WO2009015434A1 - Assessment of mycobacterium infection - Google Patents

Abstract

Individuals with a mycobacterium infection tend to have higher levels of expression of particular proteins than individuals who do not have a mycobacterium infection. The current invention is directed to methods, antibodies, polynucleotides and kits for assessing the likelihood of an individual having a mycobacterium infection and related pathologies based on assessment of the expression level of the proteins.

Description

Assessment of mycobacterium infection

Field of the invention

The invention relates to detecting mycobacterium infections and related pathologies, especially ovine Johne's disease.

Background of the invention

Johne's disease (JD) is a chronic, insidious wasting disease of livestock caused by infection with Mycobacterium avium subsp. paratuberculosis which is transmitted in faeces to young animals by infected adults. The disease is characterized by intestinal lesions of granulomatous enteritis, lymphadenitis and progressive emaciation of the animal. The disease has been reported in cattle in Australia since 1925 but only recently recognized in sheep. The first report of JD in sheep in Australia was in 1980 and the first outbreak reported in New South Wales in 1981. Since then, JD has been recognised as being widespread in both domestic and wild ruminants in Australia.

JD typically progresses through three distinct stages of disease, the division of each stage being based on the detection of host immune responses to paratuberculosis antigen, detection of faecal shedding of the organism and the existence of clinical signs. In the early subclinical stages, animals are infected although asymptomatic, and can remain so for many years without developing signs of clinical disease. Subclinical^ infected animals can transmit the infection via faecal shedding of the causative organism into the environment, although in the very early stages of infection it cannot be detected by culture. Shedding has also been found to be intermittent. As animals progress through the late sublinical phase and into clinical disease, they begin to shed high numbers of bacteria into the environment, which can be detected by culture and antibody response to Mycobacterium avium subsp. paratuberculosis antigen.

Current diagnostic tests for JD include indirect detection of the host immune response to infection or direct tests to detect the microorganism by culture. During the early stages of infection the host response is primarily cell mediated, with little or no humoral immune response. Therefore, serologic tests of host immune response such as enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) assay which reply on detection of antibodies produced during the humoral response are more useful at later stage of infection. In addition, the sensitivity of these tests vary with the stage of disease.

In the early stage of infection, no single diagnostic test can definitively diagnose infection although the gamma interferon test (Bovigam ™) has shown some promise for early detection in cattle and sheep but with moderate sensitivity and specificity.

The faecal culture test remains the definite diagnostic test for JD infection and is the only method that can transverse both sub-clinical and clinical stage of disease.

However, this is limited to detecting animals that are already shedding the organism into the environment and are in mid-late subclinial stage of disease. In addition, culturing of

Mycobacterium avium subsp. paratuberculosis requires a lengthy incubation time to demonstrate growth. Current methods for the culturing from faecal samples require 8 to 16 weeks on solid medium or up to 8 weeks in liquid medium using the Bactec detection system.

Detection of Mycobacterium avium subsp. paratuberculosis DNA by PCR amplification, particularly the application of real-time PCR, nested PCR and immuno-magnetic bead separation assays have made the detection of Mycobacterium avium subsp. paratuberculosis more rapid and semi quantitative. However, the majority of tests are based on amplification of the Mycobacterium avium subsp. paratuberculosis IS900 element which has been reported to have moderate specificity due to the high degree of similarity between Mycobacterium avium subsp. paratuberculosis and environmental mycobacteria that contain IS900-like insertion sequences.

There is a need for a new diagnostic test for detecting of Mycobacterium related organisms, especially those involved in the aetiology of JD, that has greater sensitivity and specificity than current diagnostic tests, and particularly one which has a better capacity to detect animals in the early stage of infection before they begin to shed large numbers of organisms into the environment. Summary of the invention

The invention seeks to at least minimise one or more of the above identified limitations and in one embodiment provides a method for assessing the likelihood of an individual having a mycobacterium infection including:

-selecting an individual;

- assessing the level of expression of a target protein described in Table 1 herein in the individual;

thereby assessing the likelihood of the individual having a mycobacterium infection.

Preferably the individual is an ovine or bovine individual.

In other embodiments there is provided a use of an antibody specific for a target protein described in Table 1 herein in a method for assessing the likelihood of an individual having a mycobacterium infection.

In another embodiment there is provided a use of a polynucleotide for detecting a nucleic acid that encodes or controls the expression of a target protein described in Table 1 herein in a method for assessing the likelihood of an individual having a mycobacterium infection.

In further embodiments there is provided a kit for assessing the likelihood of an individual having a mycobacterium infection including:

- an antibody specific for a target peptide described in Table 1 herein; or

- a polynucleotide capable of detecting a nucleic acid encoding or controlling the expression of a target peptide described in Table 1 herein.

As described further below, the kit may further include an infected and/or uninfected control and written instructions for use of the kit in a method as described herein. Brief description of the figures

In all Figures, "early pauci"= early paucibacillary; "inter pauci"= intermediate paucibacillary; "late pauci"= late paucibacillary; "multi"= multibacillary; "unexp"= unexposed; "unif = uninfected.

Figure 1 illustrates the results of the QPCR analysis of DD-PCR genes SAA3 like, Cathepsin K like, TOM22 like and SLAMF7 like. Numbers show mean fold change in expression relative to unexposed animal. Error bars on the column graphs show SEM of ddCT; error bars on the other graphs show SD of dCT.

Figures 2A-2C illustrate the results of the QPCR analysis of toll-like receptor (TLR) genes 1 to 8 in the ileum and jejunum. Numbers show mean fold change in expression relative to unexposed animal. Error bars on the column graphs show SEM of ddCT; error bars on the other graphs show SD of ΔCT.

Figure 3 illustrates the results of the QPCR analysis of TLR gene 1-8 expression in the ileal lymph nodes. Significant increases in expression of TLR1 , TLR2, TLR3, TLR6 and TLR8 were found in the ileal lymph nodes of infected animals relative to uninfected animals. Error bars show standard error. Numbers show mean fold change in expression relative to unexposed animals calculated using ΔCt. a-significantly different from the uninfected animals, b-significantly different from paucibacillary animals. For illustrative purposes, the y-axis is reversed to show the direction in fold change obtained by 2 ^"^ analysis.

Figure 4 illustrates the mean ΔCt of TLR1-8 expression in the jejunal lymph nodes of sheep infected with M.ptb. Significant increases in expression were found in the jejunal lymph node of infected animals relative to uninfected animals for TLR1 , TLR2, TLR3, TLR6 and TLR8. Error bars show standard error. Numbers show mean fold change in expression relative to unexposed animals calculated using ΔCt. a-significantly different from the uninfected animals, b-significantly different from paucibacillary animals. For illustrative purposes, the y-axis is reversed to show the direction in fold change obtained by 2 ^"MCt analysis. Figure 5 illustrates the mean ΔCt of TLR1-8 expression in PBMCs of sheep infected with M.ptb. None of the TLR genes analysed were significantly altered in the PBMCs of infected animals relative to uninfected animals. TLR1 , TLR6, TLR7 and TLR8 showed trends to increases in expression in uninfected animals relative to the unexposed group. Error bars show standard error. Numbers show mean fold change in expression relative to unexposed animals calculated using ΔCt. a-significantly different from all exposed animals. For illustrative purposes, the y-axis is reversed to show the direction in fold change obtained by 2 ^"MCt analysis.

