat diet without increasing the metabolic HP. In the dairy cow experiment, a lower rectal temperature and higher milk yield was found in cows fed corn diets that were associated with reduced HI and metabolic HP, and higher glucose availability than in cows fed untreated wheat. It was also demonstrated that rumen temperature is a sensitive predictor of core body temperature and physiological and productive variables negatively affected by HS. Thus, this thesis demonstrated that the use of slowly fermentable grains is a viable nutrition management to improve thermo-tolerance of ruminants under high heat loads.
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ItemBacterial enteritis in young Merino sheep.( 2017)Diarrhoea during winter is a common problem of sheep in the high rainfall regions of south-eastern Australia. Gastrointestinal nematodes are the primary cause of this problem, especially in young Merino sheep (those <16 months old). However, a syndrome of persistent diarrhoea (‘winter scouring’) is also relatively common in young sheep with low or moderate nematode burdens, with outbreaks usually occurring between June and October. Affected animals have profuse, watery diarrhoea, and often show signs of abdominal discomfort. Before this study, the cause of this syndrome and the epidemiology and risk factors for outbreaks and faecal shedding of the causative organisms, were not well understood. To determine the primary cause of the syndrome, 45 outbreaks of ‘winter scouring’ were investigated on 24 farms across south-eastern Australia. Necropsies were conducted and faecal samples were collected from animals in affected flocks. Faecal and tissue samples were cultured for Yersinia, Salmonella and Campylobacter species, and faecal WECs conducted to assess nematode parasitism. The sensitivity of Yersinia isolates to four common antimicrobial compounds was also assessed. Yersinia pseudotuberculosis serotype III and virulent Y. enterocolitica were the most frequently isolated bacteria, with outbreaks typically occurring when more than 10% of the mob were shedding either of these organisms. Campylobacter and Salmonella did not play a role in any of the outbreaks investigated. Over 64% of all Yersinia isolates were resistant to sulphonamides, but 98% were susceptible to oxytetracycline. These results highlighted the need to investigate the epidemiology and risk factors of yersiniosis in more detail to support the development of more effective control strategies. As a first stage of this process, a longitudinal study was conducted on four farms to evaluate the patterns of faecal shedding of Yersinia and assess some potential risk factors for outbreaks of yersiniosis, including live-weight, average daily weight gain and WEC. Faecal shedding of both species occurred predominantly during the winter, but shedding of Y. enterocolitica also occurred at other times. Therefore, in addition to scouring in winter, Y. enterocolitica can also cause diarrhoea at other times. In contrast to the outbreak investigations, the prevalence of faecal shedding of Yersinia species was often very high (>30% of animals within a mob) without any clinical disease. This suggests that complex interactions between risk factors contribute to yersiniosis, and that simply monitoring the prevalence of faecal shedding of Yersinia species will not be a reliable predictor of an outbreak of yersiniosis. High WECs (>300 eggs per gram) were associated with an increased risk of shedding each Yersinia species, and so effectively controlling gastrointestinal nematodes should also decrease the risk of yersiniosis. Animals that gained weight were less likely to shed Y. enterocolitica, but more likely to shed Y. pseudotuberculosis. Despite this, the current strategy of ensuring weaner sheep (those 3-12 month of age) continue to gain weight during autumn and winter is still recommended. The effect of an autogenous bacterin against Y. pseudotuberculosis serotype III was assessed on two farms that consistently had severe outbreaks of yersiniosis. There was no outbreak in the year of the study (2014), and vaccinated weaners had similar live weight, growth, faecal shedding of both Yersinia spp. and antibody titres to Yersinia outer membrane proteins to unvaccinated controls. Consequently, further studies to evaluate the efficacy of vaccines during an outbreak of yersiniosis are required.
