General Summary of the 2022 Johne’s Disease Fecal Proficiency Test Page | 1  

2022 Johne’s Disease Fecal Proficiency Test

General Summary

October 31, 2022

Overview

A total of 53 laboratories ordered panels during the 2022 Johne’s Disease Fecal Proficiency Test (6

Canadian, 3 European Union, 1 Australian, 1 Japanese and 42 USA laboratories). Table 1 details the

number of individual and pooled panels shipped and the overall pass/fail status for each method. A total

of 134 panels were requested.  None of the individual or pooled panels were reported to be incomplete

or missing individual samples.  Results were not returned for 5 individual and 4 pooled panels. Upon

receipt of results, labs were notified of their preliminary pass/fail status. If preliminary results indicated

the laboratory failed, the laboratory was given the opportunity to retake the proficiency panel provided

the results were submitted by September 30th, 2022.  The results provided in Table 1 include the retests.

Laboratories that used reagents for DNA isolation and PCR from a single manufacturer are listed by

manufacturer. Laboratories that use either in‐house reagents or mixed commercial reagents for DNA

isolation and PCR are listed under the “In‐House” category. Proficiency panels used for liquid culture are

divided into two categories TREK and Other (in‐house made liquid media or other commercially

available liquid culture systems). Laboratories that purchased solid media or made their own solid media

for Johne’s culture are grouped together as “HEY Solid Media.” All samples were considered valid

because “... at least 70 percent of the laboratories participating in the fecal culture ...” reported the

same result.   

Table 1. Summary results of the 2022 Johne’s Disease Fecal Proficiency Test. In order to pass, results must meet the

criteria listed in the 2010 Uniform Program Standards for the Voluntary Bovine Johne's Disease Control Program.
   

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Individual Panel Description  

Each individual panel consisted of 25 blinded samples and 1 positive control. Positive samples were

collected from naturally infected cows and negative samples were from individual animals residing in

uninfected herds. When possible, approximately 4 liters of fecal material was collected rectally per

animal, shipped to the National Veterinary Services Laboratories (NVSL), aliquoted into individual vials,

and stored at ‐70°C until panels were distributed. Fecal material from moderate shedding animals could

not be obtained for use.  To approximate moderate shedding animals, three samples were produced by

diluting feces from a high shedding cow with material from a culture negative cow. These samples are

18‐05419A (NE), 18‐05419C (NE), and 18‐05419D (NE). The name reflects the sample ID of the positive

material used. Panels were assembled in lots of 20 with each lot having a different order of samples.

(See Appendix 1 at the end of this report for the key). Table 2 shows the categorical (positive/negative)

performance for each identification method by animal ID.  According to the 2010 Uniform Program

Standards, a laboratory receives a passing score when: all samples from non‐shedding and high‐

shedding animals are correctly classified; and 70% of the remaining samples (low and moderate

shedding animals) are correctly classified. All samples performed as expected.  

Table 2. Composition of the 2022 Johne’s Disease Fecal Proficiency Panels, and the overall categorical summary

results per cow for each method performed by laboratories.
   

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Samples from 13 animals were also used in the previous year’s panel.  Their performance is compared in

Table 3 showing the respective year panels’ performance for each identification method.  These samples

performed similarly between years.

Table 3. Comparison of animals used in the 2021 and 2022 Johne’s Disease Fecal Proficiency Panels with the

overall results for each method performed by laboratories.

Table 4 shows the average values reported for each of the testing methods summarized by animal. It is

interesting to note that the Tetracore method of PCR showed a 5 Ct value difference on average for the

sample 20‐08637 (NE) compared to the Thermo Fisher or In‐House methods otherwise performed

comparably for the rest of the samples.  This is a similar result to last year’s testing on this sample.
   

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Table 4.  A comparison of the averaged result values among the three methods for shedding animals.  Total panel

numbers for each method are listed below the method title.

The performance of each method was further evaluated by determining the number of samples that

were misclassified (Figure 1). The TREK system sample classification declined since last year, decreasing

13%.  The other liquid culture systems were not used last year but were 100% in 2020. Laboratories

using solid media correctly classified 100% of the samples, a large increase over last year (67%). The

performance of all the direct PCR methods remained constant compared to last year.  

Figure 1. Percentage of 2022 Johne’s disease fecal proficiency panels by number of samples misclassified for the

three culture (solid media, TREK liquid media, and other liquid media) and three direct PCR (Tetracore, Thermo

Fisher and In‐House) methods. A panel consisted of 25 fecal samples.
   