Figure 6 illustrates the mean ΔCt of Fas, Bax and Bcl-2 expression in the terminal ileum of sheep exposed to M.ptb. ΔCt values are expressed as the mean ΔCt value +/-SE. Numbers show mean fold change in expression relative to unexposed animals. The y- axis shows ΔCt plotted in reverse to reflect the direction of the fold change by 2 ^"^⁰¹ analysis relative to unexposed animals.

Figure 7 illustrates the mean ΔCt of Fas, bax and Bcl-2 expression in the ileocaecal lymph node of sheep exposed to M.ptb. ΔCt values are expressed as the mean ΔCt value +/-SE. Numbers show mean fold change in expression relative to unexposed animals. The y-axis shows ΔCt plotted in reverse to reflect the direction of the fold change by 2 ^"^⁰¹ analysis relative to unexposed animals.

Figure 8A-C is the change in expression of satbi , Thymosin beta 4 and calmodulin dependent PK gene respectively in the ileum of M.ptb infected animals. Data are dCT predicted means with standard errors (left panel) and mean fold change relative to the uninfected group expressed as ddCT (right panel in 8A and 8B). In Figure 8C, the mean fold change is shown as a numeral above the bar. dCt values with different superscripts are significantly different.

Figure 9A-9C illustrates the results of QPCR validation of potentially differentially expressed genes in the lymph nodes. Comparisons were made between unexposed (n=8), unifected (n=6), early paucibacilliary (n=6), late paucibacilliary (n=4) and multibacillariary animals (n=6). Error bars show standard error. Numbers above bars show mean fold change in expression relative to unexposed animals. ^a significantly different from the uninfected animals. ^b significantly different from early paucibacillary animals. ^c significantly different from late paucibacillary animals. ^d significantly different between unexposed and exposed groups of animals.

Figure 10A-10B illustrates the results of an analysis to determine whether a selection of the genes illustrated in Figure 9A-9C are also differentially expressed in PBMCs in sheep infected with M.ptb. Error bars show standard error. Numbers above bars show mean fold change in expression relative to unexposed animals. ^a significantly different from the uninfected animals. ^b significantly different from early paucibacillary animals (3a). ^d significantly different between unexposed and exposed groups of animals.

Detailed description of the embodiments

The inventors have found that individuals having a mycobacterium infection tend to have a higher level of expression (for example, more molecules per cell and/or more cells expressing a molecule) of target proteins described in Table 1 than do those individuals who do not have a mycobacterium infection.

Table 1 : Target proteins

Importantly, the higher level expression or in other words, increased or up-regulated expression of these target proteins has been observed in individuals having an asymptomatic mycobacterium infection and in individuals having a sub clinical mycobacterium infection.

Thus in one embodiment there is provided a method for assessing the likelihood of an individual having a mycobacterium infection including:

-selecting an individual;

- assessing the level of expression of a target protein described in Table 1 in the individual;

thereby assessing the likelihood of the individual having a mycobacterium infection.

Typically the level of expression of the target protein in the individual is assessed by the steps of:

- measuring the level of expression of the target protein in a tissue of the individual; and - comparing the measured level with an uninfected control that describes the level of expression of the target as observed in the same tissue of a subject that has been determined as not having a mycobacterial infection;

thereby assessing the likelihood of the individual having a mycobacterium infection.

An "uninfected control" may be derived by measuring the level of expression of the target in a tissue of a subject that, but for an absence of a mycobacterium infection, is generally the same or very similar to the tissue of the individual selected for assessment of likelihood of mycobacterium infection (the latter otherwise known as the "test sample").

In certain embodiments, the uninfected control is derived by measuring the level of expression of the target in a cell line or an engineered tissue.

In certain embodiments, the method includes measuring the level of expression of the target in the uninfected control to compare the measured level in the test sample with the level in the uninfected control.

In other embodiments, an internal standard is applied. This may be used to ensure that the method operates within accepted decision limit quality control criteria. The assay then provides a value/number/result or other output for each test sample. That output is then deemed to represent infection or non infection based on independent data derived from frequency distributions of results from an uninfected control. This control may describe distributions of results obtained from more than one uninfected subject, for example, from an uninfected population which may be of the same species, geographic origin, age, sex as the test sample. A positive -negative cut -point for the method is determined from these distributions to provide defined levels of diagnostic sensitivity and specificity for the method.

The internal standard or reference may obviate the need to physically provide an uninfected control in the form of uninfected cells or otherwise to physically measure the level of the target in an uninfected control. Where the internal standard or reference is used, the level of expression in an uninfected control has been predetermined and may be provided for example in the form of written information that is supplied with a diagnostic kit.

The measurement of the level of expression of the target in the tissue of the subject for deriving the uninfected control is generally done using the same assay format as that that is used for measurement of the target in the test sample. However, it is not necessary to use the same assay when an internal standard that can be used to compare data obtained from different assay formats is or has been applied.

Generally the subject from which the uninfected control is derived and the individual selected for assessment of likelihood of infection are of the same family. For example, where the individual to be assessed for likelihood of mycobacterium infection is an ovine, the negative control is general derived from the measurement of the level of expression of the target in an ovine. In one embodiment, the individual selected for assessment of likelihood of infection and the subject from which the negative control is to be derived are from the same species. It is not necessary that they be of similar age or sex or have been exposed to similar environmental influence.

In another embodiment, the level of expression of the target protein in the individual is assessed by the steps of:

- measuring the level of expression of the target protein in a tissue of the individual; and

- comparing the measured level with an infected control that describes the level of expression of the target as observed in the same tissue of a subject that has been determined as having a mycobacterial infection;

thereby assessing the likelihood of the individual having a mycobacterium infection.

An "infected control" is generally derived by measuring the level of expression of the target in a tissue of a subject that is generally the same or very similar to the tissue of the test sample. The measurement of the level of expression of the target in the tissue of the subject for deriving the infected control is generally done using the same assay format that is used for measurement of the target protein in the test sample. In certain embodiments, the method includes measuring the level of expression of the target in the infected control to compare the measured level in the test sample with the level in the infected control. However, again an internal standard may be applied that obviates the need to provide an uninfected control or otherwise to measure the level of the target in an uninfected control.

The measurement of the level of expression of the target in the tissue of the subject for deriving the infected control is generally done using the same assay format as that that is used for measurement of the target in the test sample. However, again it is not necessary to use the same assay when an internal standard that can be used to compare data obtained from different assay formats is or has been applied.

Generally the subject from which the infected control is derived and the individual selected for assessment of likelihood of infection are of the same family. For example, where the individual to be assessed for likelihood of mycobacterium infection is an ovine, the infected control is generally derived from the measurement of the level of expression of the target in an ovine. In one embodiment, the individual selected for assessment of likelihood of infection and the subject from which the positive control is to be derived are from the same species. It is not necessary that they be of similar age or sex or have been exposed to similar environmental influence. Further it is not necessary that the infected control be derived from a subject that has otherwise been confirmed as having an asymptomatic or sub clinical infection. For example the infected control may be derived from a subject having later stage disease.

In one embodiment the method includes

- measuring the level of expression of the target protein in a tissue of the individual; and

- comparing the measured level with an uninfected control and an infected control.

Typically the target protein is directly detected to assess the likelihood of the individual having a mycobacterium infection. This is otherwise known as a "direct detection" of the target to measure the level of expression of the target. In these embodiments, the target protein described in Table 1 , or a peptide or fragment derived from it may be detected. In certain embodiments, the level of expression of a molecule, the expression of which is modulated in accordance with the up-regulation of the target is measured. This is otherwise known as an "indirect detection" of the target to measure the level of expression of the target.

