Research Projects - Sheep

Professor Kym Abbott, Charles Sturt University
Novatis Australia Ltd
Merial Australia Pty Ltd

Sheep were exposed to different levels of M ptb, from birth to weaning and/or from weaning onwards. The different levels of exposure were high (H), medium (M) and low (L) with the level of exposure in the H groups about 10 times higher than in the M groups. The L groups were not deliberately exposed to M ptb but accidental contamination at very low levels did occur. The experiment was replicated.

The study demonstrated that careful management of young sheep can reduce the level of OJD in the flock and reduce the death rate. Steps taken to limit the degree of exposure of pre-weaned lambs to infection from pastures will lead to reduced rates of severe infection in those sheep in later years. Continuous exposure to OJD bacteria throughout early life results in higher infection rates than exposure which is limited to either the pre-weaning or the post-weaning period alone. A critical factor in management is to provide 'low risk' pastures to young sheep at weaning to give them a break from exposure to infection which occurs in the lambing paddocks.

Producers who successfully limit the infection will find themselves in an improved position. Additionally, by using the library of samples collected during the experiment, many more insights will be gained into the epidemiology of OJD, including the relationship between age at first exposure and the time before excretion of the organism, seroconversion, sub-clinical disease effects, clinical disease and death. These insights may allow the development of additional management options and systems which restrict the impact of OJD in flocks and will also improve the overall understanding of the pathogenesis of the disease, interpretation of existing and new diagnostic tests and the value of emerging research tools.

Vaccination with Gudair® vaccine in OJD infected flocks has commenced in Australia. To date, use of vaccine has been recommended to be limited to lambs in an attempt to protect sheep before OJD pathology becomes advanced. As vaccination is considered by many graziers to be their best option for reducing losses and managing the disease, careful documentation of the effects of vaccination in a high prevalence infected flock and in older sheep will enhance knowledge and understanding of the benefits of the whole-flock vaccination strategy.

The proprietors of a farm reported a very high mortality believed to be due to OJD. Early estimates suggested that 25% of the flock had died in 1999. A preliminary investigation by The University of Sydney supported the owner's initial suspicion, with post mortem findings suggesting an annual mortality rate of 18.1% (+/- 12%). Vaccination of all sheep was favoured as the quickest way to reduce the OJD-contamination of pastures, the incidence of OJD and the death rate due to OJD.

The study concluded that vaccination combined with management changes led to a significant decline in the risk of OJD mortality and effective control of OJD in a heavily infected flock. Findings also suggested that vaccination may be beneficial in sheep as old as 8 months, even when exposed to a heavily contaminated environment since lambing.

This information will have immediate application to a large number of affected producers in NSW who have chosen or are considering vaccination as their major or initial method of OJD control.

This study provided the first estimate of flock mortality due to OJD based on objective data and was central to explaining the economic impact of OJD in high prevalence flocks. This information assisted development of rational control programmes.

Anecdotal reports of the extent of mortality due to ovine Johne's disease ranged from less than 1% to over 20% of adult sheep per year, but there were no objective data. The aim of this study was to estimate the annual mortality rate due to OJD on twelve affected sheep flocks in four different regions of NSW using the methods developed in project OJD.015.

OJD mortality estimates were derived from farm records (livestock inventories) and quarterly farm visits (necropsy inspections). A most likely cause of death was determined for 362 sheep on the basis of findings related to the environment, clinical signs, gross pathology and histopathology. OJD was most likely to have contributed to the death of 250 of these sheep. OJD mortality increased from 1 year of age (10.4%) to peak at 4 years of age (35.6%) and was very similar between wethers (49.6%) and breeding ewes (50.4%).

On the 12 farms, the average OJD mortality rate based on inventory records was 6.2% (range 2.1% to 17.5%), more than twice that considered acceptable (from all causes) in sheep flocks in southern Australia. The OJD prevalence in 2-year old sheep based on pooled faecal culture ranged from 0.7% to > 23% on the 12 farms and was found to be associated with OJD mortality rate.

The average decrease in gross margin due to a farm being infected with OJD was 6.4% (range 2.2% to 15.4%) and the average estimated cost of OJD losses on the 12 farms over the 12- month study period was $64,100 ($15,569 to $154,083). The average estimated cost of annual OJD losses/DSE was $7.68 ($0.84 to $20.51) while annual OJD losses/ha were $65.92 ($6.75 to $244.80).