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ItemImpacts of heat stress on the biomarkers of oxidative stress in sheep and potential nutritional strategies for amelioration( 2015)Heat stress (HS) is a multi-billion dollar global problem as it impairs animal performance during the summer. Heat stress has been implicated in promoting oxidative stress either through excessive production of reactive oxygen species (ROS) or decreased antioxidant defence. Excessive ROS production overwhelms the antioxidant defence, and this leads to oxidative damage of biological molecules including proteins, lipids and DNA, which in turn disrupts normal metabolism and physiology. Therefore, a robust antioxidant network capable of preventing oxidative damage of biological molecules holds promise for improving the health and performance of animals during heat stress. This thesis investigated the impacts of heat stress on the oxidative stress biomarkers, elucidated potential role and optimized the dose of vitamin E and selenium required to ameliorate heat stress in sheep. The first study investigated the impact of HS and dietary antioxidant supplementation on the oxidative and physiological status of sheep. This study showed that heat stress negatively affects the oxidative status of sheep along with the physiological responses; however some of these affects can be ameliorated through dietary vitamin E (Vit E) and selenium(Se) supplementation at supranutritional concentrations. To understand the molecular mechanisms involved in heat stress ameliorative action of Vit E and Se, mRNA expression of heat shock proteins (HSP) and pro-inflammatory genes were investigated. High dietary antioxidants proved to modulate skeletal muscle expression of HSP, pro-inflammatory cytokine and NFĸB transcription factor, which may protect against HS in sheep. The potential role of high dietary Vit E or Se in amelioration of HS in sheep, and changes in acid base balance and respiratory oxidative stress biomarkers along with systemic biomarkers, were investigated in the second study. This study suggested that the hydrogen peroxide concentration in exhaled breath condensate (EBC) can be utilized as a novel biomarker to demonstrate respiratory oxidative stress induced by HS in sheep. The study also highlighted the potential of Se to reduce respiratory oxidative stress. While Vit E improved the physiological responses of heat stressed sheep; combined supplementation of Vit E and Se was required to prevent the acid base imbalance in heat stressed sheep. Finally, optimization of dietary levels of Vit E and Se to improve the performance and oxidative status of lambs finished during hot conditions was undertaken. This study suggested that supranutritional levels of dietary Vit E and Se increased average daily feed intake and the average daily gain in lambs during finishing and maintained the oxidative balance during exposure to HS. Thus it is suggested that heat stress leads to oxidative stress in sheep and supranutritional supplementation of dietary Vit E and Se can be used as a nutritional strategy to ameliorate negative effects of HS in sheep. Further research is required to elucidate the effects of dietary Vit E and Se on retail colour stability and shelf life of meat obtained from lambs finished under hot conditions.
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ItemInduction of tolerance versus immunity following pulmonary vaccination( 2014)When targeting mucosal pathogens, it is generally accepted that vaccination at mucosal sites will generate a more efficient mucosal immune response. As influenza is an infection of the airways, a vaccine that targets the actual site of infection should result in a more efficient immune response as compared to the conventionally delivered vaccines. The lungs are continuously exposed to environmental antigens and allergens, and the induction of immune responses against these molecules would result in continuous immune stimulation, which would eventually harm the host. As a result, immune responses in the lungs need to be tightly regulated to prevent a hypersensitive response leading to inflammation. Exposure of antigens to the lung often leads to induction of tolerance rather than immunisation, therefore, the lung may not be the most obvious site for induction of mucosal immunity. For a pulmonary vaccine to work, this tolerance first needs to be understood and overcome so that a protective immune response can be generated. In order to develop a pulmonary vaccine, the challenge of inducing immunity rather than tolerance must be overcome. The specific aims of this thesis were to i) determine if mucosal tolerance is induced in the lung following exposure to influenza antigen without an adjuvant, and if so what this mechanism is, and ii) determine the mechanism responsible for avoiding this tolerance leading to an immune response after pulmonary vaccination with an influenza ISCOMATRIX(TM) vaccine. Pulmonary vaccines were delivered to sheep, and antibody titres in both the lung and the blood were determined through ELISA. Tolerance-associated markers and cytokines were also measured after vaccination through the use of qPCR. It was found that if sheep were vaccinated with an initial dose of influenza antigen followed by an influenza ISCOMATRIX(TM) vaccine, the antibody response generated was significantly reduced compared to animals that received an influenza ISCOMATRIX(TM) vaccine only. Results suggest that this initial exposure to influenza antigen may cause a tolerizing effect in the lung. In contrast, the expression of the regulatory T-cell marker FOXP3 and regulatory cytokines (associated with the induction of tolerance) were significantly reduced in vaccinated animals in the presence of adjuvant, indicating that regulatory T-cells may need to be suppressed in order to generate an efficient immune response. This decrease in a regulatory response should indicate the production of either cell-mediated or a humoral immune response, however, it was found that neither a Th1, Th2, or Th17 response was generated following pulmonary vaccination with an influenza ISCOMATRIX(TM) vaccine. In the long-term, it is hoped that these improvements might contribute towards making lung-delivery of influenza vaccines a more feasible option in humans.
