Gamma interferon responses to proteome-determined specific recombinant proteins in cattle experimentally- and naturally-infected with paratuberculosis

Accepted Manuscript Gamma interferon responses to proteome-determined specific recombinant proteins in cattle experimentally- and naturallyinfected with paratuberculosis

Valerie Hughes, Jim McNair, Samuel Strain, Claire Barry, Joyce McLuckie, Mintu Nath, George Caldow, Karen Stevenson PII: DOI: Reference:

S0034-5288(16)30571-9 doi: 10.1016/j.rvsc.2017.04.018 YRVSC 3313

To appear in:

Research in Veterinary Science

Received date: Revised date: Accepted date:

14 November 2016 19 April 2017 20 April 2017

Please cite this article as: Valerie Hughes, Jim McNair, Samuel Strain, Claire Barry, Joyce McLuckie, Mintu Nath, George Caldow, Karen Stevenson , Gamma interferon responses to proteome-determined specific recombinant proteins in cattle experimentallyand naturally-infected with paratuberculosis, Research in Veterinary Science (2017), doi: 10.1016/j.rvsc.2017.04.018

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Gamma interferon responses to proteome-determined specific recombinant proteins in cattle experimentally- and naturally-infected with paratuberculosis.

Valerie Hughes1*, Jim McNair2, Samuel Strain2†, Claire Barry2, Joyce McLuckie1, Mintu Nath3‡,

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Pentlands Science Park

Agri-Food and Biosciences Institute

James Clerk Maxwell Building

Greycrook

Bush Loan

Stoney Road

The King's Building

St Boswells

Penicuik EH26 0PZ

Stormont, Belfast BT4 3SD

Edinburgh EH9 3JZ

Roxburghshire TD6 0EQ

United Kingdom

United Kingdom

United Kingdom

United Kingdom

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Biomathematics & Statistics Scotland

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Veterinary Sciences Division

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SRUC

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Moredun Research Institute

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George Caldow4 and Karen Stevenson1.

Running Title: Diagnostic markers for bovine paratuberculosis

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gamma release assay

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Key words: Mycobacterium avium subspecies paratuberculosis, Johne’s, cell-mediated, diagnosis, interferon

Abbreviations: IFN-γ, interferon- γ; IGRA, Interferon Gamma Release Assay; JD, Johne’s disease; Maa, Mycobacterium avium subspecies avium; Map, Mycobacterium avium subsp paratuberculosis; PCR, Polymerase *

Corresponding Author: FAX: [email protected]

+44

(0)131

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6111;

TEL:

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†

(0)131

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5111;

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mail:

Currently: Animal Health and Welfare N. Ireland, Box 10 1st Floor, Dungannon Business Cube, 5 Coalisland Rd, Dungannon, Co. Tyrone, BT71 6JT ‡

Currently: University of Leicester , University Road , Leicester, LE1 7RH, UK

ACCEPTED MANUSCRIPT 2 Chain Reaction; PPD, Purified Protein Derivative; HRF, High-Risk Farm; LRF, Low-Risk Farm; IRF1,

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Intermediate-Risk Farm 1; IRF2 ,Intermediate-Risk Farm 2.

ACCEPTED MANUSCRIPT 3 Abstract Johne’s disease (JD), is a fatal enteritis of animals caused by infection with Mycobacterium avium subspecies paratuberculosis (Map). Diagnosis of subclinical JD is problematic as test sensitivity is limited. Th1 responses to Map are activated early, thus detection of a cell-mediated response, indicated

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by measuring interferon gamma (IFN-γ) stimulated by mycobacterial antigens, may give the first

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indication of sub-clinical infection.

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Crude extracts of Map (PPDj) have been used to detect the cell-mediated response in infected cattle. More specific, quantifiable antigens may improve test specificity and reproducibility. Map-specific

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proteins, MAP_3651c and MAP_0268c, raised a cell-mediated immune response in sub-clinically infected sheep. Results presented in this manuscript demonstrate these proteins elicit a cell-mediated

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response in experimental and natural infections of cattle.

Individual ranked IFN- responses of experimentally infected calves to PPDj showed a high, statistically

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significant association with ranked responses of recombinant Map antigens. Responses of infected

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animals were higher than the control group. Threshold values determined using data from an experimental infection were applied to naturally infected animals. Some animals exhibited responses above these

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threshold values. Responses to MAP_3651c on a farm categorised as high- risk for JD showed strong

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evidence (p<0.001) that responses were significantly different to lower-risk farms. The IGRA test may prove to be an additional tool for the diagnosis of JD, and inclusion of specific antigens a refinement

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however, understanding and interpretation of IGRA results remain challenging and further investigation will be required to determine whether the IGRA test can detect exposure and hence predict clinical JD.

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Introduction

Johne’s disease (JD), or paratuberculosis, is a fatal, chronic granulomatous enteritis of animals caused by Mycobacterium avium subsp. paratuberculosis (Map), characterised by severe

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emaciation and in some species diarrhoea. Young animals may be more susceptible to infection

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and the disease is mainly spread through the ingestion of contaminated faecal material.

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Following infection, there is a long sub-clinical phase during which no sign of disease is apparent but the animal may shed Map intermittently and be infectious [Sweeney et al., 1992;

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Chiodini, 1996; Toman et al., 2003].

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During the sub-clinical phase of JD, diagnosis is problematic as the sensitivities of commercially available tests are limited. Sub-clinical animals may not shed at the time of sampling or may

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shed very low numbers of Map in their faeces, which may not be detectable by PCR or

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bacteriological culture. Circulating antibodies against Map may be absent or too low to allow differentiation of infected and uninfected animals when currently available commercial ELISA Thus eradication programs are hampered by the inability to detect most

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tests are applied.

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infectious sub-clinical animals.

