General Summary of the 2020 Johne’s Disease Fecal Proficiency Test Page | 1  

2020 Johne’s Disease Fecal Proficiency Test

General Summary

November 2, 2020

Overview

A total of 60 laboratories ordered panels during the 2020 Johne’s Disease Fecal Proficiency Test

(7 Canadian, 3 European Union, 1 New Zealand, 1 Australian and 48 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 with 2 pooled panels reported to be incomplete but did not need

replacements. Results were not returned for 6 Individual panels and 1 Pooled panel. 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, 2020. The results provided in Table 1 include these retests.

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. The laboratory that used in‐house liquid

culture reagents to test for Johne’s is grouped with the laboratory using the MGIT system. Laboratories

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

Table 1. Summary results of the 2020 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.

   

2020

# passed 

1st attempt 

(%)

# failed 

1st attempt 

(%)

# passed 

2nd attempt 

(%)

# failed 

2nd attempt 

(%)

# Panels 

not 

retested

Total 

Shipped

Total shipped in 

2019 (%change)

Individual Panel

Direct PCR (all) 52 (96%) 2 (4%) 2 (100%) 63 68 (‐7%)

Tetracore 16 (94%) 1 (6%) 1 (100%) 17 18 (‐6%)

Thermo Fisher 27 (96%) 1 (4%) 1 (100%) 28 28 (+0%)

In‐House 18 (100%) 16 19 (‐16%)

Liquid Systems (all) 11 (100%) 12 13 (‐8%)

MGIT 960 2 (100%) 2 3 (‐33%)

TREK 9 (100%) 9 9 (+0%)

HEY Solid Media (all) 5 (100%) 6 6 (+0%)

Individual Panel Total 68 (97%) 2 (3%) 2 (100%) 81 87 (‐7%)

Pooling Panel

Direct PCR (all) 47 (98%) 1 (2%) 1 48 51 (‐6%)

Liquid 11 (100%) 11 12 (‐8%)

HEY 3 (100%) 3 3 (+0%)

Pooled Panel Total 61 (98%) 1 (2%) 1 62 66 (‐14%)
   

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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 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 20‐00152 (WI). The

2010 Uniform Program Standards states that an animal is considered valid “by a consensus of at least 70

percent of the laboratories participating in the fecal culture check testing process.”  Animal 20‐00152

(WI) had 68% of all vials correctly classified (direct PCR and all culture methods) and had 56.3% correctly

classified by laboratories using culture methods, making it invalid. Designating the samples from Animal

20‐00152 (WI) invalid did not affect the pass/fail result of any laboratory.

Table 2. Composition of the 2020 Johne’s Disease Fecal Proficiency Panels, and the overall categorical summary

results per cow for each method performed by laboratories.  

HEY TREK MGIT Thermo F. Tetracore In-House

Cow ID 792 5 9 2 28 17 18

18-01899 (IA) 1 Critical- Neg 100% 100% 100% 100% 100% 100% 100%

18-01900 (IA) 2 Critical- Neg 99% 100% 100% 100% 96% 100% 100%

18-01901 (IA) 2 Critical- Neg 100% 100% 100% 100% 100% 100% 100%

19-00486 (IA) 1 Critical- Neg 99% 100% 100% 100% 100% 94% 100%

20-00152 (WI) 2 Low 68% 40% 56% 100% 63% 76% 81%

20-00154 (WI) 2 Low-Mod 98% 100% 94% 100% 100% 100% 94%

20-00153 (WI)3 2 Low-Mod 100% 100% 100% 100% 100% 100% 100%

18-05419B (NE)4 2 Mod-High 100% 100% 100% 100% 100% 100% 100%

18-05419C (NE)4 1 Mod-High 100% 100% 100% 100% 100% 100% 100%

18-05419A (NE)4 1 Mod-High 100% 100% 100% 100% 100% 100% 100%

18-05419D (NE)4 2 Mod-High 99% 100% 94% 100% 100% 100% 100%

18-06468 (NE) 2 Critical- High 100% 100% 100% 100% 100% 100% 100%

18-05422 (NE) 2 Critical- High 99% 100% 100% 100% 100% 94% 100%

12-03432 (ND) 2 Critical- High 100% 100% 100% 100% 100% 100% 100%

18-05419 (NE) 2 Critical- High 100% 100% 100% 100% 100% 100% 100%

2

Number of proficiency panels submitted per method.

