Paratuberculosis in sheep and goats

G Model VETMIC 7038 No. of Pages 9

Veterinary Microbiology xxx (2015) xxx–xxx

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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic

Paratuberculosis in sheep and goats P.A. Windsor Faculty of Veterinary Science, The University of Sydney, Camden, NSW, 2570, Australia

A R T I C L E I N F O

Keywords: Disease control programs Goat Health Paratuberculosis Sheep Small ruminants Vaccination

A B S T R A C T

Paratuberculosis is a chronic insidious, often serious, disease of the global small ruminant industries, mainly causing losses from mortalities and reduced productivity on-farm, interference in trading and, in Australia, profound socio-economic impacts that have periodically compromised harmony of rural communities. The pathogenesis, diagnosis, impacts and disease management options for ovine and caprine paratuberculosis are reviewed, comparing current controls in the extensive management system for sheep in wool flocks in Australia with the semi-intensive system of dairy flocks/herds in Greece. Improved understanding of the immune and cellular profiles of sheep with varying paratuberculosis outcomes and the recognition of the need for prolonged vaccination and biosecurity is considered of relevance to future control strategies. Paratuberculosis in goats is also of global distribution although the prevalence, economic impact and strategic control options are less well recognized, possibly due to the relatively meagre resources available for goat industry research. Although there have been some recent advances, more work is required on developing control strategies for goats, particularly in dairy situations where there is an important need for validation of improved diagnostic assays and the recognition of the potential impacts for vaccination. For all species, a research priority remains the identification of tests that can detect latent and subclinical infections to enhance removal of future sources of infectious material from flocks/herds and the food chain, plus predict the likely outcomes of animals exposed to the organism at an early age. Improving national paratuberculosis control programs should also be a priority to manage disease risk from trade. The importance of strong leadership and communication, building trust within rural communities confused by the difficulties in managing this insidious disease, reflects the importance of change management considerations for animal health authorities. Although concerns of vaccine efficacy, safety and issues with diagnosis and administration persist, vaccination is increasingly recognized as providing a robust strategy for managing paratuberculosis, having made important contributions to the health of Australian sheep and the lives of producers with affected properties, and offering a mechanism to reduce risk of infection entering the food chain in ovine and caprine products. ã 2015 Elsevier B.V. All rights reserved.

1. Introduction Paratuberculosis (‘Johne’s disease’) is a chronic bacterial disease of global importance in mainly domestic and wild ruminants, caused by Mycobacterium avium subsp. paratuberculosis (Windsor, 2014). The disease was first reported in cattle in Europe in 1895 and has spread throughout the developed and parts of the developing world, particularly in the bovine dairy industries. The infection in small ruminants is also considered of worldwide distribution, diagnosed in sheep and goats in many countries. For ovine paratuberculosis, these include southern hemisphere’s Australia, New Zealand and South Africa, numerous northern hemisphere’s countries, particularly Great

E-mail address: [email protected] (P.A. Windsor).

Britain, Norway and Austria, as well as para-Mediterranean countries, e.g., Greece, Spain, Portugal, Morocco and Jordan (Benazzi et al., 2010; Djønne, 2010; Hailat et al., 2010). Caprine paratuberculosis has been diagnosed in Turkey, France, Norway, Switzerland, Croatia, Canada, the USA and Chile (Barkema et al., 2010). Unlike the clinical signs in cattle, which are readily recognized as profuse watery diarrhea and weight loss, the disease is more insidious in small ruminant flocks/herds, with affected animals eventually displaying progressive weight loss and exercise intolerance termed as ‘an increase in the tail to the mob’, with soft faeces in some animals (Windsor, 2014). Persistence of the causal organism in the environment and lack of sufficient application of vaccines that are presumed to have less than the desired efficacy, have complicated on-farm control (Sweeney et al., 2012).

http://dx.doi.org/10.1016/j.vetmic.2015.07.019 0378-1135/ ã 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: Windsor, P.A., Paratuberculosis in sheep and goats. Vet. Microbiol. (2015), http://dx.doi.org/10.1016/j. vetmic.2015.07.019

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This paper reviews current knowledge on paratuberculosis in sheep and goats, including pathogenesis, diagnosis and disease impacts, and also discusses the various disease control options that have been successfully used in these species, particularly vaccination and disease awareness extension programs. A change management framework enabling reflection on national control programs in Australia and consideration factors of relevance to improving paratuberculosis control by animal health managers (Windsor, 2014) is briefly discussed, including: drivers and motivation for change, resistance to change, knowledge management, farming systems dimensions and leadership of change management in paratuberculosis control. 2. Pathogenesis of paratuberculosis and aspects of relevance to disease control Ovine and caprine paratuberculosis involves chronic inflammatory lesions of the intestinal and lymphoid organs, caused mostly by one or other of the ‘S’ (sheep), ‘C’ (cattle) or ‘Bison type’ strains of M. avium subsp. paratuberculosis (Windsor, 2014; Kumar et al., 2010). In Australia, the S strain mostly causes paratuberculosis in sheep or fibre-producing goats, the C strain is mostly involved in dairy goat infections and to date the ‘Bison strain’ has only been detected in Brahman cattle (unpublished observations). This most likely reflects the largely separate geographical locations of the sheep and fibre-producing goat populations from dairy cattle, goat or sheep and tropical beef cattle enterprises farmed in this country. Although sheep are susceptible to the more common C strain and it has been a common finding in sheep in Europe, it has been suggested that distribution and abundance of the S strain may have been underestimated, due to more challenging requirements for mycobacterial culture (Begg and Whittington, 2010). Cattle appear relatively resistant to infection and disease with the S strain, although, in Australia, calves in contact to heavily infected sheep have become infected (Moloney and Whittington, 2008). In a longitudinal evaluation of diagnostic tests in a cattle infection model for paratuberculosis, including determination of adult infection status by post-mortem examination and tissue culture of sheep, goats or cattle infected as young animals, cattle were found to be less susceptible to the C and S strains of the organism than goats and sheep, with goats considered the least naturally resistant (Stewart et al., 2007). A recent study examining experimental infections of C and S strains in 1.5-month-old Assaf lambs found that specific antibody and interferon gamma (IFNg) production was significantly greater in animals infected with the C strains, with no consistent IFNg responses observed in animals infected with S-type strains (Fernandez et al., 2014). Lambs infected with S strains had granulomatous lesions restricted to the lymphoid tissue, with no differences in lesion intensity over time; C strain lesions were more severe at 150 dpi, while at 390 dpi lesions, characterised by well-demarcated granulomas with fibrosis, tending to decrease in severity as infection progressed. It was concluded that the strain of M. avium subsp. paratuberculosis has a strong influence over immune and pathological responses developed by the host. M. avium subsp. paratuberculosis infection mostly results from faceal-oral route exposure, with entry via the intestinal tract then to the lymphatic system, where it resides in M cells overlying Peyer’s patches in the ileum. Faceal-oral route exposure may occur as described below.  Ingestion of faecal material from an infected animal, particularly on the teat of an infected dam, plus exposure to manure contaminated pasture, water, supplements or hay contaminated with faecal material from infected adult animals (Windsor and Whittington, 2010).

