Editors: L.P. Kahn and D.L. Watson

SUSTAINABLE CONTROL OF INTERNAL PARASITES OF SHEEP (SCIPS) A SUMMARY OF RECENT AND CURRENT RESEARCH ON CONTROL OF INTERNAL PARASITES OF SHEEP Editors: L.P. Kahn and D.L. Watson Table of Contents Introduction...2 Improved diagnostic techniques...4 Flow cytometry and fluorescent staining...4 Faecal antigen detection...4 Specific PCR assay...5 Pasture larval counts of gastro-intestinal parasites of sheep...6 Review of pasture larval counts...6 Application of pasture larval counts in South Australia...6 Parasite management software...7 Pharmacokinetics and metabolism of anthelmintics...8 Prevalence of anthelmintic resistance...9 A New South Wales perspective...9 A Western Australian perspective...9 A South Australian Perspective...10 Research on macrocyclic lactone resistance...11 Development of macrocyclic lactone resistance...11 Genetics of avermectin resistance...11 Scouring in sheep...13 Partial flock drenching to minimise selection for drench resistance...14 Smart Grazing...15 Effects of nutrition on resistance to infection...16 Protein supplements...16 Non-protein nitrogen supplements...16 Trace elements...16 Breeding worm resistant sheep...18 Nemesis project...18 Genetic markers for parasite resistance...18 Blood antibody levels the use of the Host Resistance Test...19 Relationship between resistance to worms and lice...19 Vaccines...21 Worm vaccines based on concealed antigens...21 Worm vaccines based on conventional antigens...21 Irradiated larval vaccine...22 Boosting immunity in young lambs...23 Biological control of parasitic nematodes...24 Copper oxide wire particles for Haemonchus control...25 Condensed tannins to assist worm control...26 Acknowledgments...27 Appendix Contact details for contributors...28

INTRODUCTION In late 1998 The Woolmark Company (now Australian Wool Innovation Ltd, or AWI) commissioned a thorough reappraisal of its projects on internal parasites of sheep and the preparation of a comprehensive business plan for future endeavours in this area. The widespread, and increasing, emergence of anthelmintic resistance to all known classes of anthelmintic drugs was the single most important factor driving the synthesis of a program focussed on sustainable, cost-effective control of internal parasites. After extensive consultation with woolgrowers, researchers, consultants and extension workers, a plan was developed and adopted. Known as SCIPS (Sustainable Control of Internal Parasites of Sheep), the program of research, education/training and technology transfer was offered by tender and contracted out in 1999, and work began in early 2000. The SCIPS projects that are currently in progress are: Project 1. Identification of the most costeffective methods for accurately diagnosing internal parasite infestations (CSIRO Livestock Industries, Agriculture Western Australia, NSW Agriculture). This is a shortterm research project with a finishing date of June 2001. The objectives of the project are to quantify the cost, accuracy and turnaround time of several technologies with the potential to supersede or complement current techniques for diagnosing parasite burdens; and to develop national quality assurance guidelines for parasite diagnosis. Project 2. Development and field validation of computer simulation models of internal parasite infestations (CSIRO Livestock Industries, Agriculture Western Australia). Work has just commenced on adapting the single species simulation model Wormworld into a combined three species model (Haemonchus, Ostertagia and Trichostrongylus). Field validation of the model is also underway in a winter and a summer rainfall environment. The project is due for completion in February 2003. Project 3. Development of a national information database and website to serve as an information/advice resource for sustainable control of internal parasites (University of Sydney & regional collaborators). This is a communication and education/training module for SCIPS, based on a comprehensive update of technical resource material on sheep worm control in Australia. Considerable progress has been made in establishing the website, and although it is by no means complete it is accessible at www.sheepwormcontrol.com. Additionally, a discussion forum has been established for use by professionals involved in worm control (www.vetsci.usyd.edu.au/cgibin/scips/ultimate.cgi). Project 4. Increased knowledge of sustainable control of internal parasites through targeted education/training courses (Veterinary Health Research Pty Ltd, Agriculture Western Australia, and University of New England). This is an education and training module. A series of Professional Research Update seminars, delivered throughout Australia, has recently been completed. In 2001 training courses on sustainable parasite control will be offered to drench resellers in woolgrowing regions across the country. A similar series of seminars/courses for woolgrowers is also being planned. Project 5. Accelerating adoption of Nemesis technology (breeding parasite resistant sheep) by Australian woolgrowers (CSIRO Livestock Industries & regional collaborators, University of Melbourne). This is predominantly a technology transfer module designed to increase the adoption of breeding for worm resistance throughout the industry. CSIRO has appointed a Technology Transfer Specialist for the project and is working with collaborators in each of the States. Staff at the Mackinnon Project (University of Melbourne) are using a push-pull strategy to increase the availability of rams sold with faecal egg count estimated breeding values in Victoria, working with leading commercial woolgrowers and studs. Project 6. Characterising the relationships between scouring and genetically-based resistance to internal parasites in various environments (Agriculture Western Australia, CSIRO Livestock Industries). Work has just commenced on this research module, which aims to determine (in a winter and a summer rainfall environment) whether breeding sheep with enhanced genetic resistance to worms increases the problem of scouring/ diarrhoea/ dagginess. The research phase of the project is due for completion in May 2003. This document, A Summary of Recent and Current Research on Control of Internal Parasites of Sheep, is being distributed as a supplement to the recent SCIPS Professional SCIPS page 2