This study provided the first objective data on the true impact of OJD and the findings are generally applicable to sheep flocks in southern Australia. Industry groups claiming that OJD does not present a threat on-farm can now be provided with accurate figures on direct losses attributable to OJD within the endemic area of NSW. There was a wide range of impacts, with some very high mortality rates. The data can be used to justify vaccination programs, other control options and the general concept of disease control and prevention.

The challenge for industry is to use the scientific findings from this study and other recent research to prepare education and extension material to address issues of misinformation about OJD and to develop cost effective strategies for the future control and management of OJD.

September 2001 - October 2005

The aim of this project was to determine whether pasture contamination rates and the age of sheep when they are first exposed to infection influence the occurrence of ovine Johne's disease. The outcomes were targeted to improve understanding of the development of OJD and will facilitate development of control strategies based on pasture management.

The principle conclusions from this study were that post-weaning lambs were highly susceptible to infection with M. paratuberculosis and if exposed to high levels of contamination a proportion will develop severe infection leading to clinical disease and death. Hoggets and adult ewes are less likely than lambs to develop clinical disease after exposure to M. paratuberculosis. Nevertheless, even adult ewes may become infected and later act as a source for transmission of the disease. Lateral spread of OJD is a serious threat; it is not necessary for infected sheep to be present in a paddock for transmission of infection to occur if infected sheep are present in neighbouring paddocks. Conventional wire strand fences do not prevent spread of infection. For diagnosis on a flock basis, pooled faecal culture is more effective than the agar gel immunodiffusion assay for detection of the infection at relatively early stages in young sheep. Pooled faecal culture detected infection in sheep only 6 months after first exposure to contaminated pasture, when they were 11 months of age.

Samples were stored from sheep in this project for later research, for example in project OJD.031. In addition, blood samples were provided regularly to CSIRO for validation of the gamma-interferon assay for diagnosis of OJD.

The results of this study will have immediate impact on the management and control of OJD as they provide objective data to support and extend current recommendations for livestock grazing management.

November 2001 - June 2005

Ovine Johne's disease is a chronic and intractable problem. Spread of the disease has continued despite stringent regulatory measures, and in the absence of compensation for affected producers has led to severe division within the industry. Vaccination and risk-based trading have been accepted as an interim approach to limit further spread of the disease.

There is clearly an urgent need for better diagnostic tests. The main requirement is for a test that can detect infection in young sheep before the onset of faecal shedding. The test needs to be sensitive, specific, accurate, cost effective and able to distinguish an active infection from one that has died out. New automated technology platforms will be needed if tests are to have wide application in the sheep industries.

None of the work on OJD to date has included basic research. However, the need for this has been recognised and there are opportunities to take advantage of new technologies. Consequently the aims of this program are to research fundamental aspects of OJD including host-pathogen interactions at the cellular level. Protemics, genomics and advanced immunology techniques will be applied to in vivo and in vitro models to study the early stages of infection and contrast these with events later in the disease process. The aim is to discover new pathways for disease development and expression that can be exploited later for development of diagnostics, vaccines and chemotherapeutics.

September 2002 - December 2007

The purpose of the project is to validate the widespread use of Gudair® vaccine for the reduction of bacterial shedding in medium/high prevalence flocks and the prevention of increased shedding in low prevalence flocks. The major outcome from the project will be to allow producers to predict the infectivity of flocks over time following the commencement of a vaccination program.

There is much producer interest, both at an individual and industry level, in the use of Gudair® vaccine to control the impact of OJD in flocks varying in disease prevalence from very low to very high. This project will observe changes over time in the prevalence of mycobacterial shedding following the commencement of a Gudair® vaccination program in flocks varying in initial OJD prevalence. Up to four flocks, each with high, medium or low OJD prevalence at the commencement of a vaccination program will be sampled over a six-year period to estimate changes in the prevalence of shedding as the proportion of vaccinates in the flock increases. The relevant industry questions being addressed in this project are as follows:

• how long will it take for a vaccination program to reduce Mptb shedding to a level where safe trade in low risk sheep can occur?
• can the disease be eradicated by long term vaccination?
• how effective will vaccination be in low prevalence flocks in the control zone?
• will vaccination prevent the increase in losses commonly seen in long term infected flocks?
• can healthy sheep be vaccinated on arrival at an infected property so that infection and shedding is prevented?