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ItemMortalities in sheep transported by sea( 1995)Major animal welfare concerns existed about mortalities which occurred among the two million sheep transported by sea from Victoria, Australia to the Middle East each year during the 1980s. Research was undertaken to better define the extent of the mortalities, to establish the causes of death, and to prioritise and methodically unravel the underlying causes and risk factors involved. An analysis of five years of existing industry data on 100 sheep shipments from Victoria (1984/85-1988/89) showed that mortality rates ranged mostly between 1% and 4%, with an average of around 2 ½%. These analyses also showed that sporadic episodes of sudden, high mortality occurred in association with hot and humid conditions in Middle Eastern waters. Heat stress was the presumptive diagnosis. While the occurrences were rare, they accounted for 9% of all deaths. Observational studies undertaken on 8 research voyages with sheep to the Middle East showed that two other causes of death – an inanition syndrome and salmonellosis – accounted for the majority of all mortalities. Together, these two conditions caused around three quarters of all deaths. The following risk factors associated with these two major diseases were identified: poor feeding behaviour in the assembly feedlot; physiologic stress at the point of arrival of sheep at the feedlot; and the physical location of sheep on board ships, whereby sheep in the upper tier of pens on every deck of a ship suffered substantially higher mortality rates than those in the lower tier. Specific studies on the inanition syndrome showed that affected sheep were persistent poor consumers of the pelleted shipboard ration. A problem of severe weight loss was detected in an additional 5% of sheep, which appeared to be associated with inanition. Sheep with the syndrome still had an appetite for familiar food such as hay. An hypothesis was developed that the syndrome may be caused by the failure of affected sheep to recognise or accept the pelleted ration as food. The epidemiology of salmonellosis outbreaks in the live sheep trade was elucidated. The outbreaks occurred as rise and fall epidemics starting with a large increase in faecal salmonella excretion from sheep immediately after their arrival in the assembly feedlot near the port of embarkation. Epidemics of mortalities from salmonellosis peaked one to two weeks later, then declined before the voyage was completed. Evidence was obtained that salmonella infections persisted in the feedlot environment between consecutive batches of sheep initiating new epidemics in subsequent consignments. The differences in shipboard mortality rates between upper and lower tier pens (the “tier’ effect) was investigated with observational studies and a major field trial. An hypothesis that the effect was caused by higher light intensity in upper tier pens was tested and rejected. A remaining hypothesis is that the sheep’s fear of elevation in upper tier pens may be the cause. Overall, the research has advanced knowledge substantially on animal health in the live sheep trade. The findings will influence the debate on animal welfare in the trade, they will be of practical use to the industry, and they have focussed research direction for the future.
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ItemThe effect of time of shearing on wool production and management of a spring-lambing merino flock( 2006)Choice of shearing time is one of the major management decisions for a wool-producing Merino flock and affects many aspects of wool production and sheep health. Previous studies have investigated the effect of shearing on only a few of these factors at a time, so that there is little objective information at the flock level for making rational decisions on shearing time. This is particularly the case for flocks that lamb in spring, the preferred time in south-eastern Australia. A trial was conducted in a self-replacing, fine wool Merino flock in western Victoria, from January 1999 to May 2004, comparing ewes shorn annually in December, March or May. Within each of these shearing times, progeny were shorn in one of two different patterns, aligning them with their adult shearing group by 15–27 months of age. Time of shearing did not consistently improve the staple strength of wool. December-shorn ewes produced significantly lighter and finer fleeces (average 19.1 μm, 3.0 kg clean weight), whereas fleeces from March-shorn ewes were heavier and coarser (19.4 μm, 3.1 kg). Fleeces from ewes shorn in May were of similar weight to fleeces from March-shorn ewes (3.1 kg), but they were of significantly broader diameter (19.7 μm). In young sheep, beneficial changes in some wool characteristics for each shearing group were offset by undesirable changes in others. Shearing ewes in March or May, and weaners in March, May or June, significantly increased the risk of post-shearing mortality about three- and four-fold, respectively, compared to unshorn sheep. Substantial, highly significant associations in young sheep between post-weaning mortality, bodyweight and growth rate were also quantified using various survival analysis techniques. For example, the lightest 20% of weaners at weaning contributed 31% of all deaths in the year following weaning, and increasing average growth rate over summer and autumn from 250 to 500 g/month reduced the risk of death by 74%. These results could be used to develop supplementary feeding systems that efficiently reduce weaner mortality, which is a significant animal welfare issue in many Australian Merino flocks. Mortality effects were incorporated into estimates of the total value of wool produced by the different shearing times between birth and culling at 6¼ years of age. Using median historical (1991–2006) wool prices, shearing ewes in March and their progeny first in June, or October (weaner)-December (ewe) shearing produced the greatest total value of wool ($111/head). March (weaners)-March (ewes) shearing had a wool value of $107/head and December (weaners)-December (adults) shearing $103/head. May-shorn ewes produced the smallest value of wool, irrespective of whether their progeny were first shorn in May or July ($93–96/head). No shearing time consistently improved all animal health measures. May-shorn ewes had significantly more fleece rot in late autumn than the other shearing groups (odds ratio 2.5) and were up to 0.4 condition score lighter during winter, although they had a lower cost of dag (average $0.64/head) and significantly less breech strike risk in spring, compared to December-shorn ewes (odds ratio 0.18). December-shorn ewes had the greatest cost of dag ($1.50/head). March-shorn ewes had an intermediate cost of dag ($1.03/head) but significantly less breech strike than May-shorn ewes (odds ratio 0.38).Overall, December and March shearing were shown to be appropriate alternatives for a self-replacing Merino flock in south-eastern Australia, whereas May was an undesirable shearing time.