Research Update seminars. It is not a formal literature review, rather a synopsis that has been compiled to provide industry professionals with a snapshot of the research frontiers in the late 1990's/early 2000's in the area of internal parasites of sheep, with particular emphasis on the Australian industry. For those seeking more detailed information or an entree to the scientific literature the Appendix provides a comprehensive list of key personnel and their contact details. Further information on SCIPS can be obtained from Dr Dennis Watson SCIPS page 3

IMPROVED DIAGNOSTIC TECHNIQUES Faecal egg counts (FEC) and larval differentiation are the basis for diagnosing the severity of and species present in gastro-intestinal nematode infections. Nevertheless, the techniques used in diagnosis have not really improved over the 40 years since the development of the McMaster slide. There are a number of disadvantages associated with FEC and larval differentiation, including considerable variation in FEC between faecal samples taken from the same sheep, and variation between nematode species in their development during culture. Furthermore, FEC are unable to indicate a worm burden until egg laying commences at three to four weeks after infection, by which time the worms are well established and the host may already be suffering adverse effects. The increasing spread of anthelmintic resistance on Australian sheep farms has resulted in a change from the large region-based drenching programs to drenching strategies devised for individual farms. Decisions on when to drench, therefore, depend increasingly on FEC of sheep from the individual farm. Thus, many more FEC are being done. In addition, FEC provide the basis for genetic selection programs for resistance to internal parasites. This section provides a summary of a number of different diagnostic procedures currently being developed and evaluated with the aim of providing a superior diagnostic test to FEC. Flow cytometry and fluorescent staining Current research funded by AWI through its SCIPS program and conducted by Dr Leo Le Jambre and Dr Ian Colditz, CSIRO Livestock Industries, is comparing enumeration and species differentiation of nematode eggs in faeces by flow cytometry with the conventional McMaster light microscope method and larval culture. The advantages of flow cytometry over conventional methods include greatly increased sample size, thus reducing variation between samples in FEC, and decreased time taken for species differentiation since differentiation is performed on eggs rather than larvae. Sample preparation for egg counting by flow cytometry initially uses the McMaster salt flotation method but is followed by centrifugal separation of eggs and addition of an internal standard. Egg counting is performed mechanically by the flow cytometer. Species differentiation of nematode eggs is performed using a similar process with the exception that eggs are specifically stained prior to analysis. In results so far, flow cytometry compares favourably with conventional faecal culture and larval differentiation. There is also an opportunity under the SCIPS project to develop a protocol for a national interlaboratory self-assessment program for worm diagnostic techniques. The NATA-accredited Parasitology Laboratory at Agriculture Western Australia has developed standard procedures for Quality Assurance Assessment of worm egg counts, while NSW Agriculture s EMAI has provided an inter-laboratory proficiency testing program for faecal egg counts, larval identification and liver fluke counts, by co-ordinating the preparation, distribution and analysis of single species and mixed species faecal cultures. The Standing Committee on Animal Health Laboratory Standards (SCAHLS) has indicated that standards need to be established in parasite egg counting and larval differentiation. NSW Agriculture has recently offered to co-ordinate a national assessment program for parasitology testing to SCAHLS. The SCIPS Project will allow self-assessment techniques to be standardised between NSW Agriculture and Agriculture Western Australia by the exchange of assessment protocols between laboratories and the development of a validated standard operating assessment technique. Faecal antigen detection The presence of worms in the gut of a sheep results in the passage of a range of proteins in faeces that would not otherwise be present. The detection and characterisation of such proteins offer the prospect of improved methods of worm diagnosis. A prototype faecal antigen detection test has previously been developed by Agriculture Western Australia (current contact: Dr Dieter Palmer) with support from AWI. The test is an antigen capture ELISA designed to identify and quantify burdens of the major nematodes, hence removing the need for worm egg counts and larval differentiation. The test showed promising results when evaluated on penned sheep infected with different parasite burdens. However, it did not prove as encouraging when evaluated in the field, with results varying from acceptable to poor against different worm genera. Investigation of the problems with the test is continuing, although considerable further development will be needed before a useful field test could become available. Dr Khosse Mitri and Dr Mark Sandeman, La Trobe University, have been following a similar approach. Protein separation procedures clearly show many antigens specific to faecal samples taken from sheep infected with H. contortus or O. SCIPS page 4