 Drinking of contaminated colostrum or milk, as M. avium subsp. paratuberculosis is also excreted in the colostrum and milk of cattle (Windsor and Whittington, 2010) or sheep/goats (Lambeth et al., 2004; Nebbia et al., 2006). Pre-natal infection has also been well described, with prevalence in cows with subclinical or clinical disease estimated to be 9% (95% confidence intervals: 6–14%) or 39% (5% confidence intervals: 20–60%), respectively (Whittington and Windsor, 2009). In a study of 142 late-pregnant ewes and their fetuses from two heavily infected flocks, all five ewes with clinical disease had infected fetuses, but only one of 54 ewes with subclinical disease had an infected fetus, resulting in <1% (95% confidence intervals: 0–5%) of all fetuses diagnosed as infected (Lambeth et al., 2004). There is little information on the consequences of pre-natal infection and whether such animals progress more rapidly to clinical disease, commence shedding of M. avium subsp. paratuberculosis earlier, or are relevant to disease management. However, as in utero infection may occur frequently in sheep with clinical paratuberculosis, the immediate culling of reproductively active female animals with clinical signs is strongly advised to mange this risk (Lambeth et al., 2004; Whittington and Windsor, 2009; Windsor, 2014). Culling of clinical cases is also of importance to managing environmental contamination with the causal organism as sheep with clinical paratuberculosis shed huge numbers of the organism in their faeces, estimated at 108 organisms g 1 of faeces (Reddacliff et al., 2006). This is of concern to disease control programs as M. avium subsp. paratuberculosis may persist in the environment, potentially surviving for around one year, although this is dependent on a range of environmental factors, particularly shade (Whittington et al., 2004, 2005). In Australia, the initial paratuberculosis control strategy was regulation and quarantine of infected properties, with destocking of infected sheep properties from the beginning of one summer to the end of the next advocated. This program failed, most likely because of the difficulties of identifying ‘clean’ replacement stock, despite the early development of a market-assurance scheme to encourage studs to regularly test and provide evidence of their likely uninfected or low risk status (Windsor, 2014). Of continuing concern has also been the potential zoonotic link between paratuberculosis and human inflammatory bowel diseases, including Crohn’s disease. If M. avium subsp. paratuberculosis is eventually confirmed as a zoonotic pathogen, it is expected that public confidence in products from bovine and potentially small ruminant dairy industries will very likely decline. With increasing certainty of a role for M. avium subsp. paratuberculosis in Crohn’s disease, efforts to diminish or remove the organism from the human food chain should be encouraged. Three distinct forms of paratuberculosis have been observed in sheep: multibacillary disease, paucibacillary disease and asymptomatic infection (Gillan et al., 2010). The multibacillary lesions of chronic granulomatous enteritis and lymphadenitis (particularly involving the mesenteric lymph nodes) in paratuberculosis is characterised by accumulation of epithelioid macrophages containing numerous M. avium subsp. paratuberculosis in the lamina propria and submucosa of the intestine. The paucibacillary lesions are typically more lymphocytic in nature with M. avium subsp. paratuberculosis being far less numerous (Windsor, 2014). The enteric lesions typically develop within 6–12 months following initial detection of M. avium subsp. paratuberculosis infection and although some sheep may develop severe lesions within 12 months of infection, others progress from mild and paucibacillary to severe and multibacillary at variable rates, potentially fluctuating in severity or in the character of the inflammatory infiltrate over a period of years (Dennis et al., 2011). Clinical signs generally become evident in the months following development of severe

Please cite this article in press as: Windsor, P.A., Paratuberculosis in sheep and goats. Vet. Microbiol. (2015), http://dx.doi.org/10.1016/j. vetmic.2015.07.019

