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February 2009 Issue

Overview of Mycobacterium avium subsp. paratuberculosis
(MAP, M. pt. or Johne’s Disease)

Paratuberculosis is chronic enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The disease was first described by H. A. Johne and L. Frothingham in 1895. Paratuberculosis can affect all ruminants and has been observed on every continent.

Economic Impacts

Direct economic losses from paratuberculosis are caused by decreased milk production, reproductive dysfunctions, poor feed conversion, shortened production age and increased predisposition to other diseases. Some studies have estimated the losses to the U.S. dairy industry at $100 million annually.¹

Clinical Signs

Clinical signs of paratuberculosis include a slowly progressive wasting and diarrhea (in bovines), which is intermittent at first, then becoming progressively more severe, until it is constantly present. Diarrhea is less common in small ruminants.

Disease Transmission

Paratuberculosis in cattle spreads by ingestion of MAP from the contaminated environment and persists after the introduction of infected animals. The primary source of infection in calves is milk from infected cows or milk that is contaminated with the feces of diseased cattle. Infection can be spread vertically from cow to fetus. Infected semen can also spread the disease. Animals with subclinical signs may shed MAP with colostrum and milk.

Herd certification programs are used to limit the spread of Johne’s disease and to protect buyers from purchasing infected animals. Certification programs classify herds according to the herd’s degree of paratuberculosis prevalence.

Testing for MAP Requires Highly Specific Assays

Because MAP organisms grow very slowly, the sensitivity of MAP assays is often less than 60%. Sensitivity is linked to animal disease status: the more infection in the tested animal, the higher the sensitivity of the test.

Because the overall sensitivity for MAP assays is low, an effective MAP assay must be highly specific. A highly specific assay ensures that if any positive results are detected, they are true positives. False positives lead to wasted time, effort and expense trying to confirm a diagnosis.

Animals with subclinical signs can be detected by testing for the presence of specific antibodies (using a test kit such as the IDEXX Pourquier* M. pt. Antibody Test Kit), by culture from feces or tissues collected at necropsy, or by the demonstration of cell-mediated responses.

Reliable, Simple, Fast Detection of Paratuberculosis Antibodies

The IDEXX Pourquier* M. pt. Antibody Test Kit is an indirect ELISA designed to detect antibodies against Mycobacterium avium subsp. paratuberculosis (MAP). Kit specificity approaches 100% in tested populations. The IDEXX Pourquier M. pt. Antibody Test Kit is available outside the U.S. and Canada. For a test available within the U.S. and Canada, please read about the IDEXX HerdChek* M. pt. Antibody ELISA at www.idexx.com/view/xhtml/en_us/livestock-poultry/ruminant/map.jsf.

Preabsorption with Mycobacterium phlei Provides High Specificity

The IDEXX Pourquier* M. pt. Antibody Test Kit protocol includes a step in which samples are diluted in a buffer containing M. phlei extract and then incubated. The Pourquier* M. phlei preabsorption step—from 15 minutes to 2 hours—eliminates cross-reactions with atypical mycobacteria. This step is responsible for the high specificity of the assay observed in several international studies. High specificity provides confidence in test results and limits the amount of time and effort spent confirming paratuberculosis diagnoses.

Specificity and Sensitivity Studies

To assess the specificity and sensitivity of the IDEXX Pourquier* M. pt. Antibody Test Kit, IDEXX collected serum and fecal samples from 796 presumed-negative animals and from 247 presumed-positive animals. The samples were tested using both fecal culture and the IDEXX Pourquier ELISA. Results are summarized below.

The IDEXX Pourquier* M. pt. Antibody Test Kit demonstrated excellent specificity (99.4%) and sensitivity (51.4%) for the tested population.

In addition, multiple international studies have demonstrated the performance of the IDEXX kit. The following tables summarize the sensitivity and specificity results.

Use with Multiple Sample Types, Including Milk

The IDEXX Pourquier* M. pt. Antibody Test Kit can be used to test both individual and bulk bovine milk samples, as well as serum and plasma samples.