circumcincta. The isolation and sequencing of a range of these proteins reveal that most are parasite-derived with some pathologyassociated, host molecules also present. Therefore, these parasites apparently release a wide range of proteins that are able to survive passage through the gut. These proteins are associated with different developmental stages of the parasite and with damage to host tissues at various points during the infection. The diagnostic value of these antigens was tested by ELISA using antibodies specific and sensitive to the target copro-antigens. Results indicate that several of the copro-antigens are potential candidates for the development of specific and sensitive ELISAs for the early detection of haemonchosis or ostertagiosis. The ELISA is able to detect infection as early as five (Haemonchus) and eight (Ostertagia) days post oral infection during the prepatent period and well before the appearance of parasite eggs in faeces. The response also appears to be quantitative. Specificity has been tested for one of the Haemonchus antigens, and the ELISA was found to be highly specific for this species in sheep. Initial work with Trichostrongylus spp suggests hat the same techniques are applicable and should be able to detect specific faecal proteins for a wide range of parasites and hosts. Specific PCR assay Faeces passed from infected sheep also contain nucleic acids from the parasitic nematodes present. Dr Robin Gasser, Dr Ian Beveridge and Dr Neil Chilton, University of Melbourne, have been investigating the application of a highly sensitive DNA test system (i.e. the polymerase chain reaction, or PCR) to detect the DNA of individual species of nematode. The potential advantages of this technique are increased rapidity, and sensitivity and specificity of detection, particularly as infections can be detected before nematode eggs appear in faeces. The specific PCR assay obviates the need for faecal culture and larval differentiation and is potentially able to semi-quantify the level of infection. Although this methodology is not yet available commercially, it provides an alternative prospect for improved diagnosis in dedicated service laboratories. SCIPS page 5

PASTURE LARVAL COUNTS OF GASTRO- INTESTINAL PARASITES OF SHEEP Review of pasture larval counts A review of recent literature and a survey of veterinary diagnosticians was recently undertaken for AWI by livestock industries consultant, Dr Dennis Watson, to evaluate the practicality and cost-effectiveness of pasture larval counts (PLC) as a diagnostic and predictive tool for sheep nematode parasite burdens. Interest in PLC has arisen from the recognition that numbers of infective larvae on pasture may be a useful management tool for integrated parasite control. Pasture larval counts involve direct human sampling of pasture herbage by plucking (designed to simulate the grazing animal) in a designated pattern across a chosen area of pasture. Pooled herbage samples are then subjected to extraction procedures, and nematode larvae recovered, identified and quantified. From the literature reviewed it is clear that PLC have been successfully used as a research tool and for this purpose they have merit. Even as a research method, however, there remain serious concerns about their accuracy and validity - first in terms of the pasture samples collected being representative of the herbage ingested by sheep, and second, in the laboratory in terms of the reliability of recovery of larvae from pasture and accuracy of larval identification and counting in the presence of free-living nematode species. There is little or no encouragement in the scientific literature to extend PLC from its research use to a more widely available diagnostic/predictive tool for use as a management aid on grazing properties. failing dramatically within 1-2 years due to heavy worm infections. In a study over 2 years on 4 pivot systems, lambs were introduced in December after paddocks had been destocked of sheep for at least 3 months. Immediately prior to introduction sheep were drenched with moxidectin and held aside for worm eggs to clean out. Thereafter no drenches were given to lambs grazing the pivot area. Cattle grazed the pivot either together with or following the sheep. Sheep were removed from all systems within 3-6 months and replaced by cattle. Pasture larval counts and worm egg counts remained consistently low throughout both summers, demonstrating the importance of restricting contamination in the previous spring and ensuring that lambs carry no residual infection on to the pasture. Levels of sheep worm larvae on pasture rose in autumn, by which time the sheep had been marketed. Levels remained stable for 2 months and then declined. The study demonstrated that profitable, sustainable production of prime lambs under pivot irrigation over the summer months is possible, provided very strict conditions of pasture preparation and flock sanitation are followed. The PLC review will shortly be available on the SCIPS website (www.sheepwormcontrol.com), or alternatively, it can be obtained from Dr Dennis Watson. Application of pasture larval counts in South Australia One supporter of pasture larval counts is Dr Ian Carmichael, of SARDI Livestock Systems, South Australia. Dr Carmichael has recently applied the technique to evaluate the viability of pivot irrigation systems in south-eastern SA. Pivots operate between November to March to enable prime lambs to be grown out at a time of market shortage. The single most important constraint to the current and future success of pivot farming systems is an inability to control Trichostrongylus vitrinus, with most systems SCIPS page 6

PARASITE MANAGEMENT SOFTWARE Field trials to evaluate new or modified methods for the control of internal parasites of sheep are extremely time-consuming, expensive and virtually impossible to replicate in a wide range of environments. To overcome these difficulties, a simulation model of the population dynamics and genetics of worms ( Wormworld ) has been developed by Dr Robert Dobson and Ms Liz Barnes, CSIRO Livestock Industries, with support form AWI. The development and survival of the free-living infective stages of the parasite on pasture are controlled by climatic data such as rainfall, temperature and evaporation. This information is made available to the model on a daily basis from weather files for the locality in which it is desired to test the control program. The model can be used to explore the potential effectiveness of control technologies still under development, such as genetically resistant sheep, worm vaccines or biological control, as well as modifications of current strategies, including drenching programs, nutrition, grazing management, stocking rates, and lambing and weaning times. The model could be used, for example, to predict the efficacy of worm control and the development of drench resistance over a 20-year period in Albany, WA, under a one or two summer drench worm control program. (Another example is discussed in the section on Research on Macrocyclic Lactone Resistance below.) Currently, there are three separate models, for each of Trichostrongylus colubriformis (Black Scour Worm), Ostertagia spp (Small Brown Stomach Worm), and Haemonchus contortus (Barbers Pole Worm). A new project under the SCIPS program, with AWI funding, will see the three species combined into a single model. Two-year field trials at Chiswick (Armidale NSW) and Albany WA, to further validate the model, are also underway as part of the project. After validation testing, it is likely the predictive model will be made available to users as a module in CSIRO s Farmwi$e software. When used together, Farmwi$e will provide Wormworld with data on feed intake and herbage density and the worm model will provide Farmwi$e with data on inappetence and deaths attributed to worms. In this way the software will provide better estimates of production benefits under different grazing management systems. SCIPS page 7