In one embodiment, a nucleic acid contained in the individual selected for assessment for likelihood of mycobacterium infection that encodes a target protein, or that is complementary to a nucleic acid that encodes a target protein, is measured. In this embodiment, the nucleic acid may be one which can be used to determine the presence of a given protein, or level of expression of a given protein in a host as per a molecular genetic approach. One example is where a polynucleotide that is complementary to a nucleic acid (DNA, RNA, cDNA) that encodes a target protein is hybridised to the nucleic acid and hybridisation is detected. One example is quantitative PCR. Others include quantitative Northern and Southern blotting, and microarray. An example is shown in Examples 1 and 2. As is well known in the art, hybridisation of nucleic acid molecules may be controlled by the type of buffer used for hybridisation and the temperature of the buffer. "High stringency conditions" are conditions in which the buffer includes about 0.1 x SSC, 0.1% SDS and the temperature is about 60⁰C.

In another embodiment, the method includes the step of detecting a target protein, or peptide or fragment thereof in the individual to assess the level of expression of the target in the individual. The presence of a given protein, or level of expression of a given protein in a host can be detected by any number of assays. Examples include immunoassays, chromatography and mass spectrometry. The immunoassay may be one wherein purified antibody specific for the target protein is used to detect the presence or level of expression of the target protein in the host. For example, purified antibody is bound to solid phase, host tissue or samples derived from host tissue are then applied followed by another antibody specific for the target protein to be detected. There are many examples of this approach including ELISA, RIA. One example of an immunoassay that is particular preferred is FACS.

In preferred embodiments the level of expression of a target protein in a peripheral blood mononuclear cell (PBMC) of the individual is measured to assess the level of expression of the target protein in the individual, thereby assessing the likelihood of the individual having a mycobacterium infection. Other tissues include those likely to be exposed to mycobacterium including epithelial tissue and neighbouring interstitial tissue. Ileal and jejenal tissue and the appropriate draining mesenteric lymph nodes (ileal lymph nodes and jejunal lymph nodes) are examples, as is lung tissue.

It will be understood that the level of expression of the target protein may be measured by obtaining a sample of tissue from an individual selected for assessment and determining the level of expression of the target in the sample. Alternatively, the level of expression could be determined in vivo, for example by providing labelled antibodies to the individual which can be visualised in vivo.

In one embodiment the mycobacteria is Mycobacterium avium subsp. paratuberculosis (M.ptb). Examples include the M.ptb sheep (S) strains and the M.ptb cattle (C) strains. The former are distinguished from the latter by a point mutation in IS1311 and a major genomic deletion including the deletion of mmpL8.

It will be understood that in certain embodiments, the bacteria may be another species or sub species of Mycobacterium. Examples include M.tuberculosis, M.avium, M.bovis, M. avium-intracellulare-scofulaccum complex, M.ulcerans, M. leprae. M.kansasii, M.gordonae, M.celatum. M.abscessus, M.africarum, M.asiaticum, M.avum, M.chelorae, M.flavescers, M.fortiutum, M. gastri, M.haemophilum. M.intracellulare, M. interjectum, M. intermedium, M.karsasii, M.malmoense, M.marirum, M.non- chromogenicum, M.phlei, M.shimodei, M.imiae, M.smegmatis, M.szulgai, M. terrae, M.trivale, M.ulcerars and M.xerzopi.

Typically, the host is an ovine, such as a sheep, or a bovine, such as domestic cattle. Particular sheep breeds include: Merino, Rambouillet, Romney, Lincoln, Drysdale, Herdwick, Suffolk, Hampshire, Dorset, Columbia, Texel, Montadale, Coopworth, Corriedale, St. Croix, Barbados Blackbelly, Mouflon, Santa Inez and Royal White and genetic crosses between these breeds. It will be understood that in certain embodiments, including where the mycobacteria is other than M.ptb, the host may be another mammal, such as a bovine, especially cattle and other or a human, goat, deer, antelope, ruminants and carnivores such as foxes, ferrets, and some non-ruminant herbivores such as rabbits.

In one particular embodiment, the mycobacterium is M. tuberculosis and the host is a human being, such as an individual having an acute or chronic, asymptomatic or sub clinical infection of M.tuberculosis.

As generally understood, a host having an asymptomatic infection is one which does not show obvious signs or symptoms of disease caused by mycobacterium infection. For example, an asymptomatic host having Johne's disease generally does not display any symptoms of the disease that are apparent in animals exhibiting clinical manifestations.

As generally understood, a host having an early sub clinical infection is one without clinical manifestations of the infection or pathology accompanying it. For example, an early sub clinical host having Johne's disease generally does not display intestinal lesions of granulomatous enteritis, lymphadenitis or progressive emaciation. However, the host may display a very mild symptom of a infection, such as an elevated interferon level in response to challenge with M.ptb or extracts thereof. Late sub clinical infection may be accompanied by pathology such as intestinal lesions of granulomatous enteritis, lymphadenitis, prior to clinical signs of progressive emaciation becoming apparent.

In certain embodiments, the method may be useful for assessing a response in of an individual to administration of a protein or substance representing part or all of a mycobacterium. In these embodiments, the protein or substance is administered to an individual and the level of expression of a target protein described in Table 1 herein is assessed to determine a response to the mycobacterium protein or substance. The substance may be a mycobacterium stress antigen or other antigen known in the art, such as those in the form of purified protein derivatives including Johnin, avium PPD or tuberculin. Typically the response is assessed 48 to 72 hours after administration of the mycobacterium protein or substance to the host. In another aspect of the invention there is provided a kit for assessing the likelihood of an individual having a mycobacterium infection. In one embodiment the kit may include an antibody specific for the target peptide, such as those described in Table 1 herein, or a polynucleotide capable of detecting a nucleic acid encoding or controlling the expression of a target peptide, such as those described in Table 1 herein.

In certain embodiments, the kit may also include infected and/or uninfected controls which may be used with each use of the kit with a test sample. In alternative embodiments, the kit may include an internal standard or reference. Where the kit contains an internal standard or reference, the level of expression in an uninfected control has been predetermined.

The kit may further include written information in the form of instructions for use of the kit in a method in accordance with the invention for assessing the likelihood of an individual having a mycobacterium infection.

Examples

Example 1 : Determination of Reference Genes

Long distance differential display PCR (DD-PCR) is one technique available for comparison of gene expression between 2 or more mRNA populations. As well as using DD-PCR to identify differentially expressed genes, it can be used to identify a suitable, stably expressed gene to be used as a reference in comparative qPCR to validate the expression of the isolated DD-PCR genes.

A. Materials and Methods.

Animals. '

24 Merino sheep aged 2-3 years were obtained from 3 properties in New South Wales and housed in a pen at Camden for two weeks prior to collection of samples. They were fed a mixture of lucern chaff and lupins, and had ad libitum access to water. The disease status of the animals was determined by Perez histological classification (as either uninfected, early paucibacilliary, late paucibacillary or multibacillary) culture, AGID and other standard methods. Table 2 summarises the disease status of each test animal.

Table 2: Disease status of test animals

Sample Collection Tissue samples

Intestinal and corresponding draining lymph node samples were taken from the terminal ileum (adjacent to ileo-caecal junction) and terminal jejunum (3m proximal to the ileo- caecal junction) at time of necropsy. Sections for RNA preparation were snap frozen on 5 dry ice and stored at -8O⁰C until required. Sections for histological analysis were fixed in formalin, embedded in paraffin, sectioned and stained with Ziehl-Neelson stain. Tissue and faecal samples for M.ptb culture were taken at the time of necropsy and processed.