3

The positive control was one of the two from this animal.

4 Positive sample diluted with negative material.

Percent of Samples Correctly Classified

# Vials 

/Panel

Shedding 

Status1

Liquid Media Direct PCR

1

In order to pass, laboratories must correctly classify critical samples. A critical sample is any negative sample or 

a sample that is identified as a heavy shedder by more than 50% of the laboratories using solid media. 

All 

Panels
   

General Summary of the 2020 Johne’s Disease Fecal Proficiency Test Page | 3  

Samples from 5 animals were also used in previous years.  Their performance is compared in Table 3

showing the respective year panels’ performance for each identification method.  

Table 3. Comparison of animals used in the 2019 and 2020 Johne’s Disease Fecal Proficiency Panels with the

overall results for each method performed by laboratories.  

Table 4 shows the averaged values reported for each of the testing methods summarized by animal.  

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

HEY TREK MGIT Thermo F. Tetracore In-House

2019 831 6 9 3 28 18 19

Cow ID 2020 79 5 9 2 28 17 18

18-01899 (IA) 2019 3 Critical- Neg 98% 100% 100% 100% 96% 100% 98%

18-01899 (IA) 2020 1 Critical- Neg 100% 100% 100% 100% 100% 100% 100%

18-05419A (NE) 2019 2 Moderate 99% 100% 100% 100% 96% 100% 100%

18-05419A (NE) 2020 1 Mod-High 100% 100% 100% 100% 100% 100% 100%

18-05419B (NE) 2019 2 Mod-High 99% 100% 100% 100% 96% 100% 100%

18-05419B (NE) 2020 2 Mod-High 100% 100% 100% 100% 100% 100% 100%

18-05419C (NE) 2019 1 Mod-High 100% 100% 100% 100% 100% 100% 100%

18-05419C (NE) 2020 1 Mod-High 99% 100% 94% 100% 100% 100% 100%

18-05419D (NE) 2019 2 Mod-High 99% 100% 100% 100% 98% 100% 100%

18-05419D (NE) 2020 2 Critical- High 100% 100% 100% 100% 100% 100% 100%

1

Number of proficiency panels submitted per method.

Panel 

Year

# Vials 

/Panel

Shedding 

Status

Percent of Samples Correctly Classified

Liquid Media Direct PCR All 

Panels

HEY1 TREK MGIT Thermo F. Tetracore In-House

Colonies 

per Tube

Days to 

Positive

Days to 

Positive Ct Ct Ct

Cow ID 5 9 2 28 17 18

20-00152 (WI) Low 1.3 27 16 35.8 34.2 34.4

20-00154 (WI) Low-Mod 16.9 28 22 29.8 28.6 29.4

18-05419B (NE) Low-Mod 24.9 23 24 30.0 28.4 29.2

18-05419C (NE) Mod-High 37.0 26 15 29.3 28.1 28.1

18-05419D (NE) Mod-High 44.1 25 18 28.8 27.0 28.1

20-00153 (WI) Mod-High 17.8 20 14 28.3 27.9 27.2

18-05419A (NE) Mod-High 17.5 20 12 28.1 26.2 27.5

18-05422 (NE) Critical- High 26.0 18 12 26.0 24.6 25.4

18-06468 (NE) Critical- High 40.0 16 11 24.6 23.2 23.5

12-03432 (ND) Critical- High 7.5 18 12 23.5 22.0 22.7

18-05419 (NE) Critical- High TNTC 16 11 22.6 20.7 21.7

Average Result Values for Shedding Animals

Liquid Media Direct PCR

Shedding 

Status

1

Results shown include reported values only. Reports that do not include Ct values for direct PCR, days-topositive for Liquid culture, colonies per tube or list Too-Numerous-To-Count (TNTC) for solid culure are not 

included; this skews the values down for the solid culture of high-shedding animals.
   