Infected cattle initially develop an early and effective pro-inflammatory Th1 immune response to

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Map [Stabel, 2000; Sweeney, 2011]. Thus detection of cell-mediated immune response by measuring the levels of interferon-γ (IFN-γ) produced in response to mycobacterial antigens may give an early indication of infected animals [Wood et al., 1989]. Recently, Map recombinant proteins were substituted for Protein Purified Derivative (PPD) in an attempt to improve the specificity of the response in cattle [Mikkelsen et al., 2011]. However, it was noted that a cocktail of antigens may be required as a PPD substitute because not all animals

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respond to the same antigen stimuli. Thus, increasing the variety of immune-reactive antigens for use in an IFN-γ release assay (IGRA) should boost the sensitivity of the test. Proteome determined Map–specific antigens were shown to elicit a cell-mediated response in subclinically-infected cases of ovine paratuberculosis [Hughes et al., 2013]. This study focuses on

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two of these Map recombinant proteins encoded by MAP_0268c and MAP_3651c: the first,

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MAP_0268c encodes a protein listed in NCBI’s conserved domain data base [Marchler-Bauer et

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al., 2015] as a S-adenosyl-L-methionine-dependent methyltransferase, with a general predicted function in secondary metabolite biosynthesis, transport and catabolism; the second,

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MAP_3651c encodes a protein listed as a member of the acyl-CoA dehydrogenase superfamily

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and is related to the alkylation response protein AldB involved in lipid transport and metabolism. The ability of these recombinant antigens to elicit a Th1 response and their potential to detect

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early Map infection in cattle when incorporated into an IGRA was investigated. From the results

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of the experimental infection, it was possible to determine a threshold value for the IGRA, which was then applied to naturally infected animals from farms with different seroprevalence in order

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to assess the animal responses to these recombinant proteins and their potential for diagnosis.

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Materials and Methods

Procurement of JD-free animals for experimental infection All animals (male Friesian-Holsteins) were sourced from farms with no history of clinical JD or

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bovine tuberculosis in Northern Ireland. The calves were screened using a JD serum ELISA

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(Idexx, following the manufacturer’s recommendation) and faecal culture. The Bovigam TM

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(Celtic Diagnostics Ltd, Dublin, Ireland) IGRAs were also performed, on whole blood stimulated with PPDa. IGRAs were performed according to the manufacturer’s instructions. Animals

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producing low levels of IFN-γ in response to PPDa were purchased and transported to Veterinary

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Services Division of AFBI. Experimentally infected animals were housed in a secure, level 2 containment facility in groups of 6 animals and cared for until the end of the study. Control

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animals were housed in a secure farm facility. All experimental procedures were assessed and

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approved by an Ethics Committee and authorised under the Animals (Scientific Procedures) Act

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1986.

Calf experimental infection models

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Briefly, 12 calves (age range 15-18 weeks) were infected with Map (108cfu administered orally in a single dose, suspended in PBS) and monitored (IGRA responses to PPDs, and Poke Weed Mitogen (PWM)) for 39 weeks post infection before being euthanized.

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12 calves (age range 17-19 weeks) were infected with Maa (109 cfu administered orally in a

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infected and served as controls for both experimental infections.

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single dose, suspended in PBS) and monitored for 8 weeks post infection. Six animals were not

IGRA responses to recombinant proteins were measured at 28 and 37 weeks post-infection for

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Map-infected calves and at 5 and 7 weeks post-infection for Maa-infected calves.

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Blood collection

Incubation of blood with antigen

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Blood (200µl) was incubated with either PPDj (kindly provided by Douwe Bakker) and/or PPDa (AHVLA Weybridge) (added in a volume 50 µl to a final concentration of 20 µg/ml) or purified recombinant protein (added in a volume of 50 µl to a final concentration of 4 µg/ml). In addition to this, a positive control for stimulation was used (PWM at a final concentration of 10µg/ml or SEB at a final concentration of 1µg/ml). Media controls (PBS) were also included. Stimulation of blood was performed in tissue culture plates and incubated in 5 % v/v CO2 for 24 hrs. Plates

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were centrifuged (500g, 5 min at room temperature) and supernatant removed and stored at 70oC until required.

Bovigam TM IGRA

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IGRAs were performed essentially as described by the manufacturer. IFN- (kindly provided

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by Sean Wattegedera and Gary Entrican, supported by the Scottish Government RESAS and by

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BBSRC [grant number BBI019863/1], standards (ranging from 0-8000 pg/ml in the experimental

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infection or 0-50 000 pg/ml for the naturally-infected animals) were included on each ELISA plate analysed. The standard curves of this data enabled calculation of the absolute concentration

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of IFN-γ released during stimulation of blood. Working with concentrations interpolated from standard curves covering the range of interferon concentrations likely to be encountered in an

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IGRA, provides a robust normalisation of data. Alternatively, ODs were adjusted according to

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the OD-values of the positive and negative controls supplied with the BovigamTM kit on the

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respective plates as described previously [Jungersen et al., 2002] to minimize effects of plate to plate variation.

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Data presented in the first two figures was normalised by the method of Jungersen et al [2002], all other data was interpolated from standard curves and reported as absolute IFN-γ

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concentrations.

Diagnosis of paratuberculosis by serum antibody ELISA test Serum samples from blood were tested for antibody against paratuberculosis using ID Screen Paratuberculosis indirect ELISA (IDvet). The procedure was carried out in accordance with the manufacturer’s protocol.

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Diagnosis of paratuberculosis by faecal culture Faecal samples were collected and decontaminated by a slightly modified double incubation method [Shin, 1989]. Briefly, 2 g of faeces were mixed with 35 ml sterile deionised water and

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resuspended. Particulate material was allowed to settle for 30 min and the top 5 ml of the liquid

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fraction was removed and treated with hexadecylpyridinium chloride in half strength Brain Heart

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Infusion Broth (Oxoid Ltd., Hampshire, UK) to a final concentration of 0.7 % (w/v) at 37 oC for 18 h. The suspension was centrifuged 1500 g, 30 min. The resulting pellet was resuspended in 1

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ml para-JEM AS (ThermoFisher Scientific, Paisley, UK, reconstituted in accordance with the

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Manufacturer’s protocol and diluted 15 fold in Brain Heart Infusion Broth) and incubated for a further 18 h at 37 oC. This was then used to inoculate broth (reconstituted from para-JEM

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Reagent Kit w/Blue, in accordance with the manufactures protocol) and cultured using the TREK

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ESP II system for detection of Map [Woods et al., 1997]. Samples signalling positive were submitted for Ziehl-Neelsen staining to confirm the presence of acid-fast bacteria. To confirm

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the presence of Map in positive faecal cultures, the culture was diluted 1:3 and boiled for 10 min

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to lyse the cells. DNA in the cell lysate was amplified by PCR using primers 90 (5'GTTCGGGGCCGTCGCTTAGG-3') and 91 (5'-GAGGTCGATCGCCCACGTGA-3' ) as

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described by Sanderson et al. (1992).