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ItemThe therapeutic use of mesenchymal precursor cells in an ovine collagen induced arthritis model of rheumatoid arthritis( 2013)Rheumatoid arthritis (RA) is an autoimmune disease that affects 1% of the population and causes joint destruction, deformity and substantial disability. The current treatment of the disease alleviates the clinical symptoms to some extent but does not affect a cure. Also, the patient’s response to current therapeutic options is highly variable and often incomplete, reflecting the complex involvement of multiple pathways in RA disease progression. Furthermore, continuing anti-inflammatory treatment may suppress immune responses essential in host defense. Thus, despite current therapies, novel therapeutic approaches to RA are required. Mesenchymal stem cells (MSC) are self renewable, multipotent, non-hematopoietic progenitor cells that have the capacity to differentiate into various lineages like adipocytes, osteocytes and chondrocytes. Additionally, they have immunomodulatory properties that are considered as a potential therapy for immune mediated diseases and for RA in particular. To examine the therapeutic effect of these cells, animal models that can mimic the human disease are required in order to investigate the immunomodulatory effects of these cells. Collagen induced arthritis (CIA) is the most studied and used RA model in animals, as it shares many pathological and immunological features of the human disease. Although CIA has been examined in a number of species including mice, rats and monkeys, it has not been fully investigated in a large animal model that might offer more advantages over the current rodent models and which may more closely resemble humans in joint size and degree of load bearing. The aims of this study were first to develop the CIA model of RA in sheep and confirm its reproducibility and capacity to provide efficient assessment of disease activity before investigating the efficacy of allogeneic ovine mesenchymal precursor cells (MPC) which had been immune-selected and culture-expanded to reduce arthritis in this model. MPC are a later developmental stage of MSC and because of their purification techniques through the isolation using stro1 and stro3 antibodies, they are more homogeneous population of cells than MSC. Two subcutaneous (s.c.) injection of bovine collagen type II (BCII) in freund’s adjuvants followed with intra-articular (i.a.) injection of the same protein in saline in the hock joints has resulted in establishment of CIA in sheep with arthropathy displaying much of the classical pathology associated with human RA. Further, it provided clinical, histopathological and immunopathological features that can be used in testing biological therapeutics for the treatment of RA, such as stem cells. Following induction of arthritis with collagen, clinical signs of lameness and swelling were evident in all sheep and gross thickening of the synovium surrounding the hock joint and erosion on the cartilage surface was evident at necropsy (2 week and 6 week time points). Leukocyte cell counts and levels of antibodies to BCII were increased in serum and synovial fluid (SF), and there was synovial hyperplasia, thickening of the intimal layer, inflammation and marked angiogenesis in the synovial tissue. There was a large influx of monocytes and both T and B lymphocytes to the synovial tissue, many of which appeared to be in active phases of their cell cycle. After CIA establishment in sheep, the therapeutic effects of allogeneic ovine MPC were investigated in the early stages of CIA by Intravenous (i.v.) administration of MPC one day after arthritis induction. The development of arthritis was followed for two weeks before the sheep were euthanized. Sheep in the MPC treated group showed significantly reduced clinical signs throughout the trial and also showed significant reductions in the pro-inflammatory markers, IL-6 and acute phase proteins (APP) in blood. Activin A levels in both blood and SF of the treated group were also significantly lower than in the control group. Conversely, blood levels of the anti-inflammatory cytokine IL-10 showed a large spike following the administration of MPC. MPC treatment also lowered the neutrophil response in blood following arthritis induction, and the influx of CD4+ cells and monocytes into synovial tissues. All of these effects were accompanied by a significant reduction in the severity of histopathology in the synovial membranes of the treated hock joints compared to the control ones. These results indicate that the immunomodulatory features of MPC have a dramatic effect on acute inflammatory processes involved in arthritis and could become a first line treatment for reducing inflammation and destruction in the joints of people suffering from severe RA. The second MPC study in this thesis was to investigate the effects of MPC on later stages of arthritis using three different MPC doses and