Peripheral Blood Mononuclear Cells (PBMCs)

PBMCs were extracted from whole blood taken 1 week prior to necropsy. 24 mL of

0 blood was taken into EDTA vacutainers (Becton Dickinson) and buffy coats were isolated by centrifugation (750 xg, 10 min, 19⁰C). Buffy coats were aspirated with

Pasteur pipettes, mixed with 2ml PBS and layered carefully onto 3 mL of Ficoll-Paque

PLUS (Amersham Biosciences) before centrifugation to harvest the PBMCs (750 xg, 30 min, 19⁰C). The PBMCs were carefully aspirated with a Pasteur pipette and washed by

5 the addition of 10 mL of PBS. PBMCs were harvested by centrifugation (230 xg, 10 min,

19⁰C), the supernatant removed and taken immediately for RNA extraction with the

RNeasy mini kit (Qiagen) as described below.

Extraction of RNA from intestinal tissues

Total RNA was extracted from 30mg frozen intestinal tissue or PBMC samples using the !0 Mini RNeasy extraction kit (Qiagen) according to the manufacturer's instructions. RNA quality assessment and quantification were done by visual examination on a 1% non- denaturing agarose gel in Tris-borate-EDTA buffer stained with ethidium bromide and spectrophotometry at 260 nm using an Eppendorf BioPhotometer.

Removal of DNA from total RNA preparations

:5 Contaminating genomic DNA was removed by digestion with 10 μl of RQ1 DNAse (1 U/μl, Promega) at 37⁰C for 2 hours. Following digestion, RNA was precipitated by the addition of 0.5 μl linear acrylamide (1μg/μl, Ambion), 0.1 volume of 3M sodium acetate pH5.3 (Sigma), 3 volumes of ethanol (Sigma) and incubation on dry ice for 60 minutes. RNA was harvested by centrifugation (16,000 xg, 45 min, 4⁰C), resuspended in 25 μl of RNase free water (Ambion). Free nucleotides and sodium acetate were removed using a G50 sepharose buffer exchange column (Amersham). RNA quality was checked by agarose gel electrophoresis. Removal of DNA was confirmed by qPCR using β-2 microglobulin primers (Sigma-Genosys) as described below.

cDNA synthesis

25 μg of total RNA and 4 μg of oligo dT (Sigma-Genosys) was made up to a total volume of 50 μl with RNase free water (Ambion). Samples were heated at 7O⁰C for 5 min and then placed at room temperature for 5 min to anneal the oligo dT. 40 μl of 5x reverse transcriptase buffer (Promega), 8 μl of 25 mM dNTPs (Promega), 1 μl RNaseln RNAse inhibitor (MBI Fermentas) and 98 μl of H₂O were added and mixed before adding 1 μl of M-MLV reverse transcriptase (RNAse H-point mutant; Promega). The reaction was mixed and placed at 42⁰C for 1 hour. A further 1 μl of reverse transcriptase was added, and the reaction was incubated for another hour at 42⁰C. After synthesis, the cDNA was precipitated with addition of 0.5 μl of linear acrylamide ( 1μg/μl) (Ambion), 0.1 volumes of 3M Sodium Acetate pH 5.3 (Sigma) and 3 volumes of ethanol (Sigma) and incubation on dry ice for 60 minutes. The cDNA was harvested by centrifugation (16,000 xg, 45 min, 4⁰C), resuspended in 25 μl of RNase free water (Ambion) and free nucleotides and sodium acetate removed using a G50 sepharose buffer exchange column (Amersham).

Long distance DD-PCR

DD-PCR was performed in 50 μl reactions according to the Delta Differential Display kit (Clontech) using KlenTaq LA Taq polymerase (0.4 μl, Clontech) and 1 μl cDNA labelled with σ35S-dATP (0.12MBq/reaction, Amersham) and cycled in a PXE-2 thermal cycler (Thermo Hybaid). Cycling conditions were as follows: 1 cycle of 94⁰C 5 min, 4O⁰C 5 min, 68⁰C 5 min; 2 cycles of 94⁰C 30 sec, 4O⁰C 30 sec, 68⁰C 5 mins; 23 cycles of 94⁰C 20sec 6O⁰C 30 sec, 68⁰C 2 min; 1 cycle of 68⁰C 7 min. Products (5 μl) were resolved by electrophoresis on a 5% polyacrylamide gel (Biorad), dried under vacuum (SG210D Integrated SpeedGel Slab Gel dryer, Thermo Electron) and visualised following exposure to BMR X-ray film (Kodak).

Cloning of DD-PCR products

Products that appeared to be expressed at equivalent levels across the infected and uninfected animals were excised from the dried acrylamide gel and DNA extracted by boiling in 40 μl of TE for 5 minutes. Products were re-amplified from 7 μl of extracted DNA using the appropriate primer combination, Expand DNA polymerase (Roche) and cycled as described above. Products were resolved by electrophoresis on a 1 % non- denaturing agarose gel in Tris-borate-EDTA buffer stained with ethidium bromide, purified using the PCR extraction kit (Qiagen) and cloned into pCR2.1 using the TOPO TA Cloning Kit (Invitrogen). Restriction enzyme analysis was performed on five to ten clones using Mse\ (MBI Fermentas). Sequencing was performed on clones generating unique digestion profiles.

Sequencing

Sequencing was performed on purified plasmids at the Australian Genome Research Facility (AGRF), Brisbane using oligonucleotides M13Uni (-21) and M13R (-29).

Identification of putative reference genes

Sequences were compared to the non-redundant nucleotide database (GenBank) using blastn and/or tblastx searches of the BLAST program.

Quantitative PCR

Gene-specific oligonucleotide PCR primers were designed using Beacon Designer 4 (Premier Biosoft International) and obtained from a commercial supplier (Sigma- Genosys). Quantitative PCR was performed in an MX3000p Multiplex Quantitative PCR system (Stratagene) using QuantiTect SYBR Green PCR kits (Qiagen). QPCR reactions were carried out in duplicate 25 μl reaction volumes containing 300 nM forward and reverse primers and 10 ng template using the following program: 95°C for Tl

15 min, and 40 cycles of 95°C for 20 sec, 56°C for 30 sec, and 72°C for 30 sec with fluorescence acquisition at the end of the 56°C primer annealing step. Sequence- specific standard curves were generated by using serial dilutions of cDNA and the threshold values were noted. The built in amplification-based proprietary algorithm (Stratagene) was used to set the fluorescence threshold value for each primer pair reaction based on the efficiency as determined by standard curve generation. The value of the fluorescence threshold for each primer pair was used to analyze data from experimental reactions. The number of cycles (Ct) at which the amplification-corrected normalized fluorescence (dRn) for each reaction crossed the threshold value was exported to Excel (Microsoft), PRISM (GraphPad) and GENSTAT (http://www.vsni.co.uk/products/genstat/) for further analysis. The specificity of the reaction was checked post-amplification by melt curve analysis. The DeltaDeltaC(t) (^2" ^⁰¹) method was used to determine relative gene expression changes. The estimated error (SD 2-ddCt) is given as an asymmetric range of values, reflecting conversion of an exponential variable to a linear comparison.

Determination of reference genes by standard deviation analysis

Standard deviation (SD) of Ct was determined from triplicate reactions using 10 ng of cDNA generated from 5 infected, 3 unexposed and 2 exposed, culture negative animals. Genes producing a SD of greater than 1.0 were considered to be inappropriate for use as reference genes as this reflects a 2-fold change in expression level. Ct's were observed directly to ensure that ordering of infected and non-infected samples was not occurring (i.e. uninfected samples having closely matched Ct's, and infected samples having closely matched Ct's distinct from the uninfected Ct's). No ordering was observed during the course of these experiments. The reference gene with the lowest SD of Ct was used as the reference gene for that sample type.

