Project title: Improving Diagnostics of Bovine Johne’s Disease at the Herd and Animal Level 

Project team:  Dr Sam Strain, Dr David Graham

Duration:  36 Months

Background to proposal:

Bacteria of the genus Mycobacterium are gram-positive, acid-fast organisms that include a number of significant human and animal pathogens. The mycobacterial species M. avium is currently subdivided into three subspecies, M. avium subsp. avium (M. avium), M. avium subsp. paratuberculosis (Map), and M. avium subsp. silvaticum (M. silvaticum).

Paratuberculosis (Johne’s disease) in ruminants is characterized by a chronic granulomatous enteritis caused by the slow growing and the difficult to culture bacterium Map. This chronic enteritis results in malabsorption of essential nutrients and accelerated protein loss. Clinical symptoms of the disease are decreased milk production, progressive weight loss, intractable diarrhoea, wasting, and ultimately death.  Subclinically infected animals suffer from an increased susceptibility to other diseases and decreased production (Whitlock et al., 1996). Johne and Frothingham (Johne et al., 1895) initially reported the disease in a case report of a cow suspected of having intestinal tuberculosis in Germany in 1895. However, it was not until 1912 that Koch's postulates were successfully fulfilled by growing Map in the laboratory and reproducing the disease in experimentally infected cattle (Twort et al., 1912).

Map can infect a wide range of animals including domestic and wild ruminants, wild rabbits (Greig et al., 1997), foxes, weasels, and ferrets (Beard et al., 1999). The infection is widespread throughout the world and causes substantial financial losses for the farming industry. The reported prevalence of paratuberculosis within the EU varies significantly between member states, with Denmark and the Netherlands reporting high herd prevalences of 70-80% compared to Sweden reported as being free of disease (EFSA, Scientific Opinion, 2004). However, interpretation of the available data is complicated for several reasons. There is a lack of official data, and only a limited number of countries/regions have carried out surveys aimed at determining the true prevalence of paratuberculosis. Equally important, all the available studies have used a range of diagnostic tests with different characteristics (Juste et al., 1999; Boelaert et al., 2000; Nielsen et al., 2000; Muskens et al., 2000, Viske et al., 1997).

Accurately assessing economic losses in productivity and profit caused by the disease is difficult, given the fact that the disease is chronic and likely to be under-reported. Nonetheless, the impact of this disease is estimated to be significant: losses in the United States alone are estimated to be as high as $1.5 billion to the agriculture industry every year.

Crohn’s disease (CD) is a chronic inflammation of the human gut, often located to the terminal ileum, but the whole intestine can be affected (Crohn et al, 1932). The aetiology of CD is unknown, but the close resemblance with intestinal tuberculosis led to the early assumption that CD could have a mycobacterial aetiology (Dalziel, 1913). This contention was supported by the isolation of a mycobactin dependent mycobacterium from a patient with CD that later was confirmed to be Map (Chiodini et al. 1984).  Since paratuberculosis in animals has strong similarities to CD in humans, it was considered that Map could be a plausible candidate for the aetiological agent in CD.  Since then a number of studies focussed on the detection of the organism from tissues obtained from CD patients (Baksh et al., 2004; Bull et al., 2003; Ghadiali et al., 2004; Naser et al., 2004 and Schwartz et al., 2000) as well as on immunoresponses to Map antigens (El-Zaatari et al., 1999; Olsen et al., 2001 an Rowbotham et al., 1995) have been undertaken.  Conflicting results have led to lack of clarity on the role of Map in the aetiology of CD.  Even though the issue remains controversial, it has lead to increased attention on the disease in livestock and advice to apply precautionary measures to be taken to protect consumers, both at the production level as well as the food processing level.

Conventional bacteriological culture of Map from faecal samples is regarded as the "gold standard". However, a “true” sensitivity of only 35% has been estimated (Whitlock et al., 2000). Because of the very slow growth of the organism, it is crucial to eliminate other fast-growing bacteria and fungi from the samples (Whipple et al., 1991). These decontamination procedures cause the test to be very labour intensive, time consuming and expensive, which limits the number of samples that can be handled. The long culture period prevents rapid action; detecting a positive sample means that a cow has been shedding the bacterium, and thus potentially infecting other animals, for at least several months before the animal is removed from the herd.