January 2003 - March 2008

The level of clinical disease experienced due to ovine Johne's disease (OJD) appears to vary considerably between infected sheep flocks in Australia, even for flocks in the same locality that appear to have similar characteristics. This has led to considerable speculation about the potential importance of flock management, soil type, pH and micro-nutrients. Sound understanding about factors that influence disease expression will lead to management recommendations that improve on-farm disease control.

The aim of this project was to identify risk factors for OJD expression in infected flocks and improve the understanding of the epidemiology of the infection. The project consisted of a cross-sectional study on 92 infected properties located in New South Wales, Victoria, Tasmania and Western Australia. The information obtained from each included the OJD prevalence in specific groups of adult sheep measured using pooled faecal culture, details of farm and flock management and soil analyses from paddocks on which the sheep sampled had grazed.

A total of 31 significant farm/flock/management and soil variables were found. Some were likely to be a consequence of OJD infection, but the remainder appeared to be potential risk factors for the severity of the disease. There was a strong relationship between the PFC results and the duration of flock infection, the level of OJD mortality, dam stocking rates, as well as a relation with parent soil type. There was also a consistent but statistically non-significant trend for lower OJD levels in 4-year olds compared to 3-year olds, which may be due to deaths of affected sheep from 2 to 3 years of age. Wethers had significantly higher OJD levels than ewes, which strongly supports the anecdotal observation of higher losses in wether mobs. Higher OJD prevalence was linked to measures correlated with soil fertility including cation exchange capacity, phosphorus buffer index and organic matter content of soil.

Successful completion of this project enables the development of additional recommendations for on-farm control measures for OJD to support vaccination, and may help clarify the ecological niche of M. paratuberculosis, the potential for disease spread into areas not currently affected and the likely level of disease that would be experienced in these areas.

January 2004 - June 2005

RNAi is a technique of gene silencing where individual genes can be switched off and the effects observed. If the affected worms are affected (for example, are paralysed) the gene product may be a good candidate as a control target. Cell culture allows the study of isolated worm components. Given that it is difficult to cultivate these parasites in vitro, cell culture could open up approaches to studying cell biology that are currently unavailable.

In this project we have developed phenotyping tools that will be used to measure RNAi effects and have commenced the gene knockout experiments. These are currently available in the free-living stages of the parasite, but the ultimate aim is to develop the technique for parasites in sheep. Cells recovered from worms have been grown in culture. These will be used to study defined cell types and as a platform for RNAi. They offer several potential advantages as it may be easier to deliver RNA to these cells and their responses will be simpler to interpret than responses in sheep.

The aim is to develop tools for further research. This project falls into a multi-institutional research program with the aim of discovering targets for improved parasite control.

Australian Wool Innovation

February 2004 - February 2006

Several assays are used in the laboratory to measure: muscle contraction (using a force transducer), development, motility, migration and electrical responses in the pharynx of worms. The laboratory is equipped with electrophysiology gear that can be used to measure a range of electrical responses, including patch clamp and voltage clamp.

The project aimed to understand the pharmacology of avermectin/ivermectin resistance in sheep nematodes. This information will help develop tools for molecular diagnosis in the future. The work showed that:

• drug action and resistance occurred at two distinct sites, pharynx and body muscle of worms;
• the two major drug classes act in similar ways but their receptor populations are not identical in distribution in the worms or/and the three species of parasites;
• resistance is most likely due to different mechanisms in the different species and even isolates of the same species. A single test for resistance is not likely to be found;

Australian Research Council

Pfizer Australia

August 2003 - August 2005

This project dovetails in with our existing project on the sheep/Haemonchus relationship. The emphasis in this new project is to add value by carrying out DNA microarray experiments to identify sets of sheep genes which are up or down regulated during critical events in establishing immunity to Haemonchus. Further work to validate these genes will be performed using quantitative PCR and immunocytochemistry. The aim is to identify genes which may act as future markers for selection of sheep able to mount effective immune responses to worms.