In a validation study using milk, 97 positive milk samples were diluted in MAP-negative milk and tested with the IDEXX Pourquier* M. pt. Antibody Test Kit. There was a clear correlation between the ELISA S/P ratios and the level of dilution, as shown below. In addition, the correlation between serum and milk testing approached 95%.⁵

Simple Protocols Ensure Ease-of-Use

Kit features:
• Results in less than 90 minutes
• Daytime (room temperature) or overnight incubation for all sample types
• Compatibility with the IDEXX xChek* software for automatic interpretation of results
• Suitable for automated work flows
• Five-plate kits, monophasic (screening) or biphasic (verification)
• Use with bovine, ovine and caprine serum or plasma samples and with bovine milk (both individual and bulk)

Result interpretation is the same for both the screening and verification kits. Cutoffs are shown below:

Product Information

For more information about the IDEXX Pourquier* M. pt. Antibody Test Kit, please contact your IDEXX representative.

Screening ELISA: Ref P07130
Verification ELISA: Ref P07110

*xChek, HerdChek and Pourquier are trademarks or registered trademarks of IDEXX Laboratories, Inc. in the United States and/or other countries.

How to Install User-Defined Assays on a Computer Running the Windows Vista* Operating System

This Tech Tip will be helpful if you use the IDEXX xChek* software and you would like to run user-defined assays (UDAs) on a computer running the Windows Vista* operating system.

Before you can install a UDA on any computer (no matter the operating system), you must update the xChek.ini file to include the line “allow new assays=1.” The xChek.ini file is located in the C:\\WINDOWS folder. But on a computer running the Windows Vista operating system, the WINDOWs folder is protected so that you cannot change its contents. Therefore, you must change the folder’s security settings.

To change the WINDOWS folder security settings and modify the xChek.ini file:

1. Double-click the My Computer icon on the desktop.
2. Find the xChek.ini file, located under C:\\WINDOWS.
3. Right-click the xchek.ini file, then select Properties.
4. Click the Security tab.
5. Click Edit.
6. For each user, check “allow” for each permission.
7. Click OK. A warning message appears.
8. Click OK to close the message.
9. Shut down the computer and restart it.
10. Once the computer has restarted, use a text editor or word processing program to open the xChek.ini file.
11. In the [System] section of the file, locate the line that says “AllowNewAssays” and set the value equal to 1. The line should look like this: AllowNewAssays=1
12. Save and close the file.

You can now install UDAs. Please contact IDEXX Technical Services if you need assistance.

*xChek is a trademark or registered trademark of IDEXX Laboratories, Inc. in the United States and/or other countries. Windows Vista is a registered trademark of Microsoft Corporation.

Events Around the World

• Dallas, Texas, USA—March 7–10, 2009
  American Association of Swine Veterinarians
• Stendal, Germany—March 11–13, 2009
  Stendaler Symposium
• St. Paul, Minnesota, USA—March 17–19, 2009
  Midwest Poultry Federation
• Antwerp, Belgium—March 17–21, 2009
  EAAV Conference
• Sacramento, California, USA—March 22–25, 2009
  Western Poultry Disease Conference
• Louisville, Kentucky, USA—March 31–April 1, 2009
  National Institute for Animal Agriculture

Create User-Defined Assays for the IDEXX Pourquier* M. pt. Antibody Test Kit

If you would like to use the IDEXX xChek* software to analyze results from the IDEXX Pourquier* M. pt. Antibody Test Kit, you must first enter the assay specifications into the software. This is called creating a user-defined assay (UDA).

You can create a UDA for use with milk samples and one for use with serum or plasma samples. Two UDAs are needed, because the different sample types require different assay cutoffs.

IMPORTANT: The IDEXX xChek software does not evaluate controls or results for validity when you use a user-defined assay. You must evaluate the results from each assay in accordance with Good Laboratory Practices. To evaluate your assay validity, refer to the Validation Criteria section in the insert provided with the test kit.

Creating a UDA is a simple process that requires two tasks: editing the xChek.ini file and entering the assay specifications into the IDEXX xChek software.

To edit the xChek.ini file:

1. From the task bar, choose Start > Run, then type xchek.ini into the text box. Click OK.
2. After the xChek.ini file opens, look in the [System] section for the line labeled AllowNewAssays, and set the value to 1 (or if it is already 1, leave as is).
3. Locate the section labeled [BasicGraphs], and scroll to the end of the section. Add one or two separate lines of text as follows:
1. To use the assay with serum and plasma samples, add a line
   that says MptsSP-PQ=11.
2. To use the assay with milk samples, add a line that says MptsM-PQ=11.
4. From the menu bar, choose File > Save, and then choose File > Exit.