Diagnosis within N. Ireland has largely been based upon either the microscopical examination of Ziehl Neelsen stained faecal smears, or a commercial blood ELISA test for antibodies to the organism.  However both tests have significant shortcomings.  In the case of microscopy there needs to be a large number of bacteria within the submitted sample for a positive diagnosis to be made.  This is most likely to be made in samples from clinically affected animals.  Therefore this test has limited usefulness in diagnosing pre-clinically infected animals.  Also, there is the risk of misdiagnosis as identification of the organism is based on phenotypic characteristics (i.e. what the organism looks like) alone.  The current blood based ELISA tests also have poor sensitivity in detecting pre-clinical animals.  Although these tests are highly specific it has been estimated that they have a sensitivity of approximately 15% for detecting all infected animals including during the pre-clinical stages (Whitlock 2000).  There appears to be only a substantial antibody response to the organism in advanced cases.  There is therefore an urgent need for a reliable, specific and sensitive diagnostic test particularly for pre-clinically infected animals.  The current project seeks to address these deficiencies by developing and maintaining optimised culture systems for Map.  There are several methods currently available to grow Map organism. These include solid media, liquid media and the mycobacterial growth indicator tube (MGIT) that contains a marker which fluoresces as oxygen is depleted by the growth of the organism.  It is important that an optimal culture method is available for the Northern Ireland cattle industry and so these various methods will be compared using field isolates.  Recently a variation of the liquid culture method has been found to be an effective and relatively quick method for the identification of Mycobacterium tuberculosis infection in people.  Here liquid cultures are set up in 24 well plates thus reducing the volume of media used and therefore the cost.  The wells are examined under an inverted microscope regularly to identify early growth.  This has increased the speed of detection of organism in South American patients.  To the author’s knowledge there has been no attempt to use this methodology in the diagnosis of Map in animals.

The PCR reaction could offer an attractive alternative for the cultural methods; it is rapid, there are no problems with overgrowth by other micro-organisms and, when the right set of primers is used, the PCR based on the presence of the insertion sequence IS900 is very specific (Willemsen et al., 2000). Despite these advantages, implementation of this technique as a routine diagnostic tool for large-scale usage has not been successful thus far. The main drawback of the technique is the elaborate sample preparation procedures needed to remove inhibitors of the PCR reaction, which are present in faeces and milk which reduces the sensitivity of the test.  However a number of commercial kits have recently become available which may offer the prospect of the practical field application of this technology in the very near future and so as part of this project it is proposed to test a number of PCR kits to assess their suitability as diagnostic tools on farm samples.

While it is generally accepted that individual based tests remain the preferred method to detect Johne’s disease in a herd, this does result in monitoring programs being extremely expensive in terms of test costs. Accordingly, there has been ongoing research to develop herd level testing systems. Based upon a limited set of results from Minnesota, an aggregate system based upon sampling cow alleyways and manure storage areas holds considerable promise for herd level screening (Raizman and others 2004). Similarly, the recent development of real-time PCR makes it a potentially useful tool for detecting infected dairy herds by testing bulk milk.  However it remains uncertain whether these tests are sufficiently sensitive for practical application and so a central part of this project will be to assess the feasibility of testing samples at the herd level in the context of N. Ireland cattle production.

Proposed programme:

The proposed programme can be broken down into 5 objectives

1. Comparison of culture methods for the isolation of Map.
Culture of Map from animal faeces is internationally regarded as the ‘gold-standard’ method of laboratory diagnosis.  However it is both slow and challenging.  Samples must first be decontaminated of any organisms that might overgrow the more slowly growing Map organism thus invalidating the test.  This in itself is problematical as it relies upon the relative resistance to the decontaminants of Map compared to other organisms.  However, where there may be low numbers of Map organism the standard decontamination protocols may remove these from the culture thereby reducing the sensitivity of the test.   It is intended that different decontamination protocols will be assessed in order to increase culture sensitivity.

As discussed in the introduction there are several methods currently available to grow Map organism. An important part of this project will be to compare the cultural techniques currently available.  These methods will be run in parallel in order to gauge their comparative sensitivity and specificity in detecting Map in samples both contaminated with Map.

2. Optimisation of PCR technology for Map identification.
Culture of Map is time-consuming and challenging.  Growth of the organism takes several weeks and to confirm a negative culture requires 16 weeks.  This leads to significant constraints on the usefulness of culture as a diagnostic option for the farmer.  There is an urgent need for more rapid diagnostic tests to become available.  Part of the current proposal will be to optimise the use of PCR technology as a diagnostic option.  The major advantage of this approach is the speed of result, typically 1-2 days.  However progress towards using PCR diagnostics has been hampered by the presence of inhibitors in faeces which affects the PCR reaction.  There are now a number of commercial PCR kits becoming available and it is proposed to run a validation trial on these kits in order to assess their sensitivity when compared to routine serology and culture.  There are two major steps in a Map PCR, concentration of the organism and the PCR reaction itself.  Part of this work will be to identify the best Map concentration protocol to use in conjunction with the best PCR kit reaction.

