WO2009156395A1

WO2009156395A1 - Method for the early detection of a map infection

Info

Publication number: WO2009156395A1
Application number: PCT/EP2009/057811
Authority: WO
Inventors: Rolf Bauerfeind; Philip Bridger; Hakan Bulun; Christian Menge
Original assignee: Justus-Liebig-Universität Giessen
Priority date: 2008-06-25
Filing date: 2009-06-23
Publication date: 2009-12-30
Also published as: DE102008029833A1

Abstract

The invention relates to a method for the specific and early detection of a Mycobacterium avium ssp. paratuberculosis (MAP) infection in a biological sample of a mammal by detecting cells producing interferon-gamma (IFN-γ) after incubating peripheral blood mononuclear cell cultures (PBMC) with MAP antigen preparations or Mycobacterium avium ssp. avium (MAA) and/or Mycobacterium phlei (M. phlei) control antigens.

Description

Patent application

Method for early detection of infection with MAP

The present invention relates to a method for the specific and early detection of infection with Mycobacterium avium subsp. paratuberculosis (MAP) in mammals by detecting interferon-gamma (IFN-γ) in biological samples, including blood, after incubation of cells that specifically respond to stimulation by MAP-specific antigens with IFN-γ production.

State of the art

Paratuberculosis caused by the bacterium Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic intestinal disease of ruminants, which is mainly characterized by a progressive, granulomatous inflammation of the small intestinal wall. The animals usually become infected within the first weeks of life, but show clinical symptoms only years after infection. From an unknown point in time during the long incubation period, infected animals begin to excrete the pathogen - mostly intermittently - with the feces and thus become a source of infection for other animals. Transplacental transmission to the unborn child is discussed as a further path of infection. Due to missing signs of disease during the incubation period, infected animals remain hidden from the pet owner. Another health policy aspect is that MAP is suspected to be causally involved in the disease of the human, an incurable chronic inflammatory disease of the digestive tract known as Crohn's disease or to sustain the disease. For the remediation of infected flocks or for paratuberculosis prophylaxis in the herds, it is therefore important to detect infected animals at the earliest possible stage of the infection (ie in calf and juvenile age). For the diagnosis of paratuberculosis, there are several methods known in the art: The so-called gold standard of Paratuberkulosediagnostik is the pathogen detection by means of cultural-bacteriological examination of animal excrement or Ileocaecallymphknotens (intestinal lymph node). Another method detects MAP specific DNA in samples by PCR technology. These two methods are direct methods to detect MAP infections in animals.

In addition, there are indirect methods, for example by the detection of MAP-specific antibodies in the blood serum of the animals. For example, pathogen-specific IgG antibodies are detected by enzyme-linked immunosorbent assays (ELISA). For this purpose, test kits are commercially available.

It is known that MAP infection in ruminants in the early stages of infection is characterized by an increase in blood MAP directed T cells. The quantification of IFN-γ in restimulated blood leukocytes has already been followed as a diagnostic approach to the identification of young MAP-infected cattle. For this purpose, whole blood samples were stimulated without previous separation of the cells with MAP antigens and quantified the total secreted by this heterogeneous cell mixture IFN-γ by ELISA. This detection of cellular immune reactions provides no earlier diagnosis than the faeces culture or according to §17 TierSeuG approved serum ELISA and is in principle for MAP early diagnosis of juveniles, e.g. Not suitable for calves, since there are too many cells in the cell mixture of juveniles that react nonspecifically to antigen stimulation with IFN-γ formation.

DISADVANTAGES OF THE PRIOR ART The currently available diagnostic tests are only slightly sensitive and specific, in particular with regard to detection at an early point in time of the infection (ie from birth to the second year of life of the animal), so that MAP-infected animals are insufficient can be identified with certainty.