PHARMACOKINETICS AND METABOLISM OF ANTHELMINTICS The Australian sheep industry currently relies heavily on the use of anthelmintics to control internal parasites. Broad spectrum benzimidazole (BZ) drenches were first released in 1961 and the release of other anthelmintic groups followed over the next 35 years, the most recent addition being that of moxidectin in 1995 and the re-release of naphthalophos in the same year. The release of new anthelmintic groups has been followed by the development in worms of resistance to the anthelmintic. Resistance to BZ drenches now exists on 90% of Australian sheep farms. For the foreseeable future there is little prospect of novel anthelmintic compounds with unique modes of action becoming commercially available. Therefore it is imperative that existing drenches are used in ways that preserve their efficacy for as long as possible. tract. A reduced rate of passage of digesta prolongs the time for drug absorption and recycling. Dr Hennessy has demonstrated that withholding feed for 1 day prior to drenching with BZ, and, for maximum effect for 6 h after, slows the movement of digesta through the gastrointestinal tract and increases the exposure time of parasites to the drug, resulting in a greater reduction in worm numbers. Trials with sheep infected with BZ resistant H. contortus and T. colubriformis have shown that withholding feed is more effective than doubling the dose rate in a single drench. In a practical context the effectiveness of BZ drenches against BZ resistant parasites can be greatly improved by withholding sheep from feed for one day prior to drenching. Dr Des Hennessy, CSIRO Livestock Industries, has developed strategies that improve the efficacy of BZ drenches. These improved strategies are based on detailed knowledge of the whole-body pharmacokinetics of BZ drenches. The approach taken by Dr Hennessy and incorporated into the improved drenching strategies has been to maximise the duration of exposure of the nematode to the drug. The most effective means of increasing the duration of exposure are (1) following the correct drenching technique and (2) slowing the rate of movement of the drug through the animal. Dr Hennessy has demonstrated that with BZ drenches it is the length of exposure to the drug rather than the maximum drug concentration that is the key to effective drenching. The correct drenching technique ensures placing the drench-gun tip over the tongue to direct the entire dose down the throat and into the rumen. Placement of the drug into the rumen is important because it is the rate of flow of digesta from the rumen that defines the duration over which the drug is presented to the parasite habitat. Incorrect placement of the drench-gun may result in closure of the oesophageal groove allowing some or all of the drench to pass to the abomasum and greatly reducing the duration of the anthelmintic action. Field trials have demonstrated that as many as 60% of drenched sheep may not receive the entire dose into the rumen. The second means of increasing the duration of exposure of the nematode to BZ drenches is by slowing the rate at which digesta moves out of the rumen and through the gastro-intestinal SCIPS page 8

PREVALENCE OF ANTHELMINTIC RESISTANCE A New South Wales perspective Resistance to sheep drenches is widespread, with probably 90% or more of farms having resistance problem. Following is an overview by Dr Stephen Love, NSW Agriculture, of the current resistance situation, with particular reference to NSW. once pasture growth resumes in winter, if not diluted with non-selected worms. In the dry areas, one summer drench per year is typical, and resistance to MLs has evolved on some farms after four to six years. In the higher rainfall areas, there are farmers who have used MLs for eight years in a row, two to four times per year, with no evidence of resistance developing. Drench or Drench Group Benzimidazole (BZ) or White drenches Levamisole (LEV) or Clear drenches Combination (BZ + LEV) drenches Macrocyclic Lactone (ML) drenches Naphthalophos (Rametin ) Closantel Triclabendazole (Fasinex ) Prevalence of Resistance* Approximately 90% of properties. Approximately 80% of properties. Approximately 60% of properties. No longer rare; eg in WA, a recent series of faecal egg count reduction trials (FECRTs) showed 38% of farms had MLresistant Ostertagia. In northern NSW, 10-20+ farms have MLresistant Haemonchus. ML resistance in Ostertagia is suspected on a small number of southern NSW farms. One recorded case in Australia ( Lawes strain goats, Queensland). Resistance in Haemonchus is common in northern NSW & S.E. Queensland. One strain also ML-resistant. Small number of resistant strains of liver fluke in Australia. Small number of resistant strains of liver fluke in Australia (Goulburn Valley, Victoria). It is commonly believed that drench resistance is only a problem of the higher rainfall sheep raising areas of Australia. More frequent drenching is required in these areas and this, combined with occasional under-dosing, produces greater selection for resistance in worm populations. However, there is evidence that drench resistance in western NSW may be more prevalent than previously realised, especially given the Western Australian experience suggesting that selection for resistance is stronger in dry environments (see below). A Western Australian perspective Resistance to anthelmintics affects virtually all Western Australian sheep farms, and is believed by Dr Brown Besier, Agriculture Western Australia, to reflect the heavy selection pressure favouring resistant worms, which survive in sheep after summer drenching. In environments where no or very few larvae survive on pastures over summer, any resistant worms remaining in sheep after summer drenching are the main source of future worm populations. Although the number of resistant worms is initially very low, they can increase in proportion (to susceptible worms) very rapidly Resistance to benzimidazoles and levamisole in the major parasite genera, Trichostrongylus and Ostertagia, is present on almost every farm. These drench groups are now of minimal value when used individually; test figures indicate their effectiveness to be less than 60% on 85% (BZs) and 63% (levamisole) of farms. Combination BZ-LEV anthelmintics are more than 95% effective in only about 25% of cases, but remain useful as a tactical drench on most farms. The use of naphthalophos with a BZ, LV or combination BZ-LEV drenches has increased, as other drenches fail. However, considerable variability in efficacy has been shown in tests, and poor results can occur. It is recommended that naphthalophos be used only in combination (except against Haemonchus), with an efficacy test conducted soon after the time of use. The number of confirmed cases of macrocyclic lactone (ML) resistance in Ostertagia has increased rapidly in Western Australia in recent years. The recommended test format differentiates ML efficacy (> 95% effective at a full recommended dose rate) from resistance (effectiveness of ivermectin at a half dose rate). On this basis, figures from 1999 showed ML resistance in approximately 38% of tests, and reduced efficacy at the full dose on 19% of SCIPS page 9