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multibacillary enteritis, although some sheep may be capable of temporarily or even permanently clearing the infection, especially during the early stages of M. avium subsp. paratuberculosis infection (Dennis et al., 2011). To improve understanding of the immunopathological basis of paratuberculosis, immune and cellular profiles corresponding to the three different disease states in ovine paratuberculosis have been extensively investigated and reviewed (Begg and Whittington, 2010). Sheep with paucibacillary lesions dominated by lymphocytes are more likely to have an associated Th-1 cell-mediated immune response (Clarke, 1997). In sheep with multibacillary disease, lesions are typically dominated by macrophages and these sheep tend to have a strong Th-2 humoral antibody response, features suggesting that multibacillary lesions are likely to be associated with weakened cell-mediated immune response (Dennis et al., 2011). Asymptomatic animals are those that appear to have contained or overcome infection with M. avium subsp. paratuberculosis and do not have histological lesions or are faecal or tissue-culture negative after experimental infection. Asymptomatic animals are of interest, as they express an immunological phenotype intermediate between that for non-infected control animals and those with paucibacillary lesions (Gillan et al., 2010). On a cellular level, these animals have CD4+, CD25+ T cell and B cell populations increased in the blood and the gut-associated lymph nodes, with the number of B cells in the blood and tissues increased relative to diseased and control animals. This indicates that the Th-2 humoral response may play a role in immunity to M. avium subsp. paratuberculosis infection. Moreover, as gd T cells are also increased in the gut-associated lymphoid tissues of lambs exposed to M. avium subsp. paratuberculosis, they may also play a role in the infections (Begg and Whittington, 2010). 3. Diagnosis of paratuberculosis in sheep and goats The most definitive diagnostic test for ovine and caprine paratuberculosis is post-mortem evaluation with histopathological confirmation, seeking identification of characteristic anatomical pathological changes of depletion of fat reserves, thickening of the bowel wall and enlargement of the gut associated lymphatics, including the presence of so-called ‘lymphatic cords’ on the serosal surface of the ileum and caecum (Windsor, 2014). Histopathological confirmation of the gross pathological lesions is by demonstration and classification of the microscopic changes and presence of M. avium subsp. paratuberculosis by specific staining (Perez et al., 1996, 1997; Copra et al., 2000). Culture and PCR for M. avium subsp. paratuberculosis from tissue samples assists the diagnosis. In Australia, regular surveillance of sheep at slaughter in abattoirs has been used extensively to provide pathological confirmation of new infections and provide assurance that zones not thought to be infected or at low prevalence remain so (Windsor, 2014). One of the greatest challenges for managing paratuberculosis has been the difficulty of accurately identifying animals with tests of low sensitivity. This is compounded by their inability to identify latent and subclinical M. avium subsp. paratuberculosis infections, particularly as faecal shedding of the organism can be intermittent in these animals and tends to precede serological responses. Current ante-mortem diagnostic tests for M. avium subsp. paratuberculosis infection have been reviewed (Begg and Whittington, 2010) and include serological assays for antibodies (enzyme-linked immunosorbant assay [ELISA], complement fixations test [CFT], agar gel immunodiffusion [AGID] assay), cellular assays for cell-mediated immunity (lymphocyte proliferation or transformation assay, intradermal skin testing, IFNg tests) and faecal culture and polymerase chain reaction (PCR) tests now including quantitative real time PCR (qPCR).

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Initial disease control programs depended on immunodiagnostic tests, such as ELISA, AGID, intradermal skin testing, lymphocyte transformation and IFNg assays, despite their poor diagnostic sensitivity that is affected by the stage of infection and the type of kit used (Begg and Whittington, 2010). As validation of these diagnostic assays by defining specificity and sensitivity against the gold standard tests of tissue or faecal culture has rarely been performed, there has generally been limited understanding of the operating parameters of the various assays for diagnosing M. avium subsp. paratuberculosis infection and when compared with PCR and culture, agreement with serological tests has been poor (Ikonomopoulos et al., 2007). More recent attempts at validation of M. avium subsp. paratuberculosis diagnostics have used Bayesian latent class models that allow for the continuous interpretation of test results and do not require comparison with a gold standard and provide estimates of sensitivity and specificity that account for all latent stages of infection (Angelidou et al., 2014). It was found that as both milk and serum ELISA’s have comparable sensitivities and specificities at the recommended cut-offs across dairy goat lactations, the milk ELISA could serve as the diagnostic tool of choice in paratuberculosis control programs that require frequent testing as milk sampling is a non-invasive, rapid, and easy (Angelidou et al., 2014). The development of faecal culture for the S strain of M. avium subsp. paratuberculosis greatly facilitated the diagnosis of ovine paratuberculosis in Australia, particularly as sheep with multibacillary lesions shed on average 1010 organisms per day that are recoverable in culture, even when diluted 100-fold with faeces from uninfected sheep (Begg and Whittington, 2010). It was determined that sensitivity of pooled faecal culture test for flocklevel detection was 92%, assuming a sample size of 350 sheep pooled and cultured in 7 lots of 50. This was far superior to routine serological testing for determination of flock prevalence where to achieve a similar degree of sensitivity, blood sampling of 850 sheep would be required. As described, faecal shedding of M. avium subsp. paratuberculosis is often detectable earlier than serological responses; although peripheral IFNg responses occur earlier, they are not related to protection and may simply reflect exposure to the pathogen. As clinical cases of ovine and caprine paratuberculosis are usually shedding M. avium subsp. paratuberculosis but often have variable immune and cellular responses, the pooled faecal culture test greatly assists clarification of the risk of spread and environmental contamination within and from and flocks/herds in paratuberculosis control programs (Reddacliff et al., 2006; Lybeck et al., 2011). As sensitivities of IS900-based PCR assays for the detection of M. avium subsp. paratuberculosis from both faecal and tissue samples have improved, enhanced DNA extraction techniques have enabled use of qPCR assays using primers designed to avoid detection of environmental bacteria. In a study using the qPCR on faecal samples collected at post-mortem examination from sheep with paratuberculosis, the faecal qPCR was found to be more sensitive than culture of intestinal tissues for M. avium subsp. paratuberculosis, providing more immediate information to estimate the stage of the infection, as well as the risk of transmission from infected animals (Kawaji et al., 2011). A blood-based PCR assay has also been developed to detect evidence of the presumed bacteraemic phase that occurs during infections and to overcome the problem of PCR inhibitors in faecal samples. Validation of this assay is pending (Juste et al., 2005) and issues on the use of PCR diagnostics has been reviewed (Bölske and Herthnek, 2010). In addition to the current diagnostic tests used for ovine paratuberculosis having poor sensitivity and specificity and most only being able to detect disease in the later stages of infection, it has not been possible to predict which of the M. avium subsp. paratuberculosis exposed animals are likely to become infective