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Cloning of recombinant proteins

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Two recombinant Map proteins were selected for testing in the calf infection model. These

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proteins were previously identified as potentially Map-specific by comparison of the proteomes of strains of Map compared with those of Maa [Hughes et al., 2008] and shown to elicit a cell-

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mediated response in subclinical ovine paratuberculosis [Hughes et al., 2013].

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Open reading frames of proteins of interest were amplified by PCR from genomic DNA of Map strain K10 [ATCC® BAA. 968™]. Gene sequences of MAP_0268c and MAP_3651c were

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retrieved from Kyoto encyclopedia of genes and genomes (KEGG) [Kanehisa et al., 2000].

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Primers were designed to allow an in-frame insertion of the first coding methionine into the NdeI site of vector pET 15b (Millipore Ltd, Hertfordshire, UK). The 3’ ends of DNA encoding

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MAP_0268c and MAP_3651c were cloned into the XhoI site of the vector. In the case of

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MAP_3651c, this generated a truncated product due to an internal XhoI site within its open reading frame. The constructs were initially transformed into NovaBlue (Millipore Ltd,

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Hertfordshire, UK) competent cells and then sub-cloned into the expression host BL21 (DE3).

Expression and purification of proteins BL21 (DE3) containing the MAP_0268c or MAP_3651c constructs was grown in Rich Broth containing 1% (w/v) glucose and 75µg/ml Carbenicillin. Cultures were induced at an OD600 of 0.6-0.8 using 0.5mM IPTG, allowed to grow for a further 3 h and then harvested (3080 g, 20

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min, 4°C). Supernatants were discarded and the pellets air dried. Pellets were stored at -80°C prior to lysis and purification. Expression of protein was assessed on SDS-PAGE gels [Laemlli, 1970] by comparing the induced cell protein profile with that of similarly produced un-induced cultures.

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Cell pellets obtained from 200 ml cultures were resuspended in 2.5 ml of binding buffer (50 mM

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sodium phosphate, pH7.4, 300 mM sodium chloride, 10 mM imidazole) which also contained

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DNAse (5µg/ml) and 50 l Protease Inhibitor Cocktail Set III, EDTA-Free (Millipore Ltd,

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Hertfordshire, UK ). The cells were lysed using a Precellys Dual 24 tissue homogeniser in 7 ml tubes containing 0.1-0.5 mM glass beads. Three disruption cycles at 5000 rpm for 30 s were

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performed and samples were cooled on ice for 5 min between each cycle. The resulting suspension was centrifuged (13 000 g, 5 min at room temperature) and the supernatant removed

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and re-centrifuged. The lysate was further clarified by two consecutive filtrations: firstly using a

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membrane with pore size of 0.45µm; then one of 0.20µm. Lysate was loaded onto Vivapure Maxiprep MC spin columns (Sartorius, Surrey, UK) (pre-

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equilibrated in binding buffer) in 10 ml batches with centrifugation 100 g, 30 min, 10 oC. The

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column was washed three times with wash buffer (50 mM sodium phosphate, pH7.4, 300 mM sodium chloride, 30 mM imidazole) with centrifugation (500 g, 3min, 10 oC) between each wash

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step. For MAP_3651c, an extra wash step was inserted before the elute step in order to remove endotoxin. This involved three steps of alternate washes with10mM TRIS-HCl pH8 and isopropanol, a penultimate wash with 10mM TRIS-HCl pH8 and a final one with wash buffer before elution. His-tagged proteins were removed from the column with sequential batches of elution buffer (50 mM sodium phosphate, pH7.4, 300 mM sodium chloride, 300 mM imidazole). Samples from batches of eluate were removed, mixed directly in equal proportion with SDS

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sample buffer and submitted to SDS-PAGE [Laemlli, 1970] and visualised with SimplyBlue Safestain (Life technologies Ltd, Paisley UK). Vivaspin 15R centrifugal concentrators (Sartorius, Surrey, UK) were used for diafiltration of the fractions containing protein to remove imidazole from the purified recombinant protein preparation and also to concentrate the sample.

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MAP_3651c was expressed in an insoluble form and thus it was necessary to purify it under

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denaturing conditions. The purification procedure was essentially as detailed with the

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modifications that all buffers contained 8 M urea and the column eluate was dialysed before

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Dialysis of MAP_3651c for removal of urea

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concentration as indicated below.

MAP_3651c protein was dialysed using D-Tube Dialyzer Maxi MWCO 6-8 kDa (Millipore Ltd,

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Herfordshire, UK). The dialysis membrane was first hydrated with distilled water and then the

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eluate was placed into the dialyzer tube in the floating rack. Eluate was dialysed against 6 M urea in PBS with constant stirring for 6 hrs at 4 o C. Thereafter, the dialysis buffer was diluted at

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regular intervals by the addition of fresh PBS over a three day period until the concentration of

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the urea in the PBS had reached 2 M. On the fourth day the dialysis buffer was replaced by PBS and the dialysis continued for a further 6 hrs. Dialysed protein was centrifuged at 14000 g, 20

required.

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min, 40C and the supernatant (ie soluble faction) was aliquoted and stored at -80 oC until

Quantitation of recombinant protein Concentrations of the recombinant proteins stocks were ascertained using the PierceTM BCA protein assay kit (ThermoFisher Scientific, Paisley, UK). In accordance with the kit manual, the protein concentration was determined with a calibration curve using BSA as a standard.

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Naturally-infected serum ELISA positive heifers and their calves A herd of animals was screened by serum ELISA test for paratuberculosis as part of the Premium Cattle Health Scheme (PCHS ) [www.Sruc.ac.uk/120112/premium_cattle_health_scheme]. Six

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heifers were found to be positive by this test. One animal had a score that was deemed to be

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inconclusive. Positive and inconclusive animals from this group had calves at foot and both dams

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Selection and sampling of Scottish study farms

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and their calves had three blood samples taken at approximately monthly intervals.

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The four farms selected as our Scottish study farms were regularly tested as part of the PCHS (as referenced above) at the time of this study and in accordance with this scheme, all animals tested

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were two or more years of age.