farms. ML resistance has not been detected in Haemonchus or Trichostrongylus. Closantel resistance in Haemonchus has not been detected in Western Australia. A South Australian Perspective In a study conducted by Dr Ian Carmichael of SARDI Livestock Systems, in collaboration with private veterinarians, 70 Merino cross wethers under quarantine were artificially infected with a worm isolate from a farm where worm control had collapsed despite repeated drenching with moxidectin. Faecal egg counts and total worm counts after treatment confirmed that the animals carried a strain of Brown Stomach Worm that was strongly resistant to ivermectin. Low positive faecal egg counts in 4/10 sheep drenched with moxidectin at normal dose rate and moderate counts in 8/10 sheep drenched at half dose rate confirmed developing resistance to moxidectin. Two similar separate isolates have since been confirmed experimentally and others are currently being examined. Each of the properties concerned had used moxidectin consistently. Strategies to identify the extent of the problem and to evaluate sustainable worm management practices in the presence of various levels of macrocyclic lactone resistance are being developed locally through a combination of field and laboratory research supported by industry. SCIPS page 10

RESEARCH ON MACROCYCLIC LACTONE RESISTANCE Development of macrocyclic lactone resistance To understand the factors that influence selection for avermectin/milbemycin (ML) anthelmintic resistance it is necessary to examine the impact of drug treatment, particularly persistent drugs, on all phases of the worm life cycle. Thus a study, funded by AWI, was undertaken by Dr Robert Dobson and Ms Liz Barnes, CSIRO Livestock Industries, to determine the efficacy of various ML anthelmintics against worms resident in sheep at the time of treatment, against incoming larvae (L3) post treatment, and against development of eggs in faecal culture. Homozygous and heterozygous (F1) MLresistant strains of Barbers Pole Worm (Haemonchus contortus) were used to infect sheep before or after treatment with ivermectin (IVM) oral, IVM-capsule, moxidectin (MOX) oral or MOX-injectable. Total worm count and quantitative larval culture were used to determine efficacy against parasitic and freeliving stages, respectively. For IVM-capsules selection for resistance was greatest for adult and L3 stages. It was least selective for egg and L1-L2 larval stages because residual IVM in faeces was highly effective in preventing resistant genotypes developing to L3. MOX was least selective against adult worms as it was highly effective against resistant genotypes. MOX was similar to IVM-capsules in its capacity to be highly selective for resistant L3 genotypes. MOX showed some ability to prevent development of eggs to L3 during its persistent phase. The results indicated no evidence of sex-linked inheritance for IVM resistance. Mean IVM efficacies against homozygous and heterozygous resistant adult worms were not different, and IVM-capsule efficacy against incoming L3 was approximately 70% for all resistant genotypes, consistent with a dominant trait. MOX was highly effective against the adult resistant strains and approximately 76% effective against the incoming L3 regardless of resistance-genotype, also consistent with a dominant trait. Both drugs displayed an ability to inhibit the development in faecal culture of eggs/l1-2 from worms surviving ML treatment. Genetics of avermectin resistance Studies by Dr Leo Le Jambre, CSIRO Livestock Industries, of the inheritance of avermectin resistance (AVR) in H. contortus indicate that, in contrast to BZ and LEV resistance, AVR, in this species at least, is inherited as a completely dominant trait and is not sex-linked. Inheritance of AVR as a completely dominant trait indicates that the progeny resulting from the mating of, for example, a resistant female and susceptible male will be 25% susceptible and 75% resistant. At low resistance gene frequencies, a dominant trait will result in the more rapid development of resistance than if resistance were inherited as a recessive or incompletely dominant trait. Hence, it is expected that AVR in H. contortus will evolve more quickly than even benzimidazole (BZ) resistance under the same conditions. Furthermore, increasing the dose of drug to make the heterozygote susceptible is not an option when dealing with dominant resistance genes because heterozygous and homozygous worms are equally resistant. Two AVR strains of H. contortus were used to investigate the propensity for a non-persistent anthelmintic, ivermectin (IVM), and a persistent anthelmintic, moxidectin (MOX) to select for anthelmintic resistance. Dr Leo Le Jambre and his research team investigated the response to selection for anthelmintic resistance by resident worms and by incoming larvae in adult sheep and in lambs. In these studies the adult sheep developed immunity to the trickle infection, whereas the lambs did not. A major finding of this research was that selection pressure for AVR on resident worms was lower than that on incoming larvae. Selection for AVR larvae in lambs was greater by MOX because MOX prevented re-infection after treatment. In susceptible animals such as lambs, when the anthelmintic was IVM, the resistant worms remaining after treatment were soon again in the minority because of re-infection involving a very small proportion of resistant larvae. With MOX, the resistant worms enjoyed a considerable period of advantage because of its persistency, which increased the period of selection for AVR larvae and as a consequence resistant worms remained in the majority for the period of residual activity. Nevertheless, even with the short-acting IVM, some selection for AVR larvae was apparent. Use of a persistent drug such as MOX increases the chance of matings of resistant male with resistant female worms, providing an opportunity for genetic recombination among resistant worms to develop the next stage of ML resistance. Subsequent computer modelling of Haemonchus contortus populations in a winter rainfall environment where Closantel is used in conjunction with broad spectrum anthelmintics, predicted that IVM capsules would select most rapidly for ML resistance. In the presence on SCIPS page 11