Please cite this article in press as: Windsor, P.A., Paratuberculosis in sheep and goats. Vet. Microbiol. (2015), http://dx.doi.org/10.1016/j. vetmic.2015.07.019

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later in life; a major hindrance in disease control. With improved diagnostics tests that could determine early host responses and immune profile of an animal potentially infected with M. avium subsp. paratuberculosis, a longitudinal study was conducted to investigate if a suite of diagnostic tests conducted in M. avium subsp. paratuberculosis exposed lambs at 4 months post-infection could accurately predict their clinical status at 12 months postinfection (de Silva et al., 2013). By tracking cellular and humoral responses and quantities of M. avium subsp. paratuberculosis shedding for up to 12 months post-challenge, early increases in faecal organism DNA quantity and reduced IFNg responses were found to be significantly associated with sheep becoming infectious and progressing to severe disease. Conversely, early low faecal M. avium subsp. paratuberculosis DNA and higher interleukin-10 responses were significantly associated with an exposed animal developing protective immunity. It was concluded that the combination of early elevated faecal DNA or lower IFNg response had the highest sensitivity (75%) and specificity (81%) for identifying sheep that would later become infectious, suggesting that combined test interpretation may aid in the early prediction of sheep susceptibility to M. avium subsp. paratuberculosis infection (de Silva et al., 2013). 4. Impacts of paratuberculosis that have influenced disease control strategies Adverse impacts of M. avium subsp. paratuberculosis infection may occur at international, national, state, regional or flock/herd level and, although international trade issues are unresolved and reflect differences in perceived risk of trade concerns arising from paratuberculosis, considerable attention has been given to national and farm level impacts for paratuberculosis in Australia. As a consequence of the infection in cattle progressing to clinical paratuberculosis, infected dairy cows may display reductions in milk production, body weight and fertility, eventually resulting in premature culling or death (Merkal et al., 1975). In sheep and goats, progression of M. avium subsp. paratuberculosis infection to clinical paratuberculosis can result in profound weight loss, premature culling and death, with losses approaching 20% per annum in some sheep flocks in Australia (Windsor, 2014). Negative effects on fertility have also been reported in dairy sheep (Kostoulas et al., 2006c). 5. Significance of paratuberculosis of sheep in extensive production systems Australia has over 75,000,000 sheep and wool production from mainly Australian Merinos run under extensive pastoral conditions remains the predominant production enterprise on the almost 44,000 sheep farms (Windsor, 2014). Sheep meat production and, to a lesser extent, goat meat production are increasing in importance in Australia in recent years, with locally-produced small ruminant dairy production remaining a minor commodity activity. Most wool farms use a self-replacing management system, retaining a majority of the females and annually disposing of aged ewes (usually between 5 and 7 years) and castrated males (wethers) for meat production, although some farms still retain wethers for wool for extended periods. Rams are purchased from stud enterprises for mating ewes in either autumn (March–April) for spring-born lambs (August–September) or spring mating (December–January) for autumn-born lambs (April–May), with weaning 12–16 weeks after commencement of lambing. Ovine paratuberculosis was first diagnosed in the state of New South Wales in the early 1980s, causing sporadic problems until the current epidemic emerged in the mid-1990s, almost exclusively caused by the S strain in sheep, although C strain was

recovered from a flock where there was a close association of sheep with cattle infected with C strain. The C strain dominates caprine infections. For several years in the period of the mid-1990s to about 2005, paratuberculosis was arguably the most important endemic disease of Australian sheep. This was due to escalating costs of disease control aimed at reducing the spread of the disease from the ‘infected’ to ‘clean’ areas, plus the severe impacts on producers from sheep losses, including financial costs estimated at AUD 23,000 in 1997, plus the pressure of having to change enterprise type, e.g., substitution of prime lambs and cropping for Merino wool enterprises in response to a diagnosis of the disease (Webster, 2000; Windsor, 2014). Mounting social costs on those with infection began emerging, including clinical depression from the stress of observing huge sheep mortalities, lost reputation and reputed declines in the resale value of infected properties. This led to loss of trust by farmers in the credibility of scientific institutions linked to the government (Palmer et al., 2009; Windsor, 2014). Of concern was a widespread denial of the severity of the disease by many on farms or in areas where infection was yet to be diagnosed or only occurred with imperceptible losses, with claims that as sheep of poor condition had always occurred on many properties, the disease had been around for almost a century and that occurrence of severe losses was attributable to mismanagement. Despite the earlier study on the financial impacts on properties affected by the disease (Webster, 2000), the debate on the true impact of the disease persisted until evidence from pathological studies on 12 farms in New South Wales demonstrated that losses from the disease were significant and could be differentiated from other causes of on-farm mortality (Bush et al., 2006a,b). That study identified the disease mortality rate in 2003 on the 12 farms as 8%, resulting in an average reduction in annual income of AUD 13,715 per farm, due to an average decrease in gross margin of 8.5% per farm (Bush et al., 2006a). Ovine paratuberculosis has continued to spread in certain zones in Australia, with data derived from abattoir surveillance of slaughtered sheep in 2011 indicating that of 2117 known infected flocks (5% of the national sheep flock), approximately 60% were located in New South Wales, 32% in Victoria, 30% in Tasmania, 19% each in South Australia and Western Australia and none in Queensland (Animal Health Australia, 2013c). Although variation in sensitivity of abattoir surveillance between abattoirs and states is to be expected, with concerns of poor accuracy and reported variation in disease detection competency (Bradley and Cannon, 2008), this is considered unlikely to account for the considerable variation in paratuberculosis prevalence between and within states in Australia (Windsor, 2014). Fortunately, field research has shown that in flocks where vaccination has been occurring for at least five years, there have been significant declines in within flock prevalence, with a recent study using pooled faecal culture in 37 flocks, identifying the estimated pre-vaccination median prevalence of 3% declined to a post-vaccination median prevalence of <1% (Dhand et al., 2010). Currently over 5 million lambs are vaccinated per annum in Australia. 6. Significance of paratuberculosis of small ruminants in more intensive production systems Unlike Australia, in many countries sheep and goat dairy farming is a socially and economically important animal production enterprise activity, involving mainly semi-extensive or semi-intensive systems. This is the case in Greece, where the national population of approximately 100,000 sheep and goat farms contains over 10,000,000 million sheep and almost 6,000,000 goats of various indigenous breeds (e.g., Chios, Karagouniko, Skopelos, Capra prisca) or their crosses, although few flocks have a distinct breed pattern. These animals are raised