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One problem farm was selected as having current, circulating Map infections and was referred to as the ‘High-Risk Farm’ (HRF). This herd consisted of 108 animals the majority of which

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were Luing breed with four Simmental or Simmental cross -all of mixed ages. Serum ELISA

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positive animals had been detected at every annual sampling for the previous 5 years, with four positives and one inconclusive animal at the time of sampling for this study.

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Three other farms were selected because they had herds with no serum ELISA positive animals for the past 3 or more years. However, of these, only one farm had no faecal culture positive animals at the time of sampling and this is referred to as the ‘Low-Risk Farm’ (LRF) from which 29 Aberdeen Angus animals of mixed ages were sampled. The other two farms each had a single faecal shedding animal at the time of sampling and are referred to as ‘Intermediate-Risk Farms’. Animals from ‘Intermediate-Risk Farm 1’ (IRF1) were all Luing (32 in number) of mixed ages

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and those from ‘Intermediate-Risk Farm 2’ (IRF2) were predominantly Herefords (52 in number) of mixed ages. Animals were sampled on each farm once, blood samples were taken for IGRAs (stimulated with MAP_0268c, MAP_3651c and PPDa). Faecal samples were also taken at the same time for

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culture.

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Statistical Analysis

Some of the OD data points arising in the IGRA from experimentally infected calves were

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greater than the ODs of the most concentrated standard. We used two approaches in all statistical

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analyses of data from experimental infections: data were either extrapolated past the highest standard point on the curve or assigned the highest threshold value (8000 pg) if the OD value

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was greater than that of the most concentrated standard. Both approaches gave similar results;

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the choice of which would not have altered the findings or interpretation of the study. Results presented here were those obtained without using a threshold. Non-parametric tests (discussed

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below) that utilise the ranks of the OD values were used for all statistical analyses presented here

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as data were not normally distributed, particularly for the approach using thresholds, and sample sizes were fairly small. Such statistical analyses will tend to be conservative, but are more robust

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than parametric analyses that make unrealistic assumptions. The P-values, obtained from individual analyses of IGRA responses, were adjusted using a false discovery rate (FDR) approach in order to mitigate multiple comparisons problems [Benjamini and Hochberg, 1995]. For the experimental infection, to assess the association of measures of IFN- responses to PPDa and PPDj with responses of Map antigens (MAP_3651c and MAP_0268c) at 28 and 37 weeks post-infection in Map infected animals and at 5 weeks post-infection in Maa infected animals,

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we conducted Spearman’s rank correlation () test. The group (infected or control) IGRA responses to the antigens (PPDa, PPDj, MAP_0268c and MAP_3651c) were compared separately at each bleed (at 28 and 37 weeks post-infection) in Map-infected animals using the Mann-Whitney. The group (infected or control) IGRA responses were also compared based on

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the combined data from these bleeds using a modified Mann-Whitney U test [Rosner and Grove,

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1999] including the clustering effect of animal. Data was analysed in a similar manner from

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Maa-infected animals combining the data from the bleeds 5 and 7 weeks post-infection. The

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Kruskal-Wallis rank sum test was used to investigate whether the observed ranks of MAP_3651c, MAP_0268c and PPDa values were different in four farms classified as having

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varying risk for Map infection.

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Results

Analysis of

 responses of calves to PPDs prior to experimental infection

Samples of blood were taken from prospective animals on three consecutive weeks prior to

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infection and were tested by IGRA using PPDa and PPDj and PWM as the T-cell stimulator

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positive control. Most animals had average OD readings generated for these PPDs within the

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mean and two standard deviations of the average PBS reading for the group and below the cut control. However,

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off of 0.13 used to define the acceptable range for the negative bovine

some animals had OD values slightly above this cut off for some or all of the PPDs, which may

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reflect prior mycobacterial exposure before recruitment into the study or non-specific responses

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to antigenic stimulation.

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Experimental infection of calves with Map

 responses of the animals were

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Following experimental infection of calves with Map, the

monitored weekly using PPDa, PPDb and PPDj as stimulants and PBS and PWM as controls.

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The results are shown in Fig 1. Corrected mean ODs (PBS- background subtracted and

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normalised to control for plate to plate variation) for the group of animals showed an increasing trend for the OD responses to PPDa and PPDj in the IGRA assay initiating 9 weeks after exposure and is indicative of infection in accordance with the definition of Pirofski and Casadeval [2002]. These responses were maintained at an elevated level throughout the study period until the animals were euthanised. The magnitude of the response was less for the samples stimulated with PPDb. Animals were skin tested at 37 weeks and this did not appear to

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affect the IGRA to any great extent except for the suggestion of a small, transient boosting of the

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OD response post skin-test.

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3.5

IFN- Responses to PPDs in Map-Infected Animals

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PPDB

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PPDA PPDJ

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Normalised OD

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Weeks post infection

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IFN- Responses to PWM in Map-Infected Animals

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PWM

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Normalised OD

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Weeks post infection

Fig 1. Progression of Experimental Map infection

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Average IGRA responses (with standard deviation error bars) of Map-infected animal group (n=12) stimulated with mycobacterial antigens PPDa (20 g/ml), PPDb (20 g/ml), PPDj (20 g/ml) and Positive T-cell stimulator control PWM. Experimental infection of calves with Maa

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Following experimental infection of calves with Maa, the IFN- responses of the animals were

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monitored weekly using PPDa, PPDb and PPDj as stimulants and PBS and PWM as controls.

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Plotting mean ODs (PBS-background subtracted and normalised to control for plate to plate

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variation) for the group of animals showed an increasing trend for the OD responses to PPDa and PPDj in the IGRA assay starting 2-3 weeks after infection, peaking at weeks 4-5 see Fig 2. At

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this peak, normalised responses of Maa-infected animals to PPDA were more than 2 fold higher than those of Map-infected animals at peak response (around 19 weeks post-infection). This

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observation may be because the initial inoculum was greater in the Maa infection than that used

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in the Map-infection.

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These elevated levels were maintained throughout the study period until the animals were euthanized at 8 weeks. The responses were highest for PPDa, but PPDj and PPDb also

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stimulated responses that mimicked those of PPDa.

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The skin test (applied at 7 weeks) appeared to have a slight boosting effect on responses, even though the responses in these animals were almost at their peak level (upward trends were seen in response to PPDa, j and b between time points 7 & 8).