3.  Phage Assay for Map identification
Recently an assay for human tuberculosis diagnosis has been developed.  This is based upon the use of bacteriophages (‘phages’).  Phages are viruses that target bacteria and it is recognised that the virus used in this assay also attacks Map.  In summary the method is that samples are exposed to the phages which enter any Map organisms present.  After this the sample is processed with a virucide which kill all free viruses that are not contained within bacteria.  The sample is then added to an agar plate growing a rapidly growing mycobacterium.  Any Map infected cells will then release the phage into the surrounding cells which then attack and kill these cells.  This kill off of cells is visible to the naked eye as a plaque.  The advantages of this method are the rapidity of the result (2 days) and any results are semi-quantitative giving an estimation of the amount of Map in the original sample.  As part of the current proposal it is intended to determine if this assay could be a feasible alternative diagnostic tool for Map and to compare it to culture and PCR.

4. Herd Level Tests
There can be considerable costs incurred in testing all animals over a certain age for Johne’s disease.  For many producers it may be useful to have tests available that can give an estimate of whether the organism is present and circulating on a farm or not either before embarking on a planned control program or as a means of monitoring a control program.  Using methods developed in objectives 1, 2 and 3 it is proposed to measure the usefulness of them on herd level samples i.e. bulk milk and environmental samples.  Samples will be taken from farms with confirmed Map infection and identify if these sample matrices could be used to identify Map positive farms.  If demonstrated to be reasonably sensitive these could become useful for future preliminary tests for Johne’s disease.

Milestones with proposed dates: (commencement, end of project etc.)
M1.  Selection and assessment of assay methods on faeces and milk samples spiked with known levels of Map.  (Months 0-12)
M2.  Recruitment of farms for on-farm sampling.  (Months 9-24)
M3.  Conduct on-farm sampling of animals and the environment (Months 12-24)
M4.  Process on-farm samples using assays (Months 12-32)
M5.  Analyse Lab and on-farm results (Months 24-36)
M6.  Dissemination of project conclusions (Months 32-36)

Outputs to AgriSearch (with timescales): (i.e. Progress reports, final report, farmers booklet)
D1.  Year 1 report (M12)
D2.  Year 2 Report (M24)
D3.  Final Year Report (M36)
D4.  Press Releases on Research Findings (M24-36)
D5.  Peer Reviewed Publications (M24-36)

Benefits to industry: (Non monetary)
Farmers participating in this project will benefit directly by having results reported back to them during the course of the project.  In addition participants will become more familiar with Johne’s disease and the methods of disease diagnosis and control.  In raising awareness of Johne’s disease it is hoped that a wider section of industry will consider approaches to controlling this disease.  More directly, this project will give an evidence based approach to the optimal methods for the diagnosis of this disease at the animal and herd level which can then be used in herd level control and eradication of this disease.

A significant component of the proposal will involve sampling at the farm level.  All results will be treated in the strictest confidence.  No reference to specific farms will be used in any of the publications, presentations or reports which will be produced.

Potential financial value / economic benefits to the Northern Ireland farmer:
Johne’s disease is widespread in the NI cattle industry.  The direct costs of disease are difficult to quantify accurately but conservatively it could be costing local industry £0.5M per year.  This project will improve the current diagnostics available to industry and so equip it better to diagnose disease more quickly and more reliably thereby helping to reduce the financial impact of disease on farms.
The Food Standards Agency has advised that while a link with human disease remains controversial and unproven it is important that a long-term aim should be to introduce control measures that would stop Map at source.  By attempting to control Johne’s disease it is hoped that this project will contribute to reducing the exposure of consumers to Map.  By addressing the disease directly it may help to protect consumer confidence in local produce in the face of real or perceived threats to human health.
Technology transfer: (How will this work be disseminated to local farmers?)
The findings of this project will be communicated in a number of ways including;
• Direct communication with participating farmers and their nominated veterinary practitioners
• Articles in the popular farming press
• Farmer group meetings
• Local Veterinary Practitioner Meetings
• Annual Project updates to Agri-search
• Presentations at Scientific Meetings
• Peer-reviewed journal publications