Closantel resistance MOX would be intermediate, and IVM oral would be least selective, however, if resistance to Closantel were absent, then MOX would least selective. SCIPS page 12

SCOURING IN SHEEP Diarrhoea resulting from increased water content of the faeces leads to soiling of the breech wool, commonly referred to as dags. The major problems associated with dags are increased incidence of breech flystrike and loss of value of crutch wool. Dr John Larsen, of the University of Melbourne estimates the cost of sheep dags in the high rainfall region of Victoria at $10 million per annum, and this is likely to be an underestimate during periods of high wool prices. Similar estimates have been made for the high rainfall region of Western Australia. Scouring is an important issue to sheep producers, particularly Merino producers, in both winter and summer rainfall regions. The causative factors of scouring differ between the two rainfall regions. In winter rainfall regions scouring is of two types: that associated with large infections of gastro-intestinal parasites and that due to hypersensitivity-type responses. Hypersensitivity-type responses are unrelated to level of infection and occur most commonly during late winter to early spring when pastoral conditions favour rapidly growing green herbage. In summer rainfall regions scouring is primarily caused by large infections of non-blood feeding gastro-intestinal parasites. In contrast, infections with the blood feeder, H. contortus, result in the production of hard faecal pellets. Of the two types of scouring, hypersensitivity scouring is of most concern, as the causative factors are yet to be fully identified making management to minimise scouring problematic. programs for increased resistance to worms are unlikely to result in reduced incidence of scouring. In fact, estimates from New Zealand and those calculated by Dr Johan Greeff and Dr John Karlsson, Agriculture Western Australia, suggest that scouring may slightly increase as a correlated response to selection for increased worm resistance. If this is confirmed, sheep breeders would need to include both worm resistance and dags in a selection index to ensure against any potentially undesirable genetic correlations. This issue is the subject of current research funded by AWI as part of the SCIPS program. Genetic correlations of dags with other production traits have generally been found to be in a favourable direction. It is likely that undefined factors in green pasture may be minor contributors to hypersensitivity scours; ryegrass endophytes may be one such factor. It has also been suggested that the high levels of soluble sugars in rapidly growing pastures have the potential to cause hindgut acidosis, which would increase the water content of faeces. Because hypersensitivity scouring may occur even at low levels of larval contamination on pasture, traditional worm control options are unlikely to be successful in its control. However, Dr John Larsen has demonstrated that treatment of ewes with controlled release benzimidazole (BZ) capsules can greatly reduce the incidence of severe dag but is unlikely to be cost effective for this purpose alone. Research conducted by Dr John Larsen has demonstrated that in unselected sheep flocks susceptibility to scouring, as measured by dag score and dag weight, is highly repeatable (repeatability 0.41 0.61) between years demonstrating that selection against dags is possible. Importantly, susceptibility to scouring and to worms were found to be unrelated, but in those sheep susceptible to scouring, ingestion of even low numbers of infective larvae induced diarrhoea. No differences existed between scouring susceptible (SS) and resistant sheep in protective immune response to infection, but during infection SS sheep had an increased number of eosinophils in the upper regions of the small intestine and changes to cytokine profiles. Treatment of ewes with controlled release benzimidazole (BZ) capsules has been demonstrated to greatly reduce the incidence of severe dag, indicating L3 or L4 as the likely source of worm antigen-initiated hypersensitivity responses. Because hypersensitivity scouring is unrelated to susceptibility to infection, genetic selection SCIPS page 13