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IFN- Responses to PPDs in Maa-Infected Animals 7

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Normalised OD

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PPDA PPDJ

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PWM PBS

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PPDB

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Fig 2. Progression of Experimental Maa infection

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Average IGRA responses (with standard deviation error bars) of Maa-infected animal group

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(n=12) stimulated with mycobacterial antigens PPDa (20 g/ml), PPDb (20 g/ml), PPDj (20 g/ml) and Positive T-cell stimulator control PWM.

Responses of Map infected animals to antigens MAP_3651c and MAP_0268c MAP_0268c and MAP_3651c were used to stimulate whole blood from Map-infected animals in the experimental infection group at 28 and 37 weeks post infection. At these times infected

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animals are in the phase of elevated IFN- response to PPDa and PPDj. The IFN- responses of Map-infected and control animals are recorded in supplementary data Tables S1. At 28 weeks post-infection, the ranked IFN- responses to PPDj of individual Map-infected animals showed high, statistically significant associations with the ranked responses of the

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recombinant protein Map antigens (MAP_ 0268c (=0.75, P=0.026) and MAP_3651c (=0.86,

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P=0.002). Moderate/weak associations of PPDa and both Map antigens were also observed, but

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the estimate of rank correlation was significant only in the case of MAP_3651c (MAP_3651c:

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=0.64; P=0.042) see Table 1. By 37 weeks post-infection, the associations were weakened and were no longer statistically significant. Moderate/weak associations of the PPDs and the Map

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antigens were also observed in control animals.

Despite the significant estimates of rank correlation between responses to the recombinant

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antigens with those of the PPDs, we observed the medians of the responses of infected animals

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were not significantly higher than those of the uninfected control group at 28 weeks. As expected, the medians of the infection control antigens (PPDa, PPDj) were higher than those of

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the uninfected control group at 28 weeks, but the evidence was weak (P<0.10). However, at 37

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weeks post infection, the differences in the medians of the responses of infected and uninfected animals were significant for PPDj (P=0.022), and MAP_0268c (P=0.013) but not for MAP_3651c or PPDa.

Analysis of the combined data of the two bleeds (regardless of sampling time point) suggested the medians of the responses of infected animals were higher than those of the non-infected group and were statistically significant for PPDa (P=0.039), PPDj (P=0.039), MAP_0268c (P=0.039), however, the evidence was weak for MAP_3651c (p=0.059).

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Table 1: Spearman’s Rank Correlation of IGRA responses

PPDa

PPDj

PPDa

PPDj

=0.47(P=0.21)

=0.75(P=0.026)

=0.53(P=0.09)

=0.64(P=0.06)

=0.64(P=0.042) =0.86(P=0.002)

Control Animals

PPDa

PPDj

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=0.63(P=0.02) =0.59(P=0.03)

=0.51(P=0.094) =0.59(P=0.067)

=0.48(P=0.08) =0.66(P=0.02)

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MAP_3651c

Maa infected animals

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MAP_0268c

Map infected animals

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Estimates of Spearman’s rank correlation () of responses of individual animals, control or

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infected with Map or Maa stimulated with the novel recombinant antigens, PPDa and PPDj in an IGRA at 28 or 5 weeks post-infection, respectively. The values in the parentheses show the P-

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values.

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Responses of Maa infected animals to MAP_0268c and MAP_3651c

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MAP_0268c and MAP_3651c were used to stimulate whole blood from Maa-infected animals group at 5 and 7 weeks post infection. At these times infected animals are in the phase of  response to PPDa and PPDj. The IFN- responses of Maa-infected and control

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elevated

animals are recorded in supplementary data Tables S1. Blood of animals sampled 5 weeks post-infection, showed a moderate association between the ranked responses to either of the recombinant antigens (MAP_0268c and MAP_3651c) and those of the PPD antigens but the estimates of rank correlations were not statistically significant for any combination. Analysis of combined data from infected animals bled at 5 and 7 weeks post

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infection, suggested the medians of the responses of the infected animals were higher than those of the group of non-infected animals for PPDj (P=0.002), PPDa (P= 0.002) and MAP_3651c (P=0.016) but not for MAP_0268c (P=0.104).

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Stringent and Intermediate Threshold values for indication of infection as determined by

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the experimental infection

Threshold values were derived from IGRA results of experimental animals in the negative

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control group (who did not receive a dose of Map) and were the mean IFN- concentration value plus three standard deviations for stringent analysis. The stringent threshold values determined

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for PPDa, MAP_0268c and MAP_3651c were 1.9, 3.6 and 1.2 ng /ml IFN- respectively.

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Intermediate stringency values (mean plus two standard deviations) were also determined and

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were 1.4, 2.7, 0.9 ng/ml, respectively. These threshold values were used in the subsequent field

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studies below.

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Table 2: IGRA responses to PPDa and recombinant antigens of cows diagnosed with JD and their calves.

IFN- released in IGRA (ng/ml)

0.5

5.2

7.5

10.

13.

0.7

0.4

0.2

0.1

7.9

4

2

0.4

8.1

4.0

8.6

7.6

1.0

2.0

5.5

3.0

0.7

0.1

0.2

0.5

1.6

0.8

0.5

1.8

0.4

1.5

0.4

0.3

0.2

0.3

0.4

0.2

0.6

4.3

1.0

3.3

5.3

8.3

1.3

1.0

1.6

1.6

0.1

15.

14.

15.

14.

49.

23.

21.

4

9

0

0

7

1

9

1.6

7.4

12. 0.3

0.9

7

2.0

0.1

1.2

0.7

0.2

0.4

0.4

1.3

0.2

0.4

0.0

0.9

9.5

0.9

6

2.1

2.7

40.

24.

30.

3.3

9

9

7

0.7

1.9

7.8

3.0

651c

0.6

0.8

0.4

Cc

0.3

0.5

0.9

7

8.7

9.8

11.6

3.2

3.7

5.2

0.3

0.5

0.6

1.4

0.5

0.8

0.6

0.1

0.1

0.1

2.4

0.3

1.2

0.8

0.3

0.6

1.4

0.6

0.6

0.1

18.

49.

6.4

6

7

9.7

7.6

8.9

18.