Pathogen detection is very tedious (at least 8 weeks) in detecting pathogens by means of cultural-bacteriological examination of animal feces, and false-negative findings are very frequent since pathogens are not yet or only intermittently eliminated in the early stages of the infection. A reasonably reliable statement about the MAP-free status of a single animal is therefore possible only after multiple sampling and examination with negative findings. The cultural-bacteriological examination of the ileocaecal lymph node (intestinal lymph node) has the further disadvantage, in addition to the lengthy growing period already mentioned, that the intestinal lymph nodes of the animal are only accessible by abdominal cavity surgery for such an examination. Therefore, this method currently appears to be of limited practical use.

The same applies to the direct MAP detection by means of the mentioned PCR method. Although less time-consuming than culturally-bacteriological examination, it is often less sensitive.

Special features of the pathogen-host interaction dominate the clinically inapparent phase at the beginning of the infection by the cellular immune response, while the humoral immune response is weak. Accordingly, systems for indirect detection of pathogens using ELISA technology have only low specificity and / or sensitivity (at approx. 50%), especially in the early phase of a MAP infection. The quantification of IFN-γ in restimulated blood leukocytes as a measure of the cellular immune response is already known in the art as a diagnostic approach for the identification of MAP-infected cattle (from 12 months). Strong individual differences and frequent false-positive responses to mycobacterial antigen, especially in calves, significantly reduce the specificity of this test.

This results in considerable disadvantages:

Although an animal may have been infected for a long time, its infection status can only be determined after it has itself again born one or more offspring and this may already infected.

Even at the inventory level, the diagnostic freedom available so far can not be used to ascertain the MAP freedom with sufficient certainty. Even if the serological and bacteriological examination of the animals leads to a negative result, it can not be ruled out that the offspring has already become infected with the pathogen. A reliable assur- ance of the MAP freedom of animals to be integrated into paratubercu- lose-free populations is currently impossible, but is of great importance in preventing the spread of this infectious disease. This applies in particular to the certification of breeding livestock in the context of intra-community transfers and exports, which in the future will be required of more and more countries (so far, inter alia, Austria).

There is therefore a need for a sensitive and specific diagnostic detection method that reliably detects infection with MAP already in the early infection phases, above all in animals up to the second year of life.

task

The object of the present invention is therefore to remedy the disadvantages described in the prior art and to provide a reliable and safe method for detecting MAP infection in adults and in particular in mammals up to the second year of life and a test kit suitable for this purpose.

solution

The object is achieved by a method according to claims 1-9 and a test kit according to claims 10-13.

The basis for the development of a new method was the consideration that MAP-infected animals have in their blood a measurable proportion of T-cells (CD4 ⁺ and CD8 ⁺ T-cells), which can be specifically sensitized with MAP antigen and thereby IFN- while animals that are not infected with MAP have CD4 ⁺ and CD8 ⁺ T cells, respectively, but can not be stimulated with MAP antigen. However, it has been found that when whole blood is used, cell subpopulations (especially in juveniles) are present within the immune cells, which react nonspecifically to antigen stimulation by means of mycobacterial antigens with IFN-γ formation and thus easily give rise to false-positive or uninterpretable results a test system based on it considerably question. This was considered according to the invention in the method for the detection of a MAP infection.

The inventive method has the advantage that even when using whole blood from adult mammals and mammals up to the second year of life, only the specific IFN-γ formation is measured, within the cells that are specific to stimulation using MAP-specific antigens with IFN -γ formation react and thus no more false positive or uninterpretable results arise. Thus, a specific and sensitive method for detecting an infection with MAP is available, which provides reliable results in the early stages of infection in adult mammals and especially in mammals up to the second year of life and is used in diagnostics. The specificity and sensitivity in the early stages of infection in adult mammals and above all also in mammals up to the second year of life is achieved by first isolating the mononuclear cells from the whole blood according to the invention and then measuring and quantifying the IFN-.gamma. Salary targeted within preferably the CD4 ⁺ but also CD8 ⁺ T-ZeII subpopulations occurs. It should be noted that the IFN-γ detection is separate and occurs only within the CD4 ⁺ cell populations or the CD8 ⁺ cell populations. This has the advantage that only the IFN-γ is measured, which is formed by the cells which react specifically to antigen stimulation by means of mycobacterial antigens with IFN-γ formation. The measurement of nonspecific IFN-γ production of other cell populations is thus excluded and no false-positive results are produced.