PARTIAL FLOCK DRENCHING TO MINIMISE SELECTION FOR DRENCH RESISTANCE The rapid increase in macrocyclic lactone (ML) resistance in Western Australia indicates an urgent need to review the summer drenching worm control program, to reduce the selection pressure for resistance in some environments. survival, these strategies, with the potential for lost production, may not be necessary. A number of field trials in WA conducted by Dr Brown Besier, Agriculture Western Australia, have compared the effects of traditional summer drenching with those of a modified program that aimed to ensure the survival of some nonresistant worms in sheep over summer. In each trial, either all weaner sheep in a flock were drenched with ivermectin as they went onto crop stubble in early summer, or a proportion of the flock were left undrenched, to provide a flock average worm egg count of 50 eggs per gram. Two findings consistently emerged from the trial. First, significant worm problems in winter were associated with the part-flock drenching strategy. Even low levels of autumn pasture contamination with worm larvae led to high worm burdens, high prevalence of scouring, and significant production losses. The summer drenched sheep suffered no subsequent parasite problems, and maintained good production. Second, the level of drench resistance in Ostertagia increased significantly in the standard summer drenched flock, but did not change from the initial level when some nonresistant worms were retained by partial flock drenching over summer. The trials indicate that resistance dilution strategies can effectively reduce the development of drench resistance, although careful management is necessary to avoid a significant production penalty. Some possible resistance dilution strategies include: Leaving a small part of the flock undrenched over summer Leaving all the flock undrenched, if mean worm counts are low (most likely in adult flocks) Running some undrenched adult sheep with weaners to provide non-resistant worms. Frequent monitoring of worm egg counts will be essential to ensure acceptable worm control. Tactical drenching, or preferably, a move to a less-contaminated paddock, may be advisable to keep autumn pasture contamination at a low level. Importantly, the need to modify summer drenching will depend on the environment; where there is substantial over-summer larval SCIPS page 14

SMART GRAZING Recently, Dr Paul Niven, Dr Norman Anderson and Dr Andrew Vizard, University of Melbourne, have developed and trialed a worm management system for winter rainfall areas that combines strategic drenching with rotational grazing over summer to reduce worm contamination of pasture for weaners during the following autumn. In the winter rainfall regions of Australia summers are generally hot and dry and the majority of worm larvae, at this time, are found inside the animal as opposed to on pasture (the reverse of what is commonly seen in summer rainfall regions). To capitalise on this situation, the two summer drenching program has been developed. The first drench is given at the start of summer and the second towards the end of summer. This program has been effective at reducing worm contamination of pastures during the following autumn because drenching coincides with the time when most of the worms are found inside the animal, thus resulting in the death of the majority of the worm population, and conditions on pasture are inhospitable for egg hatch and survival of larvae, thus minimising re-infection. However, summer rains can lessen the effectiveness of the two summer drenching program by allowing egg hatch, larval development and re-infection of grazing sheep, resulting in greater deposition of worm eggs on pasture. Smart Grazing aims to prevent the loss of effectiveness caused by summer rains and to reduce pasture contamination. To achieve this, dry sheep are grazed at two to three times normal stocking rates for one month after each summer drench. The paddock is destocked during the intervening period and average summer stocking rates are unchanged. Early results have demonstrated that weaners grazed on pastures prepared by Smart Grazing had reduced worm egg counts during the following autumn, winter and spring, peaking at 400epg compared to 250epg in controls. The weaners involved cut 1.93kg of 16.8 micron clean wool/head, compared to 1.75kg of 16.3 micron. The Smart Grazed weaners were also 2 kg heavier by the middle of the following spring. Further results are expected shortly from work conducted during 2000 on five demonstration/evaluation sites, in Victoria, South Australia, New South Wales and Tasmania. SCIPS page 15