0.0

2.1

6

3.2

0.2

0.6

0.9

1.8

1.5

0.6

0.2

1.1

1.3

0.6

0.2

0.2

1.4

0.6

0.8

0.4

3.8

1.9

0.5

1.2

0.4

6.6

0.6

1.4

0.2

2.9

49.

25.

1.2

5.4

6.7

9

8.5

8.7

6.0

5.3

6

5.2

15.1

3.7

0.1

0.4

0.2

0.3

2.4

1.6

1.6

0.7

0.6

1.1

0.8

0.8

0.3

0.1

0.0

0.1

1.7

1.4

0.2

0.3

0.1

3.8

0.7

0.9

0.1

0.1

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9.2

Cb

6.9

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SEB MAP_0 268c MAP_3

0.8

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32.

3.6

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PPDA

8.9

0.2

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8.6

4.1

Ca

16.

2.7

SEB MAP_0 268c MAP_3 651c Bleed 3

0.1

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1.2

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1.6

Calves of Serum ELISA negative Cows

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9.1

C 1

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SEB MAP_0 268c MAP_3 651c Bleed 2 PPDA

0.9

D 7

Calves of Serum ELISA Positive Cows C C C C C C 2 3 4 5 6 7

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Bleed 1 PPDA

D 1

Serum ELISA Positive Cows D D D D D 2 3 4 5 6

IGRA responses of serum ELISA positive cows (D1-7), their calves (C1-7) and also calves born to Serum ELISA negative dams (Ca, Cb, Cc) to PPDa and recombinant antigens. Responses which exceed the intermediate stringency threshold, are subtly highlighted, those responses which exceed the highest stringency threshold are highlighted in bold.

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Responses of Cows with natural JD infection diagnosed by the serum ELISA test and their calves Cattle on a working beef farm were screened for JD using the serum ELISA test. The results revealed six cows (designated D1-6) were test-positive for JD (OD scores ranging from 102-217

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%). One cow (D7) was categorised as inconclusive (OD score of 61 %). Faecal samples of

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these seven test positive/inconclusive animals were submitted for bacteriological culture, all

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except D3 were culture positive at 16 weeks. Of the six positive cultures, it was possible to detect the Map-specific target IS900 by PCR in all except D6. All seven cows had calves at foot

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(designated C1-7, numbers corresponding to their dams) and blood samples were taken from

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cows and their calves three times at approximately monthly intervals (Bleed 1, 2 and 3). The IFN- responses of these animals to PPDa and novel Map antigens were determined.

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Amounts of IFN- released in the assays varied over the three month period of testing,

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fluctuations in responses were seen in some animals: (D2, D3, D4, D6) and this was most

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pronounced in D4. Steady declines were observed in others (D7 and D5), whilst one animal (D1) had stable low responses. Responses of cows and calves to PPDa, MAP_0268c and

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MAP_3651c and the T-cell stimulator positive control (SEB) are recorded in Table 2. Responses

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which exceeded this stringent cut-off are highlighted in bold. Those which exceed the intermediate threshold values are highlighted subtly. Map-infected cows showed varying degrees of IFN-  responses to PPDa and the antigens. For example, in the cow group, two animals did not show a PPDa response above the threshold in any of the three month period (D1 and D3). In addition, D1 did not show responses above the threshold to either of the recombinant antigens; its response to the positive control SEB was one of the lowest in the group (2.59ng/ml). This was consistent during the three month period of

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testing with the IFN-response to SEB stimulation being at least two fold lower for this animal than the remainder of the group. However, animal D3, in contrast, showed a consistent above threshold response to MAP_0268c, and had a strong response to SEB (one of the highest in the group). Notably this animal was the only cow not found to be shedding Map in its faeces.

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Responses in the calves were generally lower than those observed in their infected dams. There

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were more calves showing above threshold responses in subsequent bleeds, which might reflect

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their increased exposure as they were nurtured by their potentially infectious dams and/or an

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increased capacity to respond with maturity. PPDa stimulated the most number of above threshold responses in the calves born to ELISA positive dams in the three experiments. In the

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early time points, (Bleed 1 & 2) the magnitude of the response to MAP_ 0268c appeared to make it the predominant antigen for calves, stimulating the highest IFN- response in most

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animals (although this was not reflected in the number of above threshold response as the cut-off

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for MAP_0268c was higher than that of the other two antigens). Calf C3 gave a remarkably high

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transient response to MAP_0268c in Bleed 2. It’s dam (D3) also showed high responses to MAP_0268c in all 3 bleeds. The response of the calves to PPDa-the most predominant antigen

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in cows surpassed that of MAP_0268c in Bleed 3.

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Most calves born to serum ELISA positive dams showed above threshold (some at the less stringent cut-off) responses to either PPDa or at least one recombinant antigen. The only calves which did not show any response were C5 and C6.

C6 (and its dam) did not exhibit any above

threshold response to the more specific recombinant antigens MAP_ 0268c or MAP_3651c. All calves born to serum ELISA negative dams (Ca, Cb, Cc) had little responses to either PPDA or the Map antigens with only one calf (Cb) expressing above threshold response to

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MAP_3651cin the first bleed. Cb also expressed an intermediate level response to both PPDa and MAP_3651c at Bleed 3. Blood samples were taken from animals kept on the high, intermediate and low risk farms on a single occasion. A sample from each individual animal was used for serum ELISA to detect

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antibody against Map and IGRAs were performed with PPDa, MAP_0268c and MAP_3651c

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stimuli to detect cell-mediated responses. Results from the IGRAs for the individual farms are

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presented in Fig 3.

As expected, the Kruskal-Wallis rank sum test showed that there was strong evidence (P<0.001

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that farms are different for the location parameters corresponding to MAP_3651c, MAP_0268c

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and PPDA values. The LRF showed lower median values for MAP_3651c, MAP_0268c and

MAP_0268c and PPDA than the HRF.