Please cite this article in press as: Windsor, P.A., Paratuberculosis in sheep and goats. Vet. Microbiol. (2015), http://dx.doi.org/10.1016/j. vetmic.2015.07.019

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under semi-extensive or semi-intensive management for milk production. Farmers select female replacements among offspring of high-yielding ewes or does, obtaining males from other flocks/ herds with mating to rams or bucks occurring in June–September and neonates born the following November–March and weaning 45–60 days after birth. The dams are milked twice daily for a 180–220 day-long lactation period. Milk is used mainly for production of ‘feta’ cheese, approximately 30% of its production being exported every year. Although the majority of flocks in Greece are endemically infected with S and/or C strains of M. avium subsp. paratuberculosis (Kostoulas et al., 2006a,b; Florou et al., 2008, 2009), control of ovine or caprine paratuberculosis is currently of low priority to Greek producers and their associations, despite other EU-countries exporting milk and dairy products now managing control programs for dairy cows. It may be possible that Greek producers and their dairy industry leadership will be confronted with the necessity of developing a M. avium subsp. paratuberculosis-infection control program for protection of exported feta cheese from sheep/goats, particularly if evidence that the causal organism may be involved in the pathogenesis of human inflammatory bowel diseases continues to emerge. 7. Strategies for control of ovine/caprine paratuberculosis The three main approaches to eradicate or reduce impacts of paratuberculosis in sheep or goat farms are to:  introduce management changes to decrease transmission of M. avium subsp. paratuberculosis,  apply test and cull practices to eliminate the sources of infection and  vaccinate replacement stock to increase their resistance to infection.

7.1. Management changes Management of risk factors for transmission of M. avium subsp. paratuberculosis and expression of paratuberculosis are potentially useful tools for controlling ovine/caprine paratuberculosis, particularly in smaller flocks/herds, where individual management of animals is possible, similar to the three-step calf program in dairy cattle (Windsor and Whittington, 2010). Care of newborns with separation from older animals, feeding of organism-free colostrum and milk, maintenance of a clean environment, adequate feeding of growing animals, raising of replacements in M. avium subsp. paratuberculosis-free locations isolated from the flock/herd of origin, coupled with improvement of general farm hygiene, are considered the most important management tools to control paratuberculosis within a flock/herd (Djønne, 2010; Windsor and Whittington, 2010). To be effective, these interventions require knowledge, diligence and investments to improve biosecurity practices and may not seem financially viable for many flocks/herds, particularly as they may yield no immediate results; they are unlikely to be widely implemented without considerable efforts in extension. In extensive production systems, the most important management tools are improved biosecurity to prevent entry of M. avium subsp. paratuberculosis and vigilant surveillance of the ‘tail of the mob’ with rapid removal of sheep of declining body condition score, hopefully before they have started increased bacterial shedding in faeces, particularly at the highest risk period at and shortly after lambing. These recommendations are supported by evidence from an Australian randomised longitudinal field trial that used natural pasture-based exposure of 840 Merino sheep in a factorial design, testing infection and disease outcomes in relation

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to age at first exposure and the level of exposure to M. avium subsp. paratuberculosis (McGregor et al., 2013). Age at first exposure was identified as a significant determinant of lesion development, with lambs having a 3-fold more odds of developing severe lesions than adults after equivalent time. Mortalities due to paratuberculosis were strongly determined by level of exposure to M. avium subsp. paratuberculosis, with sheep exposed to high doses having 18-fold more odds of death and sheep exposed as lambs having a 5-fold more odds of dying due to paratuberculosis than if exposed as adults; the study concluded that for extensively grazing livestock, to achieve reduced transmission of M. avium subsp. paratuberculosis one should focus in limiting the exposure of young animals and reducing the levels of pasture contamination with the organism (McGregor et al., 2013). 7.2. Test and cull strategies Test and cull strategies are dependent on effective and repeated diagnostic tests to detect animals with early infection with M. avium subsp. paratuberculosis, preferably prior to their commencement of faecal shedding. However, as current diagnostic tests are problematic and generally of low sensitivity, it is somewhat surprising that such programs have been so extensively promoted, especially in cattle, considering the enormous cost of testing using the current tests (e.g. ELISA) that are best used as a flock or herd screening tool (Stehman, 1996), extended incubation times with culture and PCR, the need for specialised laboratories and personnel, coupled to limited evidence of success of these strategies. The current tests and the use of pathological approaches (e.g., identification and removal of clinical cases) are confounded by their inability to detect infected animals prior to commencement of faecal shedding (Kostoulas et al., 2006a,b; Florou et al., 2009; Begg and Whittington, 2010). Considering the high cost of testing in relation to the individual value of animals, test and cull strategies alone would appear to have limited application in sheep and goat farms. However when combined with stringent management of fecal-oral transmission to prevent exposure of young stock to infection, it has been advocated that such control programs may be considered, depending on the frequency of testing required, the level of management intervention possible, and individual farm abilities, priorities, and finances (Stehman, 1996). Individual animal testing has been advocated as appropriate for owners who consider their sheep or goats as companion animals, particularly in the USA (Robbe-Austerman, 2011). In all testing programs it is important that owners understand that negative tests do not ensure freedom from paratuberculosis and that multiple tests applied intermittently over a prolonged period of time are required to obtain confidence in the true infection status of a flock or herd, as is used in Market Assurance Programs in Australia (Animal Health Australia, 2013a). 7.3. Vaccination for paratuberculosis As control of M. avium subsp. paratuberculosis infection through hygiene-management and culling of test-positive animals has generally failed to produce the expected results, a renewed focus on vaccination has been promulgated (Bastida and Juste, 2011) as it can easily achieve control of paratuberculosis, especially in small ruminants (Juste and Perez, 2011). Importantly, vaccination does not prevent infection, although it significantly reduces the occurrence of clinical cases plus organism excretion from infected animals (Reddacliff et al., 2006; Windsor 2006; Bastida and Juste, 2011; Windsor, 2014). An international perspective of the results of vaccination of small ruminants suggests with the exception of the first report