To create the UDA for use with milk samples:

1. Start the xChek software and log in.
2. From the menu bar, choose Database > Assays > All to open the Assay dialog box.
3. Click New, then enter the following information for the respective fields:
    
   Name: Pourquier Paratuberculosis Screening ELISA-Milk
   Code: MptsM-PQ
   Kit Lot: unknown
    
   Today's date is automatically entered into the Expiration field.
   NOTE: When you begin testing, update the information in the Kit Lot and Expiration fields with the actual kit lot number and expiration date.
 

 
4. Click Basic, then select or enter the following information for the respective fields:

   Case Type: Animal	Samp Filter: 450
   Template: Johnes Ab Vert	Ref. Filter: 0
   Species: Cattle	Dilution: 2
   Technology: ELISA	Wells: 1

   
   
    
   NOTE: The IDEXX Pourquier M. pt. Antibody Test Kit has a negative and a positive control configuration in the insert that is not supported by the xChek software. Therefore, this UDA is set up to have a vertical control configuration (using the template Johnes Ab Vert). When you run the assay, place negative controls in wells A1 and B1 and positive controls in wells C1 and D1.
   
   
5. Click OK to save the settings.
6. Click Calculations, then select or enter the following information for the respective fields:
   

Formulas:    "S" Part of Ratio: Sample1 - Negative    "P" Part of Ratio: Positive - Negative    "N" Part of Ratio: None    Blocking Factor: None
First Calculation:	Second Calculation:
Variable: S/P Positive Cutoff: 0.40 Suspect Cutoff: 0.31 Comparison: >=	Variable: None Positive Cutoff: 0 Suspect Cutoff: 0 Comparison: None

NOTE: The xChek software does not support the value percent (S/P %) calculation. Instead, the xChek software calculates an S/P value. The cutoff values in this UDA have been updated to match the new calculation, and the interpretation is the same.

 


7. Click OK twice.

You can now test for paratuberculosis antibodies in milk using your IDEXX xChek software.

To create the UDA for use with serum and plasma samples:

1. Start the xChek software and log in.
2. From the menu bar, choose Database > Assays > All to open the Assay dialog box.
3. Click New, then enter the following information for the respective fields:
    

   Name: Pourquier Paratuberculosis Screening ELISA-Serum Plasma
   Code: MptsSP-PQ
   Kit Lot: unknown
   

    
   Today's date is automatically entered into the Expiration field.
   NOTE: When you begin testing, update the information in the Kit Lot and Expiration fields with the actual kit lot number and expiration date.
   
    
   
 
4. Click Basic, then select or enter the following information for the respective fields:

   Case Type: Animal	Samp Filter: 450
   Template: Johnes Ab Vert	Ref. Filter: 0
   Species: Cattle	Dilution: 20
   Technology: ELISA	Wells: 1

   
   
    
   NOTE: The IDEXX Pourquier M. pt. Antibody Test Kit has a negative and a positive control configuration in the insert that is not supported by the xChek software. Therefore, this UDA is set up to have a vertical control configuration (using the template Johnes Ab Vert). When you run the assay, place negative controls in wells A1 and B1 and positive controls in wells C1 and D1.
5. Click OK to save the settings.
6. Click Calculations, then select or enter the following information in the respective fields:

   Formulas:    "S" Part of Ratio: Sample1 - Negative    "P" Part of Ratio: Positive - Negative    "N" Part of Ratio: None    Blocking Factor: None
   First Calculation:	Second Calculation:
   Variable: S/P Positive Cutoff: 0.70 Suspect Cutoff: 0.61 Comparison: >=	Variable: None Positive Cutoff: 0 Suspect Cutoff: 0 Comparison: None

   NOTE: The xChek software does not support the value percent (S/P %) calculation. Instead, the xChek software calculates an S/P value. The cutoff values have been updated in this UDA to match the new calculation, and the interpretation is the same.
 

7. Click OK twice.

You can now test for paratuberculosis antibodies in serum and plasma using your IDEXX xChek software.

To specify additional display variables (in addition to optical density) for either UDA:

1. From the menu bar, choose Reports > Analyze Cases to open the Filter Criteria for Analyze dialog box.
2. Enter the desired search criteria, then click OK. The Select Cases for Analyze dialog box opens.
3. Select the desired cases, then click OK. The Analyze Report dialog box opens.
4. Click Variables to open the Display Variables dialog box.
5. Select the desired variables, then click OK.
6. Click OK again to save the settings.

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