Specificity and sensitivity are further achieved by the selection or production of the antigen (eg, whole cell lysate from MAP, e.g., the commercially available K10 strain, ATCC No. BAA-968, or other suitable MAP strains) that provides optimal MAP expression. specific IFN-γ response in vitro triggers. This has the advantage that only the response to antigen is measured by MAP and no false-positive results, for example, by Mycobacterium avium subsp. avium (MAA) and / or Mycobacterium phlei. According to the invention, the biological sample comprises an organ or tissue sample, faeces and milk sample, and preferably blood.

According to the invention, the biological sample is derived from a mammal including a human, preferably from a ruminant, in particular cow, sheep or goat.

In detail, the method according to the invention consists of the following steps: 1. Isolation and cultivation of cells which react specifically to stimulation by means of MAP-specific antigens with IFN-γ formation. 2. Stimulation of the cells from step 1 with MAP-specific antigen

3. Restimulating the cells from step 2 by adding agents that accelerate IFN-γ production and prevent premature secretion of IFN-γ from the cells

4. Label the cells from step 3 and the IFN-γ, to measure the antigen-specific IFN-γ production

embodiments

1. Isolation and culture of cells that respond specifically to stimulation by MAP-specific antigens with IFN-γ production

The cells are isolated from a sample of a mammal, for example an organ, tissue, feces or milk sample, preferably from blood, in particular from a cattle. The blood is drawn according to general knowledge, for example by puncturing the jugulas of a mammal, eg bovine. To inhibit coagulation, the blood is collected in suitable containers, for example syringes, and mixed with an anticoagulant eg 0.2 ml of a 3.8% strength Na ₃ citrate solution per ml of whole blood. Alternatively, the blood sample is taken for example in commercially available EDTA tubes. Preparation of antigens eg MAP-specific antigens

The preparation of MAP-specific antigens is carried out as whole cell lysate (WCS), as known to those skilled in the art, e.g. after lysing bacterial cells, lysing them with ultrasound and mechanically (e.g., by bead-beating) and then filtering and solubilizing, e.g. known from J. Pollock (National Animal Disease Center, IA, USA) "Antigen Preparation-NADC." Alternatively, the preparation of mycobacteria antigen is carried out as a protein purified derivative (PPD) by cultivating bacteria under suitable culturing conditions after cultivation of bacteria Autoclaving the suspension, precipitating the proteins, filtering them and then bringing them into solution, as is known, for example, from Haagsma and Angus in "Mycobacterium bovis Infection in Animals and Humans". These methods are used for the preparation of MAP-specific antigens, but also for the preparation of control antigens, e.g. from mycobacterium avium subsp. avium (MAA) and / or Mycobacterium phlei.

For the isolation and cultivation of cells which react specifically to stimulation by means of MAP-specific antigens with IFN-γ formation, the primary monocytic cells, for example the primary bovine monocytic cells (PBMC), are first isolated from the blood sample, for example via a Ficoll® gradient according to the manufacturer's instructions. The monocytic cells are obtained from the blood sample by filtering out (eg gradient centrifugation) of the erythrocytes and granulocytes so that only lymphocytes and monocytes remain in the cell suspension. An alternative is a modification of this Gebrauchsan instruction described in Menge et al. (C.MANGE, LH.WIELER, T. SCHLAPP, G. BALJER (1999): Shiga toxin 1 from Escherichia coli blocks activation and proliferation of bovine lymphocyte subpopulations in vitro, Infect. Immun., 67, 2209-17). The cells (eg PBMC) are then used in a defined concentration, for example of 4 × 10 ⁶ cells / ml, in 12-well cell culture plates.