EFFECTS OF NUTRITION ON RESISTANCE TO INFECTION Inadequate nutrition and gastro-intestinal nematode parasitism are major constraints to livestock production. The primary effects of worm infections, apart from high levels of mortality during severe outbreaks, are to reduce feed intake, to increase loss of host protein by leakage into the gastro-intestinal tract, and to increase the requirement of the gastro-intestinal tract for protein at the expense of muscle growth. These combined effects, even in subclinical cases, may reduce production by as much as 25-35% for weight gain, 10-25% for wool growth and 20-30% for milk production. However, the nutrition of the animal strongly influences the magnitude to which infection reduces production and also the extent to which the animal can develop resistance to infection. Protein supplements A large number of pen trials and a smaller number of field trials have demonstrated that animals supplemented with a source of rumenundegradable (i.e. bypass) protein have increased resistance and resilience to gastrointestinal parasites. Increased supply of digestible protein hastens the acquisition of resistance, which allows animals to more rapidly expel established worms. Research from New Zealand has demonstrated that an increased supply of rumen-undegradable protein to young sheep and periparturient ewes reduces FEC and worm burdens, whereas increasing the supply of metabolisable energy (ME) is ineffective. When the supply of digestible protein to young sheep infected with T. colubriformis was increased by the addition of 60g crude protein (CP), worm burdens were reduced by 65%. Similarly, when the supply of digestible protein was increased to periparturient ewes infected with T. colubriformis and O. circumcincta, worm burdens shortly after lambing were reduced by 87%. Field trials conducted over two years by Dr Lewis Kahn, University of New England and Dr Malcolm Knox, CSIRO Livestock Industries, have demonstrated that supplementation of periparturient ewes with cottonseed meal (CSM) for the six weeks immediately prior to lambing lowered FEC. Cottonseed meal typically contains 36% CP of which about 50% is rumen undegradable. Supplementation with 250 g/day CSM reduced FEC by about 50% across both years. Increased protein and ME supply can increase production of infected sheep to levels equivalent to those from uninfected sheep not receiving the supplement. Of significance to the economic feasibility of using nutrition as a worm control option are the results of a trial conducted by Dr. Frans Datta and Dr. John Nolan, University of New England, that demonstrated beneficial effects of protein supplementation on resistance to infection, growth rate and wool growth for up to 15 months after cessation of the period of supplementation. Currently, Dr Lewis Kahn, with financial support from ACIAR, is investigating whether protein supplementation will be a useful strategy to avoid production losses while encouraging the acquisition of immunity in weaner sheep to gastro-intestinal parasites, and over what time period any beneficial effects are expressed. Dr Malcolm Knox is conducting similar trials with weaner ewes to determine the effects of short-term supplementation on immunity to infection, live weight gain and reproductive performance. Non-protein nitrogen supplements When livestock are grazed on low quality roughage the most critical nutritional deficiency is often nitrogen. Provision of non-protein nitrogen (NPN), such as urea, in the diet can compensate for this deficiency and stimulate feed intake, increase the supply of microbial protein and improve feed utilisation. Dr Malcolm Knox, CSIRO Livestock Industries, has demonstrated that NPN supplementation (urea as 3% of total intake) to infected animals on low-quality feed increases feed intake, live weight gain and wool production with some benefits to worm resistance. NPN supplementation to infected animals on low quality roughage can result in similar levels of production to those from uninfected animals without NPN supplementation. Urea-molasses blocks (UMB), and dry licks containing urea are popular low-cost means of delivering NPN, energy and minerals. It is suggested that low-cost nutritional supplementation with UMB or dry licks should be included in integrated strategies for the control of nematode parasites in areas or during times of the year where low quality forages are the predominant feed resource and the provision of high quality protein supplements, rich in rumenundegradable protein, is economically prohibitive. Trace elements In addition to protein and energy, dietary deficiencies or variables inhibiting the utilisation of minerals can also limit the ability of the immune system to deal with parasites. Trace elements are components of enzymes and SCIPS page 16

therefore have pivotal roles in biochemical reactions that can have widespread repercussions in animal physiology. Increased metabolic activity can induce clinical signs of mineral deficiency in animals with sub-clinical deficiency. Cells with a short half-life such as lymphocytes are particularly sensitive to trace element deficiency. Theoretically, therefore, deficiencies of most of the trace elements could affect the development of protective immunity to worms, but this is a relatively unexplored area. Dr Susan McClure, CSIRO Livestock Industries, has demonstrated that diets low in Mo impede the ability of sheep to reject a challenge infection of T. colubriformis, and other researchers have shown similar effects with H. contortus. The development of resistance to T. colubriformis in housed 8-month-old Merino weaners was assisted by a dietary intake of Mo in the order of 0.15-0.30mg Mo/kg live weight, provided by feeding diets containing approximately 6-10mg Mo/kg DM. This appears to have been an effect on the immune response to initial exposure to T. colubriformis and was associated with three- to five-fold decreases in egg and worm counts after challenge. Mo may enhance the inflammatory response to nematodes either directly, or indirectly by reducing the effectiveness of local Cudependent anti-inflammatory enzymes. Normal pastures contain 0.5 to 3.0mg Mo/kg DM, which is less than the optimal range for development of resistance. worms, and ninety others were kept worm free. Within both groups 30 animals were given cobalt bullets, 30 received a monthly injection of vitamin B12 and 30 had no cobalt supplementation. The weaners were grazed for 6 months on a cobalt deficient farm which had been destocked of sheep for more than a year and was considered to be "worm free". Cobalt deficient lupins were supplied as a field supplement. Monthly body weights of all animals and plasma vitamin B12 levels of selected animals were measured to assess cobalt status. Standard fleece measurements of all animals were done at the end of the trial. There were clear detrimental effects of cobalt deficiency, worm infection (despite worm levels being very low) and a cobalt by worm interaction in the parameters measured. There were no differences between animals that received cobalt by means of a bullet or by monthly injection of vitamin B12. A further study with a similar design is in progress using heavier weaners with greater worm burdens on a higher plane of nutrition. Field trials of supplementation with Mo have not yet been done. Further studies may indicate that interactions with other minerals affect the ideal range of Mo concentrations, and caution will need to be exercised, as supplementation of a diet already adequate in Mo or deficient in Cu may result in toxicity, and the levels quoted above are higher than maximal recommendations for long-term feeding. Cobalt deficiency can impair the immune function of sheep and this may increase vulnerability to infection with worms. Cobalt deficient soils are found in many Australian agricultural areas, including large areas of South Australia. Only one field study to date has attempted to examine the interaction between cobalt nutrition and internal parasitism. SARDI Livestock Systems Parasitology Group (contact Dr Ian Carmichael) examined the influence of cobalt deficiency on productivity in grazing weaner sheep with a low nutritional plane and sub-clinical worm burdens. Ninety merino cross weaners were artificially infected with a standard low dose of the commonest local SCIPS page 17