Please cite this article in press as: Windsor, P.A., Paratuberculosis in sheep and goats. Vet. Microbiol. (2015), http://dx.doi.org/10.1016/j. vetmic.2015.07.019

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from Norway and the recent studies from Australia, there are only a few studies published on vaccination in sheep, with isolated reports from Spain, New Zealand, Cyprus, and the United Kingdom (Juste and Perez, 2011). The use of paratuberculosis vaccine is rarely recorded in the veterinary literature, even though millions of doses are used every year (Juste and Perez, 2011). It is estimated that over 5.5 million doses of GudairTM are used annually in Australia (commercial sources). There is even less evidence of the efficacy of goat vaccination despite being used in Spain, the Netherlands, France, Norway, and India according to commercial sources, with scarce publication of results in the veterinary literature (Juste and Perez, 2011). In Australia, vaccination of sheep has been shown to provide very effective flock-level control of clinical disease and underpins the national paratuberculosis control program along with a risk-based trading system (Windsor, 2006, 2014). With mass vaccination of the affected sheep population in Iceland proving to have been very successful in controlling the disease (Fridriksdottir et al., 2000; Begg and Whittington, 2010), the testing of the imported inactivated vaccine GudairTM for potential adoption in Australia (Reddacliff et al., 2006) was commenced in late 1999. Following early success in the Australian research program, GudairTM became available for producer use in Australia in 2002 and has proven to be the most economically viable control option in most infected flocks, suitable for implementation in a range of flock management systems (Windsor, 2006, 2014; Bush et al., 2008). Paratuberculosis vaccination is recommended for use in young replacement animals. The Australian program commences with vaccination of all lambs between 4 and 16 weeks of age, continuing annually long after all sheep have been progressively vaccinated as lambs and the non-vaccinates gradually removed as cull-for-age or clinically suspect animals (Windsor, 2014). Field evidence from Australian sheep vaccination trials indicate that there might be an age threshold for vaccine efficacy in animals beyond very young lambs, including animals up to 8 months of age (Windsor, 2006). This has encouraged some flocks to commence a program by vaccinating both lambs and replacements from the previous year. However, the value of vaccination of adult animals in the face of disease-caused losses is still unresolved, as field observations have been that significant losses of adult vaccinates will continue in heavily infected flocks that may make ‘whole flock’ vaccination uneconomical. However as vaccination of adult goats and sheep in M. avium subsp. paratuberculosis infected flocks can reduce number of animals developing clinical disease (Copra et al., 2000), it may be worth considering, particularly if it empowers producers to improve their general disease control strategies, motivated by the sense that they are ‘doing all they can’ (Windsor, 2014). There are conflicting reports on the benefits of vaccination in dairy farms (van Schaik et al., 1996; Juste et al., 2009). A meta-analysis of selected papers on vaccination against M. avium subsp. paratuberculosis concluded that most studies found vaccination reduced risks of microbial contamination, reduced or delayed production losses and pathology, but failed to fully prevent infection, with the majority of effective M. avium subsp. paratuberculosis vaccines considered to be rudimentary and requiring improvement (Bastida and Juste, 2011). In Australia, paratuberculosis management under the National Johne’s Disease Control Program (NJDCP) is now dependent on widespread use of GudairTM, following the success of field trials that commenced in 1999 and are still continuing, having been recently reviewed (Windsor, 2013, 2014). The initial study that led to registration of GudairTM involved 600 vaccinated and 600 unvaccinated lambs on three farms experiencing significant losses from the disease, accounting to 5–15% annually (Reddacliff et al., 2006). It was concluded that