B. Results.

Confirmation of M.ptb infection in animals. To determine disease status of animals, blood, tissue and fecal samples were collected from animals and these were subjected to analysis by standard methods (AGID, histopathology and culture) as described in materials and methods.

Long distance DD-PCR was used to identify 10 potential ovine reference genes by selecting bands that remained unchanged in density when comparing control and M.ptb infected animals.

Following isolation, bands were cloned and sequenced as described. Sequencing results were submitted for BLASTN and/or TBLASTX searches of the non-redundant nucleotide database (GenBank) and identified. Most of the sequences isolated were found to have homology with known ovine genes.

Gene expression analysis was performed using optimized QPCR primer pairs on the same tissues from which genes were isolated.

The genes which were considered to be the most appropriate reference genes were the ones which showed the least variation (standard deviation). HK 5.1 (SD 0.43) H7-7 (SD 0.36) and GAPDH (SD 0.6) were the least variable for measurements in intestinal tissues, lymph nodes and PBMCs respectively. HK 5.1 was identified as having homology with the bovine splicing factor (arginine/serine-rich 6). H7-7 was identified as having homology with the ovine coatomer protein complex subunit gamma 2.

C. Conclusion.

These studies have shown that suitable reference genes have been identified through DD-PCR studies. HK5.1 (similar to bovine splicing factor (arginine/serine-rich 6)), H7-7 (ovine coatomer protein complex subunit gamma 2) and GAPDH showed the least variation between control and infected animals and appear to be suitable reference genes over and above known ovine reference genes for gene expression analysis in the M.ptb model of infection. Example 2: Differentially Expressed Genes

A. Results

Long Distance DD-PCR was used to identify genes involved in M.ptb infection models in the ileum of sheep mentioned in Materials and Methods above.

Genes discovered by DD-PCR in tissues from animals in trial OJD.028 and 031 A

DD-PCR gels highlighted 18 potentially differentially regulated genes when comparing control and infected animals from group OJD 028 and 031 A (data not shown). In this study only genes isolated from the ileum were examined.

Following isolation, bands were cloned and sequenced as described. Sequencing results were compared to the GenBank database as described. The identity of the genes isolated is listed in Table 3.

Gene expression analysis was performed using QPCR optimized primer pairs. Comparisons made include control (n=9) versus all infected animals (n=16), control (n=9) versus paucibacillary animals (n=10) and control (n=9) versus multibacillary animals (n=6).

QPCR analysis results reveal that four of the eighteen genes analysed showed significant changes in gene expression in at least one comparison. The greatest changes in gene expression were seen when control animals were compared with multibacillary animals. Band 7.1 (Cathepsin K), Band 3.3 (Serum amyloid A3) and Band 9.4 (19A protein/SLAMF7) showed changes of greater than 100 fold in multibacillary animals. Additionally there appears to be a substantial difference in gene expression between paucibacillary and multibacillary animals for the same genes (see Table 4 and Figure 1 ).

The sequences of the house keeping genes HK5.1 and HK7.7 and the differentially expressed genes discovered through DD-PCR in the ovine host are given below. Table 3: Identification of DD-PCR genes isolated from OJD 028 and 031 A animals using blastn searches of the non-redundant nucleotide database (GenBank)

SEQ ID Clone Tissue Identity (blastn) Species Identities(blastn) GenBank NO accession #

4 Band3.3 Ileum serum amyloid A3 Caprine 140/158 (88%) EF564270

5 Band7.1 Ileum cathepsin K Bovine 291/302 (96%) BC109853

6 Band9.1 Ileum 19A protein (SLAMF7 Bovine 411/466 (88%) XM 001256 member) 711

3 BandPB4.3 Ileum similar to Bovine 322/331 (97%) XM_587978 mitochondrial import receptor Tom22, transcript variant 1

Table 4: QPCR analysis of DD-PCR genes

Control *-ddCh vs Fold change (2 F (ANOVA) P value (ANOVA) Mean dCt significant difference

TOM 22 Infected 4.05 14.76 <0.0001 yes

Pauci 4.18 < 0.01

Multi 3.84 < 0.01

SAA3 Infected 9.86 18.09 <0.0001 yes

Pauci 6.13 < 0.01

Multi 21.81 < 0.01

Cathepsin K Infected 5.73 80.05 <0.0001 yes

Pauci 1.63 > 0.05

Multi 46.35 < 0.01

19A/SLAMF7 Infected 6.90 20.47 O.0001 yes

Pauci 4.63 < 0.01

Multi 13.44 < 0.01 HK5.1 (similar to bovine splicing factor, arginine/serine-rich 6)

CTCAGTGAACATTTTATTGAGGCAATAAAGCACAAAATAGGTTAACAAAGTGGGAGAAAGATACCAAG AGAAAAGATGGTAACAACACAGGAGTGGAACATTAAGTCACAGGGCAGGAAAGACTTGAACTGGCC ATAAAGGCACTATGGCATGTTAACACACTATCAGGTAAAAGAAAGCGGGATCCAGATTACCACACTT CTATTCTGCTCT (SEQ ID NO: 1)

H7-7 (similar to human coatomer protein complex, subunit gamma 2 (COPG2))

TGATTTATATTTTTTTTATTAGCAACTTCAAGTATAACAACTGGAATAAGTGTTTATTTTCTATTAATAAA AATGAATTTTGACAAAAGTGGACTCTGGCTCCCCCTCCCCCACCACCCCTCTGGGATAAAAGCTTTC CAACATTGTCAGGAGCTTTCAGATACACATTAAAGAATTCAGTGAAGTTAAGCAGCTGGGGTATAGG ATAGTATTTGATTTTCAAGCTCACCAAAAGCTGTACTATCATACCAAAGCTGAACAAGTGTATTAAAAA GGAACAACAACAGAAAGACATGCACAAGGACAGACGTTTGCTTGATCTGCTGGCTCAGGGCCAAAT ATTTAATTTGCTTTTTTGAAGTCATTAATTTTTTAAAGTATGATTCTGGGAAATTCATGCCAATAGCCTG AAAGCCCACCAACAGTGAAGGGTACATCAATTTCAACTTGCCCAGTAAAACCTTATCCAACAGAAGC CAAGATAACATCTACAGGTGTTCCCTCTTTGCTTCTGACAGTCACCTGCATGGTCACCCCATCTGCTA AGGCCAGCCTGGACCTCACCAATAAATCATAGCCACCTCTGTATACA (SEQ ID NO: 2)

Band PB4.3/PB5.4 (TOM22)

TATTTAAATACAAAATGTGAGCACTGACTTTTTTTCCTTTTTAGACACATAT ACACACAAGTCT AACAG CCAAAATATTGGAGGAAATGAGCTCCTGCTCTGTCCTCCAGTCCCCATCTTGTGGCTGAAGCTGCCA GCCCTCCCTCCATCCTTTCCAACATACACAGAAATTCACAGTGGCTGCTTTGCAGCAGAGCTGTAAT TAATGCATTTTGTATTTTATCTTGTGTGCACATCCCTCCCCAACATCTCCATTCCATTAACAGTCAGGA GCACATCCTTCTCCACGGCAATCAGTTCCGACCCCGGCAACGGAATAGTGAGATCTTC (SEQ ID NO: 3)

Band3.3 (SAA3)