Footrot is caused by the bacterium Dichelobacter nodosus, a parasite of the feet. This bacterium is unable to survive off the foot for more than one week.  Eradication of footrot is therefore possible if all sheep with footrot are removed from a flock.  Current techniques to achieve this are labour intensive, expensive and often take several years to achieve eradication.  Vaccination is an alternative approach. 

Current footrot vaccines contain ten strains of bacteria to provide coverage of the major D. nodosus serogroups.  These vaccines offer only temporary (12 weeks) protection against footrot, so they are used in control campaigns, rather than for eradication.  It has been demonstrated that eradication of footrot using vaccines is possible if the vaccines only target one or two groups of the bacterium at a time because immunity is long-lasting.  This project will evaluate this approach under Australian conditions. 

Objectives of the project:

1. Produce specific footrot vaccines for local (Australian) strains of the footrot bacterium.
2. Evaluate the use of these targeted footrot vaccines using one or two different antigens per vaccination in the eradication of virulent footrot in Australian sheep.
3. Demonstrate the use of these vaccines to remove virulent footrot on 12 commercial farms across areas of high footrot prevalence in southeast Australia.
4. Evaluate the minimum interval between vaccination with different vaccines to deliver an accelerated eradication program (less than twelve months between different vaccines).
5. Enable application for a minor use permit from the Australian Pesticides and Veterinary Medicines Authority (APVMA) to allow the use of these vaccines on-farm and also aid transfer of the vaccine to commercial production facilities.

Professor Julian Rood*, Monash University
Dr Leslie Reddacliff,
NSW Department of Primary Industries
Elizabeth Macarthur Agricultural Institute

* Principal Investigator

Ovine footrot is a highly infectious bacterial disease that is of major ongoing concern to the Australian wool industry, causing significant economic losses as a result of its effect on wool production, farm management, animal welfare and the cost of control and treatment programs.  The causative bacterium is Dichelobacter nodosus. 

The overall objective of this research is to develop improved methods for the control and treatment of ovine footrot.  The specific research aims are:

1. To identify D. nodosus genes that are differentially expressed in the virulent footrot lesion.
2. To determine the role of differentially expressed genes in the disease process.
3. To determine the value of whole genome based microarrays for the epidemiological analysis and diagnosis of field isolates of
   D. nodosus.
4. To identify surface or secreted D. nodosus antigens that induce the production of bactericidal antibodies in sheep.
5. To determine the vaccine potential of D. nodosus antigens that are either essential for the disease process or induce the production of bactericidal antibodies. 

The successful completion of the project should lead to the subsequent commercial development of a protective footrot vaccine, with significant cost savings to wool producers and the Australian wool industry.  It will also lead to a greater understanding of the epidemiology of footrot infections and may result in the development of improved methods for the laboratory diagnosis of ovine footrot. 

This research program represents the pre-commercialisation phase of the development of a new generation of footrot vaccines.  The successful identification of candidate antigens that can be used to develop a protective footrot vaccine will be subject to the uncertainty of dealing with a variable biological system.

The first cases of footrot in Bhutan were reported in the flock at the National Sheep Breeding Centre (NSBC) in Bumthang in 1990. This Centre supplies breeding animals to village flocks throughout Bhutan. Despite the presence of footrot at the Centre the distribution of sheep continued. In 1998 The Royal Government of Bhutan and the Australian Centre for International Agricultural Research began a joint project in footrot research. This was aimed initially at identifying the strains of Dichelobacter nodosus responsible for the disease at NSBC. Forty isolates were cultured from cases in that flock. All isolates were identified antigenically as belonging to serogroup B. Vaccine was prepared from these isolates and shown in a controlled trial to accelerate cure of cases and to prevent infection at a time when the disease spread in unvaccinated animals. The same vaccine was used to treat all sheep at NSBC for two successive years. After the first year no further cases of footrot were seen at NSBC despite close surveillance for two years after the cessation of vaccination.

Cases of footrot had been reported in village flocks soon after the disease was diagnosed at NSBC. In order to establish the distribution and prevalence of footrot in Bhutan, a national survey was designed and implemented. This survey revealed that footrot was present in nine of 13 districts surveyed, but with the exception of one district, Bumthang, the prevalence of disease was lower than expected. There was an association between the receipt of animals from NSBC and the presence of footrot.