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PPDA compared with the HRF. Interestingly, IRF2 showed higher median values for

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The ‘HRF’ had many above-threshold IGRA responses to antigens at the highest stringency: PPDA (14%), MAP_0268c (27.8%) and particularly MAP_3651c (20.4%). Interestingly the

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four serum ELISA positive animals detected did not respond with above threshold values in the

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IGRAs stimulated with the recombinant antigens, but did for PPDa (values ranging from 2.4-3.7 ng/ml). However, the inconclusive animal responded positively for both the recombinant

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antigens MAP_0268c, MAP_3651c and PPDA (values of 6.8, 4.4 and 5.6ng/ml respectively). The ‘LRF’ had fewer instances of above threshold IGRA ELISA scores: PPDA, (6.8%); MAP_0268c, (17.2%) and MAP_3651c, (0%). For MAP_3651c, there were no positives even when the intermediate stringency threshold was applied. This reflects the lower exposure rate of these animals to Map.

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‘IRF1’ had above threshold responses at the highest stringency as follows: PPDa (25%), MAP_0268c (19%), MAP_3651c (9%). ‘IRF2’ had above threshold responses as follows: PPDa (25%), MAP_0268c (44%), MAP_3651c (0%, although three animals {6%}, were positive applying the intermediate stringency threshold).

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Correlations of the responses of the animals to the recombinant antigen (MAP_0268c or

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MAP_3651c) to PPDA are presented in Table 2 S in the supplementary data.

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Fig 3.

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The stringent threshold values used previously were 1.9, 3.6 and 1.2 ng /ml IFN-

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for PPDa, MAP_0268c and MAP_3651c respectively.

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29

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Discussion Proteins MAP_3651c and MAP_0268c are recognised by calves experimentally infected with Map when incorporated in an IGRA. Responses of infected animals to MAP_3651c antigen closely mimic the responses of these animals to PPDA, at least in the initial phases of the

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infection and its ability to discriminate between Map-infected and non-infected animals is only

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just short of statistical significance. The discriminative ability of MAP_0268c was statistically

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significant. These antigens previously have been shown to discriminate natural cases of ovine JD from uninfected animals, although in this study the results for MAP_3651c were highly

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significant [Hughes et al., 2013].

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As a marker for infection, it is reassuring that the estimates of rank correlation were statistically significant for responses of MAP_0268c and MAP_3651c with those of PPDs in the

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experimental infection. A statistically significant and higher estimate of rank correlation for

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responses of MAP_3651c with PPDj could possibly be due to a much greater level of expression of the protein in Map than that of its orthologue in Maa [Hughes et al., 2008], indeed it was

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demonstrated that a peptide derived from MAP_3651c is a component of PPDj but not PPDa

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[Wynne et al., 2012]. The association between the ODs for PPDj and the recombinant proteins

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diminished at a later time point in the infection Despite MAP_3651c being ‘proteome determined Map-specific’, it appears that it can also discriminate animals experimentally infected with Maa from uninfected controls. This observation, that MAP_3651c is not totally specific for Map, is surprising given that the level of expression of its orthologue in Maa is negligible [Hughes et al., 2008]. However, paralogues of MAP_3651c exist with a high level of identity (50% for MAP_2405c its closest relative) whose orthologues may be well expressed in Maa, this could explain why MAP_3651c can identify

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Maa infections. If this is the case, then to uncover specificity of MAP_3651c, it may be necessary to search for immunogenic peptides which are not present in paralogue sequences. Despite this, it is still possible that MAP_3651c may be useful as a diagnostic tool for detection of naturally acquired infections; the antigen stimulation seen in the Maa experimental infection

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is at the peak of a strong cell-mediated response to infection (normalised peak responses of were

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more than 2-fold higher than those of Map-infected animals) and this level of infection may be

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rarely seen in a natural setting.

The magnitude of responses of experimentally infected cattle to PPDa was greater than their

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responses to MAP_3651c and MAP_0268c antigens and this is similar to our findings in sheep

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[Hughes et al., 2013]. This is not surprising as the PPDs are a complex antigenic mix capable of stimulating T-cells with a variety of specificities whereas a single protein antigen will only be

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able to stimulate a subset of those responsive T-cells. Nevertheless, these recombinant antigens

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have advantages over the PPDs because in commercial production it would be possible to generate defined protein products and they are potentially more specific for detection of Map.

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Given the scale of this experiment, it would be premature to suggest that these recombinant

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antigens could replace the complex antigens currently in use. However, the antigens show some diagnostic promise but further work looking at different breeds and farm locations will be

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required to provide evidence that these maybe be useful tools in the diagnosis of Paratuberculosis.

The inclusion of these recombinant proteins in an IGRA may form the basis of an immunological test for Map infection. In order to see how this novel test performed in naturally acquired infections on a farm, threshold values determined from the control animals in the experimental Map infection, were applied to IGRA results from animals with naturally acquired JD infections.

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In the six cows with well-established Map infections as determined by serum-ELISA positive test results, only 3 showed consistent above threshold responses for PPDa, one animal (D6) had inconsistent responses. For MAP_3651c, only one animal (D4) showed consistent above threshold values and one animal (D2) had an intermittent response. For MAP_0268c only one

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animal (D3) had a consistently high response to antigen (greater than its response to PPDa in all

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tests). This was somewhat unexpected and may indicate that this animal had a non-specific

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response to this protein antigen. Interestingly the calf of this animal C3 also had an intermittent high response to MAP_0268c in addition to above-threshold responses to PPDa and

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MAP_3651c. Notably, this animal D3 was the only faecal culture negative cow, which could be

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due to intermittent shedding. With respect to PPDa-induced responses, some animals exhibited steady declines (D7 and D5), whilst two animals (D1&D3) had stable low responses. D1 and

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D7, had low responses to the stimulation positive control SEB (less than 6 ng/ml) and this

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suggests that they were only capable of raising a very poor cell-mediated response compared to the best responders -which were producing concentrations of between 25-41 ng/ml. This may

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have been due to biological demands of recent parturition. These animals may have passed the ] resulting in a reduced capacity to

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switch phase [

produce IFN- and a predominant humoral response. Animal D6 had an interesting profile,

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despite having low responses to SEB (less than 2ng/ml) it was still able to generate an above threshold response to PPDa on two occasions. D6 was the only culture positive animal where it was not possible to detect the Map-specific target (IS900) in the culture medium. This suggests that D6 may have been exposed to environmental mycobacteria related to Map. Although intermittently it had above threshold responses for PPDa, neither it nor its calf had responses to MAP_3651c or MAP_0268c on any of the three occasions it was sampled. It seems clear that

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none of the antigens (including PPDa) performed particularly well in inducing IFN- in this group of animals, not all serum ELISA positive animals had above-threshold responses but rather the antigens detected a subset of these naturally infected cattle. This is an indication that animals with more established infections would not be the target for this test as some animals producing a

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humoral response would not be detected. Animals in this category will be missed by tests based

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on cell-mediated responses such as IGRAs, although it is clear from this study that not all

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animals with well-defined humoral responses are deficient in cell-mediated responses and some

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are capable of simultaneously exhibiting Th1 and Th2 responses, as was previously reported [Begg et al., 2011].