ATCTCATAAGCATTTATTAGATCTGCTGCCTTCTGAGGACACAGAACTCTCTAGGCCGATGTCCCTGC CCCTGGGGACTCACCGCCCCGTCTCCTGTACAGAGAGAGAGGCATCTCATTACTTGTCAAGTAAGC CAGCTAGTCTAAAGTGGTTGGGGTGCAAGATACACATGCATGCTTACTTGGGAGAGGGCTCTTCTTA ATGACTCCTGCGTAGACATCCTTCAAAGTTGAGGCGTGGCAAGGGCGTCCTTCACTTATATAGCAAT TTGCTGTATAATAAAGACATATTGGGACCCTT (SEQ ID NO: 4) Band7.1 (CATHEPSIN K)

TAAACAGCTATTTATTAAATTATAGACAAGCGATAAACACAGTTTACTTTAAAAATGCAAAGAAGGAAA AAGAAAACACATTTGTTGTATCATGTAAACTTCCACCAAAGATTTGTGAATAGATTTATTAATGGGGTA GAATTTCTACCTGGAAGATAGAGAGGTAAACATGGAGACTGAGAAGTCAGGTCACCTTAGTCCTCTC AAGTGGTTAAAGCTAACTAGTCCCTGTGAATGAGAATTTAGCCTCTCTGAGGGTCTCTAGCCTCTATA CAGCATTGGTCTCAGAACAACA (SEQ ID NO: 5)

B9.4 (19A/SLAMF7)

GATCTTAGTACCATAACCAAGGATTGAACCTGGGCCCTCAGCAGTGAGAGCACAGAGTCCTAACCAC TGGACCACATGGGAATTCCCAGATTAGGAACTCTTAACTCTGAAGGAGCATAAGGTATAGTTTATTCT GGAAGTCAGAGTTCTTCATTCTTTCTTGATGTTTTTGGTCCAGTCAGTGAATTCTTCTGGTCATTTTCT TTGGAGCACATGTCTGTTCAGATGACATTGTTATAGGTACATGATCTTGGTGTATCTGATGACATGGG CAGGGAGTGGGGTTCTGTTACCTTTGGGGGTATGTGCAAAGTGGAATACATGGTATTTGCTGGGTTT TCCTCTGGAATAACGCTATTAAAATTAGAGATTGTATCATACTCGGGCATCTCTTCAGAAATGGGAGG GAAGTGAACAGTTTCCTGATGAGTGTCCAGTTCCTTCTTCTCTTCGGGATCTCTGACACTTTTTTCCT TTCTCTTCCTAGGTGGAAAAAAACTAACACGAGTATTTGAGAGGGTTATAAGGCCGAATAGCTTAATA GTGC (SEQ ID NO: 6)

B. Discussion

A number genes were identified as being differently regulated from this study. Band 3.3 was found to be similar to the bovine serum amyloid 3(SAA3) protein. It has been found that expression of SAA3 is associated with presence of inflammatory cells and tissue parasitism in certain infection models (Chagas disease). Additionally SAA3 has been shown to be associated with the intestinal mucin gene MUC3 whereby it provides a means of increasing MUC3 in intestinal cells for the prevention of Gl diseases including infectious diarrhoea and necrotizing enterocolitis. SAA3 is also expressed and measurable in the serum of humans with tuberculosis.

Band 7.1 was identified as having homology with the bovine Cathepsin K preprotein. At least some of the cathepsin family is involved in specific cellular processes. Gene knock-out experiments have shown that cathepsins play an essential role in processes such as wound healing, antigen processing and presentation (immune disorders), prohormone processing and bone remodelling. Cathepsin K has been has been identified as a target for some therapies and compounds targeting cathepsin K (amongst other cathepsins) are being clinically evaluated. Cathepsin K in particular is found to be important for normal bone resorption.

PB 4.3 was found to be homologous with the bovine form of a protein similar to the translocase of the mitochondrial outer membrane 22 (TOM22). Specifically, TOM22 is a preprotein receptor/organizer of the mitochondrial outer membrane translocase complex that, with other TOM's, control the activity of the complex during the transfer of precursor substrates.

Band 9.4 was identified as having homology to the predicted bovine 19A protein. 19A is a member of the SLAM family member 7 group of proteins. Members of the signalling lymphocyte activation molecule (SLAM) family of receptors are expressed in multiple cells of the immune system, including natural killer cells, T cells, and B cells. SLAM receptors interact with SH2 domain-containing, cytosolic adaptor proteins such as SAP, EAT-2, and SH2D1A, and mediate tyrosine phosphorylation events that contribute to immune cell activation. The finding that mutations in SAP are associated with an X- linked lymphoproliferative disorder suggests that signalling through SLAM receptors can influence the development and effector functions of immune cells. The receptors are single-pass type I membrane proteins, and 3 isoforms have been proposed to occur from alternative splicing, lsoform 1 mediates NK cell activation through a SAP- independent extracellular signal-regulated ERK-mediated pathway and may play a role in lymphocyte adhesion, lsoform 3 does not mediate any activation. The isoform 3 is expressed at much lower level than isoform 1. SAP can bind the cytoplasmic tail of isoform 1 when phosphorylated in the presence of Fyn (in vitro). SLAMF7 proteins are expressed in spleen, lymph node, peripheral blood leukocytes, bone marrow, small intestine, stomach, appendix, lung and trachea. Expression has been detected in NK cells, activated B-cells, NK-cell line but not in promyelocytic, B-, or T-cell lines. SLAMF7 proteins contain an Ig-like C2-type (immunoglobulin-like) domain and may be involved in natural killer cell activation and natural killer cell mediated cytotoxicity.

It is feasible that these particular genes and their expression products may have important roles during the clinical stage of ovine Johne's disease, particularly when concerned with the immune response, apoptosis, cell signalling and absorption, and roles in other mycobacterial diseases.

Example 3: The expression level of TLR αenes is altered in sheep infected with M.ptb

A. Results

QPCR analysis results reveal, with the exception of TLR1 , that Toll-like receptor (TLR) genes showed significant changes in gene expression in at least one comparison, in at least one tissue type (Table 5). The greatest changes in gene expression were seen when unexposed animals were compared with animals with multibacillary lesions.

Table 5: QPCR analysis of TLR genes in the intestinal tissues of uninfected, early paucibacilliary and multibacillary animals

Ileum Jejunum

Are means Are mea

Unexp Fold F value P value significantly Fold F value P value significan

Gene VS change (ANOVA) (ANOVA) different? change (ANOVA) (ANOVA) different?