General Summary of the 2020 Johne’s Disease Fecal Proficiency Test Page | 4  

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

were misclassified (Figure 1). Laboratories using the TREK system correctly classified 56% of the samples;

whereas, 100% of laboratories using the MGIT system correctly classified all the samples. Though only 2

labs were included in the MGIT data, this is the first time in over 10 years that the MGIT outperformed

the TREK system.  Laboratories using solid media correctly classified 20% of the samples, a decrease

from last year. The performance of the three direct PCR methods improved compared to last year with

more laboratories correctly calling all samples. Sixty‐seven percent of laboratories using In‐house direct

PCR methods correctly classified all the samples compared to 21% who correctly called all the samples

last year.  

Figure 1. Percentage of 2020 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.
   

General Summary of the 2020 Johne’s Disease Fecal Proficiency Test Page | 5  

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 5. Reasons laboratories failed the 2020 Johne’s Disease Fecal Proficiency Panel.  

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.  

Figure 2. Average, and 1 standard deviation, reported Ct of 2020 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.

2020 Direct PCR 

(Tetracore)

Direct PCR 

(Thermo F.)

Direct PCR 

(In-House)

TREK 

liquid 

media

MGIT 

liquid 

media

HEY 

solid 

media

Misclassified a negative sample as 

positive

2

Missed 4 or more low / moderate 

shedders (lack of sensitivity)

Misclassified a high shedding sample 

as negative

Multiple reasons cited above 1

Total failed panels 1 (6%) 2 (7%) 0 (0%) 0 (0%) 0 (0%) 0 (0%)

Total panels tested 17 28 18 9 2 5
   

General Summary of the 2020 Johne’s Disease Fecal Proficiency Test Page | 6  

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 9 laboratories that reported Ct values on at least one negative sample. Of those 9

laboratories, only 2 failed the PT (see Table 5) by calling a negative sample positive and is a reduction

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 both negative pools and the two pools that contained a high‐shedding animal (18-

05419). Laboratories were allowed to pass even if they misclassified the other pool (17‐12249 & 17‐

01709).

Table 7. Composition of the 2020 Johne’s Disease Fecal Pooling

Proficiency Panel.

Tetracore Thermo F. In-House Total

18-01899 (IA) 1 1

18-01900 (IA) 2 2 1 5

18-01901 (IA) 1 1

19-00486 (IA) 2 2

Num. panels 

reporting Ct 5229

Cow ID

Avg. 

CFU/ tube*

1 High, 4 Negative samples 18-05419 (NE) ~4500

1 High, 4 Negative samples 18-05419 (NE) ~4500

1 Mod, 1 Low, 3 Negative samples 17-12249 (WI) 50.5

17-01709 (IA) 2

5 Negative samples

5 Negative samples

Positive sample(s) 

description

*Refers to the positive samples, not the pooled sample.
   

General Summary of the 2020 Johne’s Disease Fecal Proficiency Test Page | 7  

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 2020 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 2020 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  

   

Direct

PCR

Liquid

media

Solid

media

Identi fied the negative pool as positive

Identi fied a high ‐shedding pool as negative

Two non‐critical pools were identi fied as negative

Failed due to multiple criteria 1

One non‐critical pool was misidenti fied as negative 1

All 5 pools were identi fied correctly 45 12 3

Total Failed Pooled Panels 1 (2%) 0 (0%) 0 (0%)

Total 47 12 3

No. panels

Panels

that

failed

Panels

that

passed

2020
   

General Summary of the 2020 Johne’s Disease Fecal Proficiency Test Page | 8  

Appendix 1. 2020 Johne’s Disease Individual Fecal Proficiency Panel key by panel number. Samples are

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

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

Appendix 2. 2020 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

Mycobacteria & Brucella Section

Kimberly.Lehman@USDA.GOV

Vial # 1-20 21-40 41-60 61-80 81-100

1 20‐00152 (WI) 18‐05419B (NE) 18‐05422 (NE) 20‐00154 (WI) 12‐03432 (ND)