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According to the 2010 Johne’s Disease Uniform Program Standards, laboratories must correctly classify

all critical‐high shedding samples as positive, all negative samples as negative and correctly identify 70%

or more of the remaining, valid, non‐critical samples (can miss ~3 samples). Table 5 lists the reasons

laboratories failed to pass the proficiency panel for each method.  As in previous years the most

common reason for failure is misclassifying a negative sample as positive.  Multiple laboratories are

represented in this table.

Table 5. Reasons laboratories failed the 2022 Johne’s Disease Fecal Proficiency Panel.  

Because direct PCR is the most common detection method used, the performance of that assay across

laboratories is critical to consistent application of the program across the US. Variation in reported cycle

threshold (Ct) of the direct PCR methods was investigated (see Figure 2) by comparing the average

reported Ct for positive samples. Ct values from each panel were used in this comparison and include

samples categorized as negative, but that had Ct scores reported (e.g. negative, but a Ct of 39.9). The

overall means of all three methods for each animal were statistically similar.

Figure 2. The average and 1 standard deviation from reported Ct values were plotted for the three direct PCR

methods (Thermo Fisher, Tetracore, and In‐house). Shedding status is listed below the animal ID. Animal numbers

ending in letters “18‐05419A (NE)” are diluted samples.
   

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False positive results continue to be the most common cause of failure. Table 6 examines the number of

negative samples reported with Ct values by PCR method; this includes laboratories that reported Ct

values and correctly classified them as negative. Errors were generally distributed amongst the negative

animals that were used in this year’s panel when considering the number of vials included. There was a

total of 4 laboratories that reported Ct values on at least one negative sample, an increase from last

year. Of those 4 laboratories, 3 failed the proficiency test (see Table 5) by calling a negative sample

positive and is an increase from last year’s proficiency testing. False positive results by PCR can be

caused by a number of factors such as cross‐contamination within the lab, problems with primer/probe

design, etc. The data collected and reported here are not sufficient to determine the cause of the false

negative results.

Table 6. The number of samples from non‐infected cows reported with Ct values

(regardless of their categorical positive/negative results) by direct PCR method.  

Pooling Panel Description  

For the pooled panels, 25 individual samples were provided with instructions regarding which 5 samples

to pool together, for a total of 5 pooled samples. Table 7 lists the contents of each pool, and Appendix 2

lists the pool numbers associated with each lot. To pass, laboratories were required to correctly classify

the negative pools and the two pools that contained a high‐shedding animal. Laboratories were allowed

to pass even if they misclassified the other pool.

Table 7. Composition of the 2022 Johne’s Disease Fecal Pooling Proficiency Panel.
   

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Table 8 describes the performance of each method used to test the pooled samples. All but 1 laboratory

using solid and liquid culture passed.  Though 2 pooled direct PCR panels are reported as failed due to

calling negative pools positive, all but 1 laboratory passed the pooled panel using direct PCR.  

Table 8. Performance of each method used in the Johne’s Disease 2022 Fecal Pooling Proficiency Panel.

A total of 5 pooled samples were in each panel.  

A current listing of all the approved laboratories is available in

the NVLS web site:

https://www.aphis.usda.gov/aphis/ourfocus/animalhealth/lab‐

info‐services/sa_approved_labs/ct_approved_labs.  

Remaining sample vials from the 2022 Proficiency Panel are

available to laboratories for validation or research purposes.

Available samples can be viewed in the reagents catalog under

Johne’s positive/negative fecal samples on the NVSL web site

Reagent Catalog at

https://www.aphis.usda.gov/aphis/ourfocus/animalhealth/lab‐

info‐services/sa_reagents/ct_reagents  

   
   

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Appendix 1. 2022 Johne’s Disease Individual Fecal Proficiency Panel key by panel number. Samples are

coded by color according to shedding status as follows: Critical ‐ Negative samples, Non‐critical positive

samples, Critical – high shedding samples. Sample 26 was the positive control.

Appendix 2. 2022 Johne’s Disease Pooled Fecal Proficiency Panel key by panel number.

Any questions or comments can be directed to the Diagnostic Bacteriology and Pathology Laboratory at

515.337.7388.  

Report was prepared by:

Kimberly A. Lehman, DVM MPH DACVPM

USDA/APHIS/DB/NVSL/DBPL

Mycobacteria & Brucella Section

Kimberly.Lehman@USDA.GOV