Within these primary monocytic cells (eg the primary bovine monocytic cells (PBMC)) are cell subpopulations that respond to stimulation by MAP-specific antigens with IFN-γ production. In particular, these are the CD4 ⁺ and CD8 ⁺ T cells. 2. Stimulation of cells from step 1 with MAP-specific antigens

The stimulation of the isolated primary monocytic cells, for example the primary bovine monocytic cells (PBMC) from step 1, with antigens, for example the MAP-specific antigens and optionally at least one control antigen in a separate reaction, is carried out in a suitable concentration of, for example, 5 μg PPD / ml medium or, for example, 1, 5 μg WCS / ml PBMC medium. The stimulation takes place, for example, in a cell culture incubator over a period of 100-200 h, preferably 120-16Oh, very particularly preferably 144 h under suitable standard conditions, eg 37 ° C. and 5% CO ₂ (eg cell culture incubator).

As control antigen, e.g. a preparation of Mycobacterium avium subsp. avium (MAA) and / or Mycobacterium phlei used and this as a negative control in the process.

3. Restimulating the cells from step 2 by adding agents that accelerate IFN-Y production and prevent premature secretion of IFN-γ from the cells

The restimulation of the cells from step 2 is carried out by further incubation in the presence of an agent which accelerates IFN-γ production (eg ionomycin and / or phorbol myristate acetate (PMA)) in suitable concentration and (simultaneously) of an agent prevents the premature secretion of IFN-γ from the cells (eg Brefeldin A) in suitable concentration over a period of 2-6 h, preferably 4 h. Suitable concentrations of ionomycin are e.g. 1 μg / ml, of phorbol myristate acetate (PMA) e.g. 50 ng / ml.

Subsequently, the cells are harvested and transferred in a defined concentration into a suitable vessel, for example 1 -2 × 10 ⁵ cells / well in a 96-well plate with "V" shape bottom. 4. Label the cells from step 3 and the IFN-γ, to measure the antigen-specific IFN-γ production

To detect and quantitatively detect IFN-γ production within the cells, in particular the antigen-specific CD4 ⁺ and / or CD8 ⁺ cells from step 3, the cell subpopulations of interest (CD4 ⁺ and / or CD8 ⁺ cells ) as well as intracellular IFN-γ (eg with appropriate antibodies). Thus, the IFN-γ content can be determined separately, either in CD4 ⁺ or in CD8 ⁺ cells. A variety of radioactive, fluorescent, bioluminescent CD4, CD8 and IFN-γ-specific antibodies are available to the person skilled in the art for direct labeling. However, indirect labeling by means of radioactive, fluorescent, bioluminescent secondary antibodies, preferably by means of a biotin-streptavidin amplification step, is also known to the person skilled in the art. The person skilled in the art knows a large number of suitable fluorescent dyes, including, for example, APC and PE, which are also suitable for detection in the flow cytometer.

First, the surface antigens CD4 or CD8 of the cells from step 3 are mixed with commercially available monoclonal CD4- or CD8-specific antibodies and labeled. These CD4- or CD8-specific antibodies are then mixed with secondary antibodies such as APC-conjugated secondary antibodies and detected. Preincubation with biotin-conjugated secondary antibodies is preferably carried out, which are then treated with APC-conjugated streptavidin and labeled. This leads to a signal amplification and thus to a reliable identification of the CD4 or CD8 positive cells. Subsequently, the cells are fixed, permeabilized and treated with commercially available monoclonal antibodies against IFN-γ to label intracellularly present IFN-γ. Antibodies which have bound to IFN-γ are detected with secondary antibodies, eg with commercially available PE-conjugated secondary antibodies. Measurement and quantification of the IFN-γ content

The quantification (IFN-γ _[%] titer) is carried out, for example, in the flow cytometer, since here, on the one hand, the CD4 ⁺ or CD8 ⁺ T cells and within both populations those with and without IFN-γ formation are clearly differentiated. The proportion of IFN-γ-producing cells on all CD4 ⁺ or CD8 ⁺ T cells after incubation with MAP antigen in comparison to incubation without antigen or with control antigen, eg MAA or M. p /? / E / Antigen is chosen as a parameter for the evaluation of blood samples.