vaccination stimulated cell-mediated and humoral immune responses, reduced mortalities caused by the disease by 90% and delayed and reduced faecal shedding of the organism. Although numbers of M. avium subsp. paratuberculosis excreted by the vaccinated animals were also reduced by at least 90% at most samplings, high levels of excretion by these animals occurred on some occasions, and all 7 of the 600 vaccinated animals that died from paratuberculosis had multibacillary lesions, indicative of risks that vaccinated sheep may transmit the organism. Vaccine injection site lesions were detected in almost 50% of sheep after two months and persisted for at least 4 years in 20 to 25% of vaccinated animals (Reddacliff et al., 2006). Subsequent trials in Australia have attempted to more clearly define the efficacy of GudairTM. This is important, as, although vaccination quickly eliminated the significant mortalities in heavily infected flocks, the shedding of M. avium subsp. paratuberculosis from vaccinated sheep may persist for many years after commencing vaccination (Dhand et al., 2013; Windsor, 2013) and the retention of unvaccinated sheep (e.g., wether mobs) on the farm was of high risk to recrudescence of M. avium subsp. paratuberculosis shedding (Eppleston et al., 2010). In a study of 37 flocks where vaccination of lambs had been applied for six years, shedding persisted in the majority of flocks and a risk factor study in these flocks indicated probable associations with increased prevalence of disease to include: straying sheep, introduction of unvaccinated sheep into the flock and use of commercial ‘contractors’ to do the vaccinations (Windsor, 2013; Windsor et al., 2014). Improvement of farm biosecurity and continuing careful vaccination of all lambs until well after all sheep in a flock will have been vaccinated is required to optimise the protection offered by vaccination programs (Windsor, 2013). Arguments against vaccination of sheep and goats include issues of safety and compromise of diagnostic tests for M. avium subsp. paratuberculosis and other mycobacterial diseases. Studies on vaccine safety were conducted with GudairTM and adverse effects to vaccination occurred uncommonly, but did occur if the vaccine was incorrectly administered (Windsor and Eppleston, 2006; Eppleston and Windsor, 2007). Dissection of tissues at postmortem examination of sheep vaccinated up to a week previously with GudairTM injected intramuscularly, revealed extensive and progressive necrosis and phagocytosis of muscle tissue in a 10 cm radius surrounding the site of injection (Windsor, 2014). Despite vaccination injection lesions at slaughter occurring in 18% of adult sheep and 65% of lamb carcasses, no economic losses were incurred from the discounting of damaged carcasses due to the extensive trimming that occurs at abattoirs (Eppleston and Windsor, 2007). Incidents of human exposure have also occurred in Australia, including accidental self-injection with GudairTM vaccine requiring medical intervention and extensive surgical removal of the injected vaccine to allow drainage and avoid progression to extensive necrosis (Windsor et al., 2005; Richardson et al., 2005). Despite the less than optimal efficacy of GudairTM with prolonged bacterial shedding by vaccinated animals and the safety concerns for both humans and sheep, use of vaccination to control paratuberculosis in Australia has been of enormous benefit to the sheep industry and to rural communities (Windsor, 2013, 2014). Currently available vaccines for M. avium subsp. paratuberculosis stimulate both cell-mediated immunity and humoral immunity, so are unsuitable for differentiating infected from vaccinated animals (DIVA), if a serological test for diagnosis of paratuberculosis is used as in test-and-cull strategies or animals tested for export. This is not an issue where the diagnostic test involves faecal culture with PCR or qPCR assays. In Australia, bovine tuberculosis has been eradicated and the diagnostic test of choice

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for ovine paratuberculosis is the pooled faecal culture or qPCR, hence presence of post-vaccination serological responses is of minimal concern except for animals being tested for export. However, researchers continue to search for new vaccine candidates with improved efficacy and reduced negative side effects, including vaccines with less tissue provoking adjuvants and potentially subunit vaccines that may have the advantage of not compromising diagnostic tests for either bovine tuberculosis or paratuberculosis (Patton, 2011). Although studies on subunit vaccines have demonstrated reduced faecal shedding, a lack of interference with immunodiagnostic assays for bovine tuberculosis and paratuberculosis and less tissue provocation, it remains to be seen whether such products will be registered for widespread use and prove to be more effective than GudairTM. 8. Change management and extension in paratuberculosis control Control of paratuberculosis in Australia by regulatory, de-stocking and other approaches was generally considered to have been a failure prior to the introduction of vaccination in 2002. Vaccination was then supplemented by the introduction of the ‘sheep health statement’, a vendor declaration system to facilitate disease risk awareness during the trading of sheep, plus eventually a more effective program of biosecurity risk awareness. It is very likely that the various iterations of the national paratuberculosis control extension program have been important in improving awareness of the disease and its management, progressing from the ‘National Ovine Johne’s Disease Control and Evaluation Program’ (NOJDP) 1998–2004, the ‘National Approach to the Management of Ovine Johne’s Disease in Australia’ (NAOJD) 2004–2007, the ‘Ovine Johne’s Disease Management Plan’ 2007–2012 to the current Ovine Johne’s Disease Management Plan 2013–2018 that commenced in July 2013 (Animal Health Australia, 2013a). However, despite the enormous efforts of extension programs to address paratuberculosis disease risks, improving knowledge and attitudes towards paratuberculosis risk management by producers has been challenging and improved disease control practices have been considered most likely attributable to the introduction of GudairTM vaccination in 2002 (Windsor, 2014). As managing change for improved disease control is complex, factors most relevant to influencing producer concerns on paratuberculosis in Australia have been recently reviewed and tabled (Windsor, 2014). These include drivers and motivation for change, resistance to change, knowledge management, farming systems dimensions and leadership. Drivers that motivate changing attitudes to paratuberculosis control can be categorised as those that aim to avoid losses including risks to reputation, and those creating socioeconomic opportunities that increase returns. Although paratuberculosis control has no proven food-safety risk, the issue of M. avium subsp. paratuberculosis as a potential causative agent in Crohn’s disease persists and has not been readily dismissed (Chiodini et al., 2012). However, it was the clear economic drivers that motivated government and sheep industry agencies to support vaccine research, motivated by the socioeconomic impacts of the disease in Australia. As the initial regulatory approaches, including quarantine of infected farms to limit paratuberculosis spread, created enormous anxiety and confusion, with few options, other than de-stocking, available, regulation was abandoned in most states, due to the growing distrust of the government agencies involved (Palmer et al., 2009; Windsor, 2014). The provision of GudairTM vaccine for widespread use by Australia sheep producers, from April 2002, enabled rapid control of mortalities on-farm and has appeared to have altered epidemic dynamics that for many had been escalating beyond reserves of resilience.