TLR1 Infected 1.98 1.759 0.1796 No 2.47 1.923 0.1507 No

Exp 0.43 P > 0.05 0.91 P > 0.05

EP 1.63 P > 0.05 2.31 P > 0.05

Multi 2.41 P > 0.05 2.63 P > 0.05

TLR2 Infected 5.75 4.618 0.0102 Yes 10.54 5.316 0.0054 Yes

Exp 0.37 P > 0.05 1.74 P > 0.05

EP 3.13 P > 0.05 5.96 P > 0.05

Multi 10.58 P < 0.05 18.67 P < 0.01

TLR3 Infected 1.90 4.01 0.018 Yes 2.25 5.517 0.0046 Yes

Exp 1.00 P > 0.05 1.89 P < 0.05

EP 1.81 P > 0.05 2.14 P < 0.05

Multi 2.00 P < 0.05 2.36 P < 0.01

TLR4 Infected 1.91 4.571 0.0106 Yes 3.52 8.156 0.0005 Yes

Exp 0.21 P > 0.05 1.17 P > 0.05

EP 1.18 P > 0.05 2.31 P > 0.05

Multi 3.08 P > 0.05 5.36 P < 0.01

TLR5 Infected 2.29 4.316 0.0135 Yes 5.03 3.712 0.024 Yes

Exp 0.36 P > 0.05 2.50 P > 0.05

EP 2.26 P > 0.05 7.03 P < 0.05

Multi 2.33 P > 0.05 3.60 P > 0.05

TLR6 Infected 2.13 0.0683 No 3.80 7.547 0.0009 Yes

Exp 0.96 2.672 P > 0.05 2.29 P > 0.05

EP 1.81 P > 0.05 3.70 P < 0.01

Multi 2.50 P > 0.05 3.91 P < 0.01

TLR7 Infected 3.83 1.746 0.1823 No 15.33 3.886 0.0203 Yes

Exp 2.30 P > 0.05 5.53 P > 0.05

EP 2.29 P > 0.05 16.44 P < 0.05

Multi 6.42 P > 0.05 14.30 P < 0.05

TLR8 Infected 7.53 8.892 0.0003 Yes 7.14 6.415 0.0021 Yes

Exp 0.64 P > 0.05 1.70 P > 0.05

EP 5.71 P < 0.05 6.08 P < 0.01

Multi 9.92 P < 0.01 8.40 P < 0.01

Exp= exposed, EP= early paucibacillary, Multi= multibacillary

B. Discussion The expression of TLR3 (Figures 2A and 2B) was significantly upregulated in the jejunum of exposed, uninfected animals relative to unexposed animals (P<0.05) as well as in the subclinical^ infected animals. TLR2, TLR3, TLR4 (Figure 2A and 2B), TLR5 and TLR8 (Figure 2A and 2B) were significantly upregulated in both ileum and jejunum of infected animals, and TLR 6 and TLR7 (Figure 2A and 2C) were significantly upregulated only in the jejunum of infected animals. Error bars on column graphs in Figures 2A and B show SEM of ddCt; error bars on other graphs in Figures 2A and B show SD of dCt.

To determine if the relative expression of TLR genes is also altered in the appropriate draining mesenteric lymph nodes, cDNA was also prepared from the tissues of the animals as described above and comparative quantitation performed following normalisation to H7-7, whose expression was found to be stable across the infected and uninfected animals.

Increases in the TLR gene expression levels were less in the lymph nodes than in the intestinal tissues. Significant increases in expression were found in both the ileal (Figure 3) and jejunal (Figure 4) lymph nodes of infected animals relative to uninfected animals for TLRI (P = 0.029, 0.021), TLR2 (P O.001 , 0.01), TLR6 (P 0.01, <0.001) and TLR8 (P <0.001 , 0.022) after allowing for variation due to property. TLR4 was significantly altered only in the ileal lymph node (P = 0.003) (Fig. 3) and TLR3 only in the jejunal lymph node (P = 0.019) (Fig. 4). TLR5 and TLR7 were not significantly altered in either lymph node of any infected groups relative to the uninfected animals. In the paucibacillary group, TLR2 (P = 0.007), TLR4 (P = 0.017) and TLR8 (P <0.001) in ileal lymph node and TLR8 (P = 0.039) in jejunal lymph node were significantly different from the uninfected group.

There is recirculation of leucocytes between intestinal and lymph node tissues via peripheral blood. To determine if the expression of TLR1- TLR8 transcripts in circulating monocytes is also altered during infection, cDNA was prepared from PBMCs isolated from unexposed animals, uninfected animals and animals in various stages of diseases.

There was a significant difference in expression of TLR7 between unexposed animals and all exposed animals (P < 0.001 ). TLR1 , TLR6 and TLR8 also showed trends to increases in expression in exposed animals relative to the unexposed group, suggesting these genes may be affected by exposure to M.ptb, although none were significantly altered in the PBMCs of infected animals relative to uninfected animals (Figure 5). The expression level of TLR1 , TLR3 and TLR5 was slightly decreased in the PBMCs of infected animals relative to the uninfected animals while slight increases in expression were seen for TLR2 and TLR8, and no change of TLR6 and TLR7 within exposed animals.

TLRs are type I membrane proteins known as pattern recognition receptors (PRRs) that are characterized by a domain composed of leucine rich repeats (LRR) that are responsible for recognition of diverse pathogen-associated molecular patterns (PAMPs) such as lipids, lipoproteins, proteins and nucleic acids, and a cytoplasmic domain homologous to the cytoplasmic region of the IL-1 receptor, known as the TIR domain, which is required for downstream signaling. In mammals, the TLRs are expressed on antigen presenting cells such as dendritic cells (DC) and macrophages. The TLRs sense the molecular signatures of microbes and play a primary role in initiating innate immune responses and the subsequent development of adaptive immune responses. TLRs signal via a common pathway leading to the expression of inflammatory genes such as cytokines, chemokines, interferons (IFNs) and upregulation of co-stimulatory molecules. Each TLR induces specific cellular responses to pathogens owing to differential usage of intracellular adapter proteins. Recent studies have revealed the importance of the subcellular localization of TLRs in pathogen recognition and signaling. TLR signaling pathways are negatively regulated by a number of cellular proteins to attenuate inflammation.

TLRs are known to be activated by diverse molecular signals, but the knowledge about the spectrum of substances capable of activating specific TLRs is incomplete. TLR1 is activated by triacyl lipoproteins. TLR2 is activated by lipoprotein and gram positive bacteria. TLR3 is activated in response to double-stranded RNA. TLR4 is activated by lipopolysaccharide. TLR5 is activated by flagellin. TLR6 is activated by diacyl lipoproteins. TLR7 and TLR8 are activated by single-stranded RNA and small synthetic compounds.

Example 4: Differentially expressed apoptotic genes

There are two well recognized pathways to apoptosis, the death receptor pathways and the mitochondrial pathways. Fas/FasL interactions are one of the best characterized death receptor pathways. Mitochondrial pathways to apoptosis are regulated by members of the Bcl-2 family which include proteins involved in inducing apoptosis such as Bax, and proteins involved in inhibiting apoptosis such as Bcl-2.

Mycobacteria have been shown to be capable of modulating the expression of several pro- and anti- apoptotic proteins/genes to evade macrophage apoptosis. qPCR has been applied to determine relative fold changes of genes involved in apoptosis (Bcl-2, Bax and Fas) in unexposed sheep (controls) and sheep exposed to M.ptb (treatments) at various levels of disease progression, to determine whether pro- and anti-apoptotic genes have altered expression levels during various stages of disease progression in infected animals compared to uninfected control animals.

A. Materials and Methods

RNA from intestinal and draining lymph nodes from the terminal ileum was prepared and analysed as described in Example 1.

B. Results

Three genes involved in central pathways to apoptosis were examined, two involved in apoptosis induction (pro-apoptotic) Fas and Bax and one gene involved in inhibition of apoptosis (anti-apoptotic) Bcl-2. Expression of these genes was examined in the terminal ileum and corresponding ileo-caecal lymph node (ICLN) of animals unexposed and naturally exposed to M.ptb.

Within the terminal ileum apoptotic genes involved in inhibiting and promoting apoptosis were significantly upregulated in exposed animals relative to the unexposed animals (Figure 6).