During the survey 234 isolates of D. nodosus were cultured from affected sheep in all districts where it occurred. Once again all isolates tested proved to be of serogroup B. When examined with a series of tests they were found to be phenotypically indistinguishable from one another. Genotypically there were minor variations in OMP gene patterns among the isolates. The conclusion was reached that all the isolates studied from Bhutan were essentially the same and were probably all derived from the same source. The presence of a single strain is most unusual in other sheep producing countries.

The presence of only one strain of D. nodosus in Bhutan suggests that it could be eliminated from village sheep by using specific vaccine for two years as was done at NSBC.

Footrot was introduced into the migratory flocks of Siklis village of Kaski district in Nepal during the 1960s with imported sheep from New Zealand and formally reported by Lumle Agriculture Centre (LAC) in 1971.  Control measures were initiated during 1975 with the assistance of the United Nation's Development Programme (UNDP) and LAC.  However, by that time the disease had spread to the flocks of the adjoining districts of Lamjung and Manang.  The UNDP programme terminated in 1977 and the sole responsibility footrot eradication was handed over to LAC.   The footrot eradication programme continued in the conventional manner with the organization of campaigns to carry out foot trimming, foot bathing and removal of non-responding animals. However, despite the apparent recovery of animals at the beginning of each monsoon season, many became re-infected during their annual migration to alpine pasture.  Hence, although the problem was contained, disease eradication remained unachievable.

A footrot management project funded by ACIAR was developed. During this initial project the disease epidemiology was investigated and the strains of Dichelobacter nodosus involved in the disease in Nepal were identified.  Specific vaccines were developed based on the two infecting serotypes and these vaccines were used in a controlled field trial which was done in association with the LAC normal programme.  The results indicated that flocks treated with specific vaccine had less footrot than others treated with conventional vaccines or controls.  All previously vaccinated flocks were treated with the specific vaccine and within 2 years there was no evidence of virulent footrot in the population of sheep and goats in the study area.

The present project provided evidence that virulent footrot has been eradicated from the flocks of Kaski, Lamjung and Manang districts where the disease had persisted for nearly 30 years.

The second part of the project aimed at surveillance for virulent footrot in the endemic area and in surrounding non-endemic areas using clinical examination, microbial culture and ELISA serology. It confirmed that virulent footrot had been eradicated from the study area in Nepal. It was shown also that benign footrot persisted in the flocks and occurred in other flocks remote from the project area, and established the existence of some other important diseases in the migratory small ruminant population.

The development of an anamnestic diagnostic test which can be used for the retrospective assessment of the life experience of sheep and goats with respect to infection with virulent organisms was another achievement. This could provide a basis for field testing and certification for the freedom from virulent footrot.

Mulesing was introduced to the Australian sheep industry by J.H.W. Mules in 1931 as a measure for the prevention of blowfly strike in sheep, and in particular, the Merino. The wrinkliness and wooliness of the Merino sheep breech makes it highly susceptible to urine and faecal staining, leading to a high risk of blowfly strike. Mulesing involves the removal of skin from around the breech and tail to decrease wrinkles and increase the size of the bare area around the perineum. The result is a significant reduction in staining, with the area drier and less attractive to blowflies.

Mulesing prevents debilitating illness and death due to blowfly strike. However, it is acknowledged that sheep suffer short-term stress and pain as the operation is performed without analgesia or anaesthesia. The Australian sheep industry is trying to find effective humane alternatives to this procedure and as part of a nationwide effort, the Faculty team is studying the conformation of the breech, examining the skin and assessing the best patterns for applying chemical or other non-surgical alternatives to mulesing.

The project will apply specialist surgical skills and evaluate the skin resection pattern used in the mulesing operation. Wound healing will be examined, focusing on the microscopic, ultrastructural and molecular changes that occur. The project will focus on characterising features that are present in the normal breech skin of sheep and comparing how these change with wound healing after the mulesing operation compared to the healing that occurs with the use of chemicals or other mulesing alternatives. The systemic inflammatory response incited by surgical mulesing and its alternatives will be examined by measuring a range of haematological and biochemical parameters.

This project is one of a suite of AWI projects aimed at assisting Australian wool growers to find an alternative to mulesing as a preventative measure for flystrike.  The primary objectives are to put the mules operation on an evidence-based, scientific and quantitative footing as a foundation for investigating, devising and comparing alternative procedures.