with i.v. and i.a. routes of administration. MPC were administered two weeks after arthritis induction and the sheep were monitored for four weeks. In the control sheep, histopathological analysis of hock joints demonstrated that the left hock synovium was characterized by leukocyte infiltration, synovial hyperplasia, pro-inflammatory cytokine expression, and cartilage erosion. In comparison with saline treated controls, synovial tissue from arthritic sheep receiving a single i.v. injection of MPC showed reduced histopathology scores and reductions in the expression of the pro-inflammatory cytokines IL-6, TNF-α, and IL-17 and absence of IL-1β expression. This was accompanied by a significant reduction in infiltrating monocytes/macrophages and a slight increase in the levels of the anti-inflammatory cytokine IL-10. The i.a delivery of MPC did not appear to lessen the intensity of the arthritis. These results indicate that a single i.v. injection of MPCs in an ovine model of collagen-induced RA attenuates joint inflammation, involving inhibition of the Th17 T cell subset and monocyte-derived production of multiple pro-inflammatory cytokines. Together, these data suggest that MPCs may have great potential as a first line treatment for joint inflammation and RA disease progression.
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ItemThe pathogenesis and control of diarrhoea and breech soiling ('winter scours') in adult merino sheep( 1997)Diarrhoea and soiling of the breech with faeces (‘dag’) is one of the most prevalent problems of adult Merino sheep grazing improved pastures in south-eastern Australia during winter and early spring. This syndrome (‘winter scours’) occurs even on farms using programs which effectively control gastro-intestinal nematodes. The causes and costs of winter scours have not been well defined. Comparisons were made among groups of ewes, with and without treatment with controlled-release capsules containing albendazole. Adjusted odds ratios indicated that untreated ewes were 12 to 16 times more likely to be affected with severe dag than ewes treated with a capsule. From this, it was concluded that trichostrongylid parasites were a necessary cause of winter scours. Subsequently, comparisons were made, between affected and unaffected ewes, for faecal worm egg counts and total worm counts. There were no relationships between these indicators of adult parasitism and the occurrence of diarrhoea. The sum of these observations provided strong evidence that winter scours is associated with the ingestion of trichostrongylid larvae. Further, it was concluded that selection for decreased faecal worm egg count would not reduce the prevalence of winter scours. The importance of host factors was examined. Significant differences were detected in the mean dag scores of different strains of Merino sheep, and winter scours was highly repeatable between years (r = 0.44 to 0.61), thus demonstrating that host factors were important in determining susceptibility to winter scours. Within sheep selected as being not susceptible to winter scours, high doses of infective larvae (20,000 Ostertagia circumcincta and Trichostrongylus vitrinus L3/week) did not induce diarrhoea. In contrast, within sheep selected as being susceptible, even small doses of larvae (2000/week) initiated scouring. Sheep selected as being susceptible also had persistently higher faecal moisture levels than non-susceptible sheep, even during times when accumulation of dag did not occur. The immune responses of affected and unaffected sheep were compared. No significant differences were detected between mast cell and globule leucocyte counts, antibody levels in serum, gut tissue and mesenteric lymph nodes, circulating eosinophil counts or the dermal eosinophil response to the intra-dermal injection of mitogens and larval antigens. From this, it appeared that there was no difference in the protective immune response to gastro-intestinal nematodes of affected and unaffected sheep. A feature of the immune response of affected sheep was a hypersensitive inflammatory reaction, characterised by the infiltration of significantly more eosinophils, and changed lymphocyte populations, in the pylorus and upper jejunum. The changes in the lymphocyte populations included a reduced CD4+:CD8+ T-cell ratio, due mainly to significantly reduced numbers of CD8+ T-cells, and significantly reduced numbers of cells reacting to interferon-gamma (IFN-y) monoclonal antibody. Overall, winter scours was estimated to cost the Victorian sheep industry at least $10 million per annum. Improved strategic worm control programs are unlikely to prevent winter scours, because only low doses of infective larvae initiated diarrhoea in susceptible sheep. Phenotypic culling and genetic selection was proposed as the most suitable long-term control strategy to remove sheep susceptible to the hypersensitivity inflammatory response, and thereby reduce the prevalence of severe dag in adult Merino sheep.