Proportion of IFN-γ producing CD4 ⁺ (or CD8 ⁺ ) PBMC after incubation with MAP antigen

IFN-Y _[%] titer =

Proportion of IFN-γ producing CD4 ⁺ (or CD8 ⁺ ) PBMC after incubation without antigen (or control antigen)

In the case of a MAP-negative animal, the quotient is about 1, 0. In the case of a MAP-positive animal, the quotient is> 1, 0.

Alternatively, the quantification IFN-γ _[MFU] titer eg in the flow cytometer by delimiting the cells of lymphocytic origin (Region 1) of those monocytic or lymphoblastoid origin (Region 2) and separate analysis. Within region 2, the mean relative fluorescence intensity (MFLI) of the IFN-γ signal of all CD4 ⁺ (or CD8 ⁺ ) cells is determined. The MFLI after incubation with MAP antigen in comparison to the incubation without antigen or with control antigens eg antigen from Mycobacterium avium subsp. avium (MAA) and / or Mycobacterium phlei is chosen as a parameter for the evaluation of blood samples.

MFLI of IFN-γ of all CD4 ⁺ (or CD8 ⁺ ) PBMC after incubation with MAP antigen

IFN-Y [MFL1] titer =

MFLI of IFN-γ of all CD4 ⁺ (or CD8 ⁺ ) PBMCs

Incubation without antigen (or control antigen)

In the case of a MAP-negative animal, the quotient is about 1, 0. In the case of a MAP-positive animal, the quotient is> 1, 0. With the aid of the preceding description, it is possible for a person skilled in the art to apply this principle of detection of MAP with slight changes and adaptations, which are within the scope of his specialist knowledge, also to the detection of other pathogens, eg infections with animal or zoonotic pathogens.

One embodiment of the invention relates to an easy-to-use test kit for detecting infection with Mycobacterium avium ssp. paratuberculosis (MAP) in a biological sample comprising materials for measuring specific IFN-γ production from cells responsive to stimulation by MAP-specific antigens with IFN-γ production.

The test kit comprises an antigen preparation of Mycobacterium avium ssp. paratuberculosis (MAP), alternatively as a negative control also an antigen preparation of Mycobacterium avium ssp. avium (MAA) and / or Mycobacterium phlei (M. phlei).

In the embodiment of the invention, the test kit further comprises antibodies for binding and labeling of the cells that specifically respond to stimulation by MAP-specific antigens with IFN-γ production, e.g. radioactive, fluorescent, bioluminescent CD4, CD8 specific antibodies, furthermore antibodies which bind IFN-γ and e.g. radioactively, fluorescently or bioluminescently labeled. In another embodiment, the test kit comprises secondary antibodies for binding to the antibodies that bind to cells that specifically respond to stimulation by MAP-specific antigens with IFN-γ production and secondary antibodies for binding to the antibodies that bind IFN-γ. These secondary antibodies are radioactively, fluorescently or bioluminescently labeled secondary antibodies known to the person skilled in the art for binding the

CD4, CD8 and IFN-γ specific antibodies which are particularly suitable for analysis by flow cytometry. Because of the teachings of the present invention, as well as the general skill in the art, it is known to the manufacturer of the kit of the present invention how to formulate and store the individual components of the kit, eg, buffers.

pictures

FIG. 1 shows by way of example the evaluation of a measurement for detecting an infection with Mycobacterium avium subsp. paratuberculosis (MAP) by detecting interferon-gamma (IFN-γ) -forming T-cell subpopulations in blood after incubation of peripheral blood mononuclear cell cultures (PBMC) with MAP antigen preparations. Shown is the IFN-γ _[MFLI] titer (normalized from the samples measured after incubation with M. phlei; y-axis) CD4 ⁺ lymphoblasts from 3 MAP-infected animals (shown by filled symbols in the graph) and 3 MAP-negative animals (shown by open symbols in the graph) after incubation without antigen (Med) or with antigen preparations) of MAA (WCS-MAA) and MAP (WCS-MAP) (x-axis).

