General Summary of the 2021 Johne’s Disease Fecal Proficiency Test Page | 1  

2021 Johne’s Disease Fecal Proficiency Test

General Summary

October 21, 2021

Overview

A total of 56 laboratories ordered panels during the 2021 Johne’s Disease Fecal Proficiency Test (7

Canadian, 3 European Union, 1 Australian and 45 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 143

panels were requested.  One individual panel was lost in shipping and replaced.  7 individual panels and

2 pooled panels were reported to be incomplete, missing individual samples were sent to make the

panels complete. Results were not returned for 5 individual and 3 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, 2021. The results provided in Table 1 include these retests.

One sample, 20‐08637 (NE), was included in the individual panels as a critical positive from initial culture

and PCR testing data; however direct PCR results, submitted by participating laboratories showed a

larger spread and higher Ct values than initial testing. According to testing procedures, a valid

proficiency panel sample will be determined “by a consensus of at least 70 percent of the laboratories

participating in the fecal culture check testing process.” As culture testing did not identify a validity issue

with the sample, but because the reported PCR data had such high variation of Ct values, it was decided

to re‐classify the sample from a critical positive to a moderate positive classification instead of removing

it from the grading scheme.  The results provided in Table 1A include test results with removal of retests

from panels that would have passed if this sample was originally classified as a moderate positive

sample.  Laboratories that only used reagents for DNA isolation and PCR from a single manufacturer are

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

isolation and PCR are listed under the “In‐House” category. Laboratories that purchased solid media or

made their own solid media for Johne’s culture are grouped together.  

Table 1. Summary results of the 2021 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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Table 1A. Summary results of the 2021 individual Johne’s Disease Fecal Proficiency Test panels with sample

reclassification.

*-By reclassifying sample 20-08637 (NE), 6 panels passed that would have failed originally. Retests from 

these laboratories have been excluded from these calculations 

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 panel sample use.  To approximate moderate shedding animals, four samples were

produced by diluting feces from a high shedding cow with material from a culture negative cow. These

samples are 18‐05419A (NE), 18‐05419B (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 except 20‐

08637 (NE) as previously described. Reclassifying the sample from animal 20‐08637 (NE) allowed 6

panels to pass that would have failed with the original classification and allowed 1 laboratory to pass the

proficiency test.
   

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Table 2. Composition of the 2021 Johne’s Disease Fecal Proficiency Panels, and the overall categorical summary

results per cow for each method performed by laboratories.

Samples from 10 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.
   

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Table 3. Comparison of animals used in the 2020 and 2021 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 5‐6 Ct value difference on average for

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

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

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

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

from 56% to 88%.  Laboratories using solid media correctly classified 67% of the samples, a significant

increase over last year (20%). The performance of the Thermo Fisher and In‐house direct PCR methods

improved compared to last year with more laboratories correctly calling all samples, but the

performance of the Tetracore method decreased by 3%.  

Figure 1. Percentage of 2021 Johne’s disease fecal proficiency panels by number of samples misclassified for the

three culture (solid media, TREK liquid media, and MGIT 960 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.  Table 5A lists the reasons laboratories

failed to pass the proficiency panel for each method with sample 20‐08637 (NE) reclassified.

Table 5. Reasons laboratories failed the 2021 Johne’s Disease Fecal Proficiency Panel.  

Table 5A. Reasons laboratories failed the 2021 Johne’s Disease Fecal Proficiency Panel with

sample reclassification.  

Because direct PCR is now the most common organism detection assay used, the performance of that

assay across laboratories becomes more important. 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. Only valid Ct values from each panel were used in this comparison and include samples

categorized as negative, but that had valid 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, except for animal 20‐08637

(NE).  
   

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Figure 2. Average, and 1 standard deviation, reported Ct of 2021 Johne’s disease fecal proficiency panel animals

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.

False positive results with PCR continues 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. Also shown are the number of panels

where at least one Ct is reported. 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 3

laboratories that reported Ct values on at least one negative sample, a decrease from last year. Of those

3 laboratories, 2 failed the PT (see Table 5) by calling a negative sample positive and is the same from

last year’s proficiency testing results.  Although fewer laboratories reported Ct values for negative

samples, it continues to be an issue. 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
   

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the negative pool and the two pools that contained a high‐shedding animal (18-06468). Laboratories

were allowed to pass even if they misclassified one of the other pools.

Table 7. Composition of the 2021 Johne’s Disease Fecal Pooling Proficiency Panel.

Table 8 describes the performance of each method used to test the pooled samples. It is commendable

that all laboratories using solid and liquid culture passed.  All but one laboratory passed the pooled

panel using direct PCR.  

Table 8. Performance of each method used in the Johne’s Disease 2021 Fecal Pooling Proficiency Panel.

A total of 5 pooled samples were in each panel.  
   

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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 2021 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. 2021 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. 2021 Johne’s Disease Pooled Fecal Proficiency Panel key by kit 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

USDA/APHIS/DB/NVSL/DBPL

Mycobacteria & Brucella Section

Kimberly.Lehman@USDA.GOV