2 18‐01900 (IA) 18‐05419 (NE) 18‐01901 (IA) 18‐05419C (NE) 18‐01899 (IA)

3 20‐00153 (WI) 18‐01900 (IA) 18‐05419B (NE) 18‐01901 (IA) 18‐05419 (NE)

4 18‐05419C (NE) 20‐00154 (WI) 20‐00152 (WI) 12‐03432 (ND) 18‐01900 (IA)

5 18‐05419 (NE) 18‐05419D (NE) 12‐03432 (ND) 18‐01899 (IA) 20‐00154 (WI)

6 18‐01900 (IA) 18‐05422 (NE) 18‐01899 (IA) 18‐05419B (NE) 18-05422 (NE)

7 12‐03432 (ND) 18‐01901 (IA) 18‐06468 (NE) 18‐05419 (NE) 20‐00152 (WI)

8 18‐01899 (IA) 18‐05419B (NE) 19‐00486 (IA) 18‐01900 (IA) 18‐06468 (NE)

9 18‐05419D (NE) 12‐03432 (ND) 20‐00154 (WI) 18‐05422 (NE) 19‐00486 (IA)

10 18‐06468 (NE) 18-01899 (IA) 18‐05419 (NE) 20‐00152 (WI) 18‐01900 (IA)

11 19‐00486 (IA) 20‐00153 (WI) 18‐01900 (IA) 18‐06468 (NE) 18‐05419B (NE)

12 18-05419A (NE) 18‐06468 (NE) 20‐00154 (WI) 18‐05419D (NE) 18‐01901 (IA)

13 18‐05419 (NE) 18‐05419C (NE) 18‐05422 (NE) 12‐03432 (ND) 18‐05419D (NE)

14 18‐05419B (NE) 18‐01901 (IA) 18‐05419D (NE) 20-00152 (WI) 20‐00154 (WI)

15 18‐01901 (IA) 12‐03432 (ND) 18‐01900 (IA) 18‐05419A (NE) 18‐05419 (NE)

16 18‐05419D (NE) 20‐00152 (WI) 18‐05419A (NE) 18‐06468 (NE) 20‐00153 (WI)

17 20‐00152 (WI) 18‐05419A (NE) 20-00153 (WI) 19‐00486 (IA) 18‐05419A (NE)

18 12‐03432 (ND) 18‐01900 (IA) 18‐05419 (NE) 18‐05419B (NE) 12‐03432 (ND)

19 20‐00154 (WI) 18‐05422 (NE) 18‐05419C (NE) 18‐05422 (NE) 18‐05419D (NE)

20 18‐05419B (NE) 18‐05419D (NE) 20‐00152 (WI) 18‐01901 (IA) 18‐05419C (NE)

21 18‐05422 (NE) 20‐00154 (WI) 18‐06468 (NE) 18‐01900 (IA) 20‐00152 (WI)

22 18‐01901 (IA) 18‐06468 (NE) 18‐05419B (NE) 20‐00153 (WI) 18‐05422 (NE)

23 18‐06468 (NE) 19‐00486 (IA) 18‐05419D (NE) 18‐05419 (NE) 18‐01901 (IA)

24 20‐00154 (WI) 18‐05419 (NE) 18‐01901 (IA) 18‐05419D (NE) 18‐06468 (NE)

25 18‐05422 (NE) 20‐00152 (WI) 12‐03432 (ND) 20‐00154 (WI) 18‐05419B (NE)

26 20‐00153 (WI) 20‐00153 (WI) 20‐00153 (WI) 20‐00153 (WI) 20‐00153 (WI)

Pool Description

Panel   

#1‐20

Panel   

#21‐40

Panel   

#41‐60

Panel   

#61‐70

5 Negative samples 2431

5 Negative samples 4243

1 Mod (17-12249), 1 Low (17-01709), 3 

Negative samples 1524

1 high (18-05419), 4 Negative samples 3152

1 high (18-05419), 4 Negative samples 5315

Sample Pool Number