Claims

claims

1. A method for detecting an infection with Mycobacterium avium subsp. paratuberculosis (MAP) in a biological sample of a mammal, characterized in that the specific IFN-γ formation from cells that are on a

Stimulation by MAP-specific antigens with IFN-γ formation react, is measured.

2. Method for detecting infection with Mycobacterium avium subsp. Paratuberculosis (MAP) according to claim 1, characterized in that the infection is already detectable in the early stages of infection in the adult mammal and the mammal until the 2nd year of life.

3. Method for detecting infection with Mycobacterium avium subsp. Paratuberculosis (MAP) according to claim 1 and 2, characterized in that it comprises the following steps

2. Stimulation of cells from step 1 with MAP-specific antigen

3. Restimulating the cells from step 2 by adding agents that accelerate IFN-γ production and prevent premature secretion of IFN-γ from the cells. 4. Label the cells from step 3 and IFN-γ to measure the cells Antigen-specific IFN-γ production

4. Method for detecting infection with Mycobacterium avium subsp. Paratuberculosis (MAP) according to the preceding claims characterized in that the biological sample organ, tissue, faeces or

Milk sample, as well as blood is.

5. Method for detecting infection with Mycobacterium avium subsp. Paratuberculosis (MAP) according to the preceding claims, characterized in that the MAP-specific antigen is a preparation of Mycobacterium avium subsp. paratuberculosis (MAP).

6. Method for detecting infection with Mycobacterium avium subsp. Paratuberculosis (MAP) according to the preceding claims, characterized in that the MAP-specific antigen Mycobacterium avium subsp. avium (MAA) and / or Mycobacterium phlei (M. phlei)

7. Method for detecting infection with Mycobacterium avium subsp. Paratuberculosis (MAP) according to the preceding claims, characterized in that the labeling of the cells takes place by means of antibodies

8. Method for detecting infection with Mycobacterium avium subsp. Paratuberculosis (MAP) according to the preceding claims, characterized in that the measurement of the antigen-specific IFN-γ production takes place in the flow cytometer

9. A method for detecting an infection with Mycobacterium avium subsp. Paratuberculosis (MAP) according to the preceding claims, characterized in that the means accelerating IFN-γ production are lonomycin and / or phorbol myristate acetate and agents which prevent premature secretion of IFN-γ from the cells is Brefeldin A. ,

10. Test kit for detecting infection with Mycobacterium avium subsp. paratuberculosis (MAP) of a biological sample, characterized in that it comprises materials for measuring specific IFN-γ production from cells responsive to stimulation by MAP-specific antigens with IFN-γ production.

11. Test kit according to claim 10, characterized in that it comprises an antigen preparation of Mycobacterium avium ssp. paratuberculosis (MAP), antibodies for binding to cells specifically responsive to stimulation by MAP-specific antigens with IFN-γ production and comprising antibodies to bind IFN-γ.

12. Testkit according to claim 10 and 1 1 characterized in that it is a antigen preparation of Mycobacterium avium subsp. avium (MAA) or Mycobacterium phlei (M. phlei).

13. Test kit according to claim 10, characterized in that it contains secondary antibodies for binding to the antibodies which bind to cells which react specifically to stimulation by means of MAP-specific antigens with IFN-γ formation and secondary antibodies for binding to the antibodies, which bind IFN-γ.