The pro-apoptotic genes Fas and Bax were expressed in relative abundance within the ileal tissues, with Ct 24-36 and 22-31 respectively. In contrast the anti-apoptotic gene Bcl-2 was expressed at comparatively lower levels with Ct values ranging from 29-37. C. Discussion

There was significant up-regulation of Bcl-2 gene expression in the terminal ileum of sheep exposed to paratuberculosis compared to unexposed controls (P=O.005) (Figure 6C). Expression of the gene Bax was also likely to be increased (P=O.1) in exposed animals but did not reach the usual accepted level of significance of <0.05 because too few sheep/properties were included in the analysis leading to too few degrees of freedom (approximately 2 for the fixed effect of exposure) (Figure 6B). In the ileocaecal lymph node (ICLN) there were significant differences in expression of Bcl-2 between exposed groups of sheep, with the group with severe paucibacillary lesions having the highest level of expression and the group with severe multibacillary lesions having the lowest level of expression (Figure 7C).

For the anti-apoptotic gene Bcl-2 the fold changes in expression of this gene in relation to the pro-apoptotic gene Bax and Fas were evaluated. Fold changes (relative to unexposed animals) suggest that within the terminal ileum of M.ptb exposed sheep there are higher expression levels of Bax (Figure 6B) (ranging from 6.62-fold to 30.52- fold) compared with Bcl-2 (Figure 6C) (ranging from 1.65-fold to 5.6-fold). In contrast the pro-apoptotic gene Fas appears to be expressed at similar levels to Bcl-2 ranging from 2.86-fold to 6.10-fold (Figure 6A).

In contrast, within the ICLN, the expression of Bcl-2 (Figure 7C) and Bax (Figure 7B) remained similar in all M.ptb exposed groups. The expression of Bcl-2 in the ICLN ranged from 0.78-2.92 and the expression of Bax ranged at from 1.16-fold to 1.92-fold.

One exception to this was animals with intermediate and late paucibacillary lesions which showed a higher fold change in Bcl-2 compared to Bax. Expression levels for BcI-

2 in these animals was 1.54-fold and 2.92-fold for intermediate and late paucibacillary lesion types respectively. Expression levels for Bax in these animals were 1.22-fold and

1.92-fold respectively. Similarly to the terminal ileal tissues, the expression of Fas

(Figure 7A) and Bcl-2 (Figure 7C) remained similar in all exposed animal groups. In addition, the fold changes of all genes tested varied in the terminal ileum and ICLN. The fold changes in the expression of Fas, Bax and Bcl-2 were higher in the terminal ileum compared to the ICLN in all exposed animal groups tested.

Example 5:

Results

Further investigations of putative differentially expressed genes was undertaken on the animals from trial OJD.028 and 031 A.

In addition to SAA3, cathepsin K, SLAMF7 and TOM22 (Example 2), a number of other genes exhibited changes in their expression level in the ileum as illustrated in Figure 8A-C, and in the table below. Samples were normalised to the reference gene HK5.1.

Table 6 QPCR analysis of DD-PCR genes in the ileum of animals with historical lesions of Johne's disease relative to uninfected animals

Band 4.4 (Figure 8C) was found to have homology with Macaca calcium/calmodulin- dependent protein kinase II. This protein has a wide distribution and plays an important role in the calcium signaling pathway. It is suggested that the protein kinase is associated with the enterocyte cytoskeleton and plays a role in regulating intestinal epithelial cytoskeleton. The ileal cell brush border Na+/H+ exchange is also regulated by calcium/calmodulin dependent protein kinase Il whereby it inhibits the brush border exchange which takes part in the regulation of Na+ absorption.

Band 4.1 (Figure 8B) was found to be homologous with bovine thymosin beta 4. The thymosin peptides have the ability to alter immune functions in animal and human models. The effects of thymosin beta 4 was studied on human colonic lamina propria proliferation and was found to suppress thymidine incorporation into lamina propria proliferation. It was thought that thymosin beta 4 may have a function in modulating the human mucosal immune system. Thymosin beta 4 was also studied in the context of the intraepithelial lymphocyte network (TRC gamma delta+ and dendritic epidermal T cells) and skin inflammation. It was shown that the Thymosin beta's had anti-inflammatory activity in this context.

PB 3.2 (Figure 8A) was found to have homology with a protein similar to the bovine DNA-binding protein SATB1. SATB1 is a cell-specific nuclear protein which has been shown to regulate numerous genes and recruit chromatin-remodelling factors for controlling gene transcription. SATB1 RNA interference studies have shown that SATB1 is required not only for compacting chromatin into dense loops but also for inducing II4, II5, 1113 and c-Maf expression involved in processes of T(H)2 cell activation.

Similarly to other genes identified by the inventors, these genes could be used to identify molecules that could be used therapeutically or aid in identifying infected animals during different stages of disease, particularly asymptomatic stages. Unique expression profiles could be used as diagnostic markers of disease.

Example 6

Results

Additional analysis of differentially expressed genes in ileaocaecal lymph nodes was undertaken using DD-PCR. A number of genes found to be differentially expressed were also confirmed to be differentially expressed in PBMCs. Without being bound by any theory, it is thought that as circulation of lymphoid cells is from the gut via the blood, 01106

37

the changes observed in the gut and lymph nodes may be detected in peripheral blood. This finding may allow the identification of potential infection 'signatures' which could facilitate diagnosis of infection.

DD-PCR patterns between unexposed and infected samples were compared, isolated and analysed by QPCR in accordance with the earlier described methods. After sequencing and database alignment, the following genes were identified:

Table 7A Potentially differentially expressed genes isolated from intestinal lymph nodes of sheep

Table 7B Potentially differentially expressed genes isolated from lymph nodes and ileum of sheep

Validation was performed by QPCR, the results of which are illustrated in Figure 9.

As mentioned above, a number of genes differentially expressed in lymph nodes were analysed by QPCR for differential expression in PBMCs. As illustrated in Figure 10, a number of these genes were also differentially expressed.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Claims

Claims

1. A method for assessing the likelihood of an individual having a mycobacterium infection including:

-selecting an individual;

- assessing the level of expression of a target protein described in Table 1 herein in the individual;

thereby assessing the likelihood of the individual having a mycobacterium infection.

2. A method according to claim 1 , wherein the target protein is a toll-like receptor.

3. A method according to claim 1 , wherein the target protein is SAA3.

4. A method according to claim 1 , wherein the target protein is cathepsin K pre- protein.

5. A method according to claim 1 , wherein the target protein is TOM2.

6. A method according to claim 1 , wherein the target protein is 19A/Signalling lymphocyte activation molecule family member 7 (SLAM 7).

7. A method according to claim 1 , wherein the target protein is a pro-apoptotic or anti-apoptotic gene selected from Bcl-2, Fas and Bax.

8. A method according to any one of claims 1 to 7, wherein the individual is ovine or bovine.

9. A use of an antibody specific for a target protein described in Table 1 herein in a method for assessing the likelihood of an individual having a mycobacterium infection.

10. A use according to claim 9, wherein the method involves an immunoassay, chromatography or mass spectrometry.

11. A use of a polynucleotide for detecting a nucleic acid that encodes or controls the expression of a target protein described in Table 1 herein a method for assessing the likelihood of an individual having a mycobacterium infection.

12. A use according to claim 11 , where the method involves quantitative PCR, Northern blotting, Southern blotting or microarray.

13. A kit for assessing the likelihood of an individual having a mycobacterium infection including:

- an antibody specific for a target protein described in Table 1 herein; or

- a polynucleotide capable of detecting a nucleic acid encoding or controlling the expression of a target protein described in Table 1 herein.

14. A kit according to claim 13, further including instructions for use of the kit for assessing the likelihood of an individual having a mycobacterium infection.