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Resistance to paratuberculosis change management by Australian sheep producers manifests as defence mechanisms that enable farmers to sustain beliefs in their coping mechanisms for their problem (Windsor, 2014). The defences observed include the farmer: believing paratuberculosis is inconsequential, blaming neighbours or government services for their problem, reverting to risky behaviours (e.g., purchasing sheep from infected properties, retaining unvaccinated sheep), denying reality of true disease risk and choosing to believe in supernatural influences rather than a biological cause or technical solution to the problem. Knowledge management drives improved attitudes to disease control and led to investment decisions by farmers. Disease prevention through improved biosecurity is required to address the risks of entry of paratuberculosis and the use of ‘champion farmers’ to communicate risk messages was important in Australia. Numerous disease-affected farmers volunteered to share their experience of with losses from paratuberculosis and the positive impact of GudairTM vaccination. More recently, electronic social media, including webinars (Animal Health Australia, 2013b), newsletters and blogs have emerged to support paratuberculosis knowledge management and to help address problems of misinformation, sharing learning by researchers, producers and their advisors with those affected by more recent incursions of the disease (Windsor, 2014). Farming systems are complex and effective extension programs require an understanding of the multiplicity of factors involved in sheep and goat farm management. Paratuberculosis causes failure within the farming system, with animal production losses and declining farm income (Bush et al., 2006a,b), seeking of alternative farm enterprises and emotional strain (Windsor, 2014). Animal health decisions by producers are made within broader farm management system, requiring that promotion of a disease interventions, such as vaccination and improved biosecurity for paratuberculosis, uses extension tools reflective of the farming system; identification of the ‘break-even point’ of investing in paratuberculosis control is an example (Bush et al., 2008). Change management leadership is critical for national animal health programs to succeed, especially in Australia, where legal responsibility for managing animal health remains with state legislature. The not-for-profit company Animal Health Australia (AHA) has been formed in 1996 to facilitate responsive animal health management that is inclusive of the multiple livestock industry stakeholders concerned with national animal health. However, developing national paratuberculosis control programs has been most challenging, requiring dedicated leadership to negotiate the series of those plans. The latest iteration commenced in July 2013 (Animal Healthy Australia, 2013a), after a prolonged period of discussion as the program was initially challenged by a disaffected producer group resisting introduction of regional biosecurity management plans, aimed at discouraging trade of sheep from high and medium paratuberculosis prevalence zones to low prevalence zones (Windsor, 2014). Although there are issues with the plan that was finally accepted by all involved parties, it remains to be seen whether it will continue the effectiveness of previous plans in assisting the control of paratuberculosis in Australia. 9. Concluding remarks Ovine paratuberculosis is a global concern that has received minimal attention in many countries, due to the low value of sheep, low to moderate levels of the disease in most sheep populations and the focus on other diseases of sheep and paratuberculosis in cattle. Just as the disease moves slowly through small ruminant populations, unless management interventions are introduced, the accumulation of infection in the population will increasingly

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gather pace and as occurred in Australia, where wool-producing sheep are very important in the livestock industries, the emerging slow epidemic may result in severe mortalities and a ‘paratuberculosis crisis’. However, this epidemic did lead to considerable sheep industry-funded research in Australia, improving the pace of accumulated understanding of the pathogenesis, diagnosis, impacts and most appropriate strategies for control of M. avium subsp. paratuberculosis infection in sheep. In particular, the recognition of different immune and cellular profiles of sheep with different disease outcomes that may eventually provide a method of predicting disease outcome in an individual, as well as increased understanding of the importance of persistent vaccination for improved long-term disease control., are of relevant to global efforts to manage paratuberculosis. Although the disease in goats is also of global distribution, the prevalence and economic impact are not well recognised in this species, partly due to the low resources available for goat industry research compared to those for sheep. There have been some advances in caprine paratuberculosis, including improved knowledge of the susceptibility of goats to both the C and S strains of the organism plus some progress in diagnostic tests. However, considerably more work is required on developing control strategies for goats, particularly in dairy situations where, like ovine paratuberculosis, there is an important need for research to find sensitive and validated diagnostic assays that can detect early subclinical stages of infection or provide insights into the likely outcomes of animals exposed to M. avium subsp. paratuberculosis at an early age. Also of importance are considerations of change management factors relevant to control of paratuberculosis, as these provides insights into national and potentially international management programs. The importance of strong leadership and effective communication to build trust with rural communities confused by this insidious disease is emphasized. Although concerns of vaccine efficacy, safety and issues with post-vaccination diagnostics and administration difficulties are often discussed, the persistent use of vaccination for paratuberculosis has considerably improved both the health of Australian sheep and the lives of affected sheep producers and deserves wider recognition globally. Conflict of interest statement There are no conflicts of interest to report in this work. Acknowledgements Many colleagues over a considerable number of years have contributed to a number of the studies discussed in this review, funded by a range of Australian sheep industry agencies. Their contributions are gratefully acknowledged in papers sourced for this review from our research team, as is the generous advice of Drs J. Eppleston, N. Dhand, R. Bush and R.Whittington from the University of Sydney, and Drs L. Leontides and P. Kostoulas from Greece. References Angelidou, E., Kostoulas, P., Leontides, L., 2014. Bayesian validation of a serum and milk ELISA for antibodies against Mycobacterium avium subspecies paratuberculosis in Greek dairy goats across lactation. J. Dairy Sci. 97, 819–828. Animal Health Australia, 2013. Ovine Johne’s Disease in Australia. Available from: http://www.animalhealthaustralia.com.au/programs/johnes-disease/ovinejohnes-disease-in-australia/ (accessed 11.12.2013). Animal Health Australia., 2013. Ovine Johne’s Disease in Australia: Animal Health Australia Webinar on OJD Vaccination. http://www.animalhealthaustralia.com. au/ojd-test/ (accessed 11.12.2013). Animal Health Australia., 2013. JD News –Summer 2012. Official Newsletter of the National Johne’s Disease Control Program. Available from: http://www. animalhealthaustralia.com.au/wp-content/uploads/2011/05/JD-NewsSummer-2012.pdf (accessed 11.12.2013).

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Please cite this article in press as: Windsor, P.A., Paratuberculosis in sheep and goats. Vet. Microbiol. (2015), http://dx.doi.org/10.1016/j. vetmic.2015.07.019