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For the calves born to serum ELISA positive dams in which we might expect early exposure/infection, only one animal (C7) had above-threshold responses to both PPDa and

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MAP_3651c at the first bleed. However, more above threshold responses developed in the

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calves in the two subsequent bleeds and may indicate that the calves had increased exposure to

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antigen from their infected dams. By contrast, calves born to serum ELISA negative dams had fewer above threshold responses to either PPDa or the Map antigens. This is consistent with a

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study of Antagnoli et al. (2007) that found that cows which shed Map in their faeces were more

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likely to have a calf with a positive IFN- test, which reflects the expected greater exposure of these calves to Map. Calves from uninfected dams had no consistent above threshold responses, which may reflect their lower exposure to Map. However, one animal Cb had one above threshold response to MAP_3651c in the first bleed with further subsequent responses (at the intermediate stringency) to both PPDa and MAP_3651c. This suggests that although born to a dam that is probably uninfected, if faecal-shedding animals are present in the herd, exposure is

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very likely to occur. It was not possible to confirm the infection status by taking tissue samples for culture as the animals in this part of the study were owned by the farmer. To further investigate the performance of the test, IGRAs with recombinant antigens as stimuli were used on four farms with varying risk of infection. The ‘HRF’ had many instances of above

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threshold responses for PPDa, and both recombinant antigens. For MAP_3651c, ‘IRFs’ had

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incidences of above threshold scores but these were at a lower level than the ‘HRF’. It was a

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very compelling observation that the percentage of above threshold responses to MAP_3651c on the ‘HRF’ was 20.4%, whilst on the ‘LRF’ it was 0% (even when the intermediate stringency

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threshold was applied) and this was statistically significant (P<0.001).

The picture for

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MAP_0268c was not so clear and did not reflect the expected exposure to Map: above threshold responses were seen on the ‘LRF’ (17.2%); were highest on the IRFs (44% on IRF2); and did not

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always correlate with PPDa responses. The responses to MAP_0268c for animals maintained on

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farm were very high (with many of them exceeding the responses to PPDa) and this is highly suggestive that MAP_0268c is giving many false positive reactions in animals maintained on

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farm and would not be suitable as a test antigen in its present form. Because of the nature of the

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disease with its protracted incubation period and our inability to detect infection in the early stages, it is impossible to know whether these above threshold responses correlate with infection

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and subsequent detection of JD by classical diagnosis techniques of serum ELISA and faecal culture. A longitudinal study of IGRA test positive animals is required to determine if they shed Map or become serum ELISA positive within their commercial lifespan. Threshold values, applied in the naturally-acquired infections described in this study are much greater than those employed for detection of M. bovis using the Bovigam kit. They were derived from non-experimentally infected calves. Such young animals may have higher non-specific

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responses due to the presence of circulating NK cells [Olsen et al., 2005]. In addition, one of the animals in the control group may have had a previous exposure prior to recruitment to experiment. This particular animal consistently gave responses where PPDj-PPDa>0.1 and highlights the difficulty of sourcing JD free animals in countries where JD is endemic. Thus the

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threshold values employed in this study may be artificially high and may not be sufficiently

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sensitive for the detection of all Map-infected animals.

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There is considerable variability of antigen stimulated IFN responses from month to month in

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both experimentally- and naturally-infected animals. In the experimental infection, responses of infected animals to PPDa varied by up to 50 % during the elevated phase of response. Whilst

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technical issues associated with performing the IGRA may account for some of the variation, it is likely that this is a biological phenomenon and reflects the natural peaks and troughs of

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responses which occur as the infection progresses (or during repeated infection cycles) and may

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be indicative of an animal controlling infection. These results suggest that there are only

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windows of opportunity in which an infected animal may test positive using the IGRA. Whilst a positive IGRA test to Map antigens may be the earliest indication that an animal is infected or

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exposed, fluctuating responses pose a major challenge to the efficacy of IGRA testing as a reliable indicator of mycobacterial infection. If responses fluctuate to such an extent that

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repeated sampling of blood results in different conclusions regarding the infection status of an animal then confidence in the test results may be undermined. Thus interpretation of test results is not straightforward: a negative test is not conclusive proof that an animal is clear of infection; alternately, a positive test, although likely to indicate a present infection/recent exposure, is not necessarily an indication that the animal will shed Map in its faeces or progress to clinical JD. While the IGRA test may prove to be an additional tool for the diagnosis of JD, further work is

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required to understand the meaning of a positive test and this will direct the ways in which the

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tool can a be applied to help address the challenge of JD control.

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Acknowledgements This work was mostly funded by The Scottish Government, Rural and Environment Science and Analytical Services Division (RESAS) and the European Union (‘ParaTB tools’ contract number: FOOD-CT-2006-023106). The authors would particularly like to acknowledge the

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contribution of Douwe Bakker, both as co-ordinator of ParaTB tools and for the kind gift of

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PPDj which has proved invaluable in this and other studies. In addition, parts of this work were

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funded by Quality Meat Scotland, and Genecom. The authors gratefully acknowledge the following people who facilitated this study: Linda May; Susan Denham- staff of the

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Mycobacteria group, Moredun, for excellent technical assistance; Sarah Brocklehurst, BIOSS,

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for reading the manuscript and offering helpful comments and suggestions regarding statistical analysis; Staff of Bioservices, Moredun, for sample collection and animal husbandry; Dr Colin

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Penny, SRUC, for sourcing animals infected with Map; Farmers and their stockmen for animal

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management and graciously allowing us to sample animals on their farm.

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Highlights:

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  

MAP_0268c and MAP_3651c stimulate the cell-mediated response of Map infected cattle Experimental infections were detected using recombinant antigens derived from Map Animals with naturally acquired Map infections responded to recombinant antigen Tests of cattle with varying risk for Map infection were significantly different

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