General Summary of the 2019 Johne’s Disease Fecal Proficiency Panel Page | 1  

2019 Johne’s Disease Fecal Proficiency Panel

General Summary

October 2, 2019

Overview

A total of 60 laboratories participated in the 2019 Johne’s Disease Fecal Proficiency Panel (7

Canadian, 3 European Union, 1 New Zealand, 2 Australian and 47 USA laboratories). Table 1 details the

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

Laboratories could order multiple panels for each method and were notified of their preliminary

pass/fail status upon submission of their results. A total of 157 panels were requested, with 1 replaced

due to a defective panel. Results were not returned for 5 Individual and 3 Pooled panels. If preliminary

results indicated the laboratory failed, it was given the opportunity to retake the proficiency panel

provided the results were completed by September 30th, 2019. The results provided in Table 1 include

these retests. Laboratories that only used reagents from a single manufacturer, either Tetracore or

Thermo Fisher Sci. (used AM1840 or MagMax CORE for extraction), are listed separately. Laboratories

that use either in‐house reagents or mix commercial reagents from multiple manufacturers are listed

under the “In‐House” category. The laboratory that used in‐house liquid culture reagents is grouped

with the laboratories using the MGIT system. All laboratories using solid media were grouped together

regardless if they purchased media or used in‐house media (1 laboratory).  

Table 1. Summary results of the 2019 Johne’s Disease Fecal Proficiency Panel. 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.

   

2019

# passed 

1st attempt 

(%)

# failed 

1st attempt 

(%)

# passed 

2nd attempt 

(%)

# failed 

2nd attempt 

(%)

# Kits 

not 

retested

Total 

Shipped

Total shipped in 

2018 (%change)

Individual Panel

Direct PCR (all) 56 (90%) 6 (10%) 3 (100%) 3 68 69 (‐1%)

Tetracore 15 (88%) 2 (12%) 1 (100%) 1 18 21 (‐14%)

Thermo Fisher 24 (89%) 3 (11%) 1 (100%) 2 28 26 (+8%)

In‐House 17 (94%) 1 (6%) 1 (100%) 19 20 (‐5%)

Liquid Systems (all) 11 (92%) 1 (8%) 1 13 16 (‐19%)

MGIT 960 2 (67%) 1 (33%) 1 3 4 (‐25%)

TREK 9 (100%) 9 11 (‐18%)

HEY Solid Media (all) 6 (100%) 6 7 (‐14%)

Individual Panel Total 73 (91%) 7 (9%) 3 (100%) 4 87 92 (‐5%)

Pooling Panel

Direct PCR (all) 49 (98%) 1 (2%) 1 (100%) 51 51 (+0%)

Liquid 11 (92%) 1 (8%) 1 12 13 (‐8%)

HEY 3 (100%) 3 4 (‐25%)

Pooled Panel Total 63 (97%) 2 (3%) 1 (100%) 1 66 68 (‐3%)
   

General Summary of the 2019 Johne’s Disease Fecal Proficiency Panel Page | 2  

Individual Panel Description  

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

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

non‐infected herds. When possible, approximately 4 liters of fecal material were collected rectally per

animal, shipped to NVSL, aliquoted in to individual vials, and stored at ‐70°C until kits were distributed.

Because we lacked moderate shedding samples, five samples were produced where material from a

high shedding cow was diluted with material from a culture negative cow and thoroughly mixed. These

samples are 18‐05419A (NE), 18‐05419B (NE), 18‐05419C (NE), 18‐05419D (NE), and 15‐00471D (FL)

reflecting the animal that the positive material originated. Panels were assembled in groups, each with a

different key (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 they correctly classify 70% of the remaining

samples (low and moderate shedding animals). All samples performed as expected except 17‐03492.

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 17‐

03492 (WI) had 33% of all vials correctly classified (direct PCR and culture) and had 69.4% correctly

classified by laboratories using culture methods, making it an invalid animal. Designating it an invalid

animal did not affect the pass/fail result of any laboratory.

Table 2. Composition of the 2019 Johne’s Disease Fecal Proficiency Panel, and the overall categorical summary

results per cow for each method performed by laboratories.  

All Kits HEY TREK MGIT Thermo F. Tetracore In-House

Cow ID 832 6 9 3 28 18 19

18-01897 (IA) 2 Critical- Neg 99% 100% 100% 83% 100% 100% 97%

18-01898 (IA) 2 Critical- Neg 99% 100% 100% 100% 100% 94% 100%

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

17-03492 (WI) 2 Low 33% 58% 78% 67% 21% 19% 29%

16-01645 (IA)3 2 Moderate 99% 100% 100% 100% 96% 100% 100%

18-05419A (NE)4 2 Moderate 99% 100% 100% 100% 96% 100% 100%

18-05419B (NE)4 2 Mod-High 99% 100% 100% 100% 96% 100% 100%

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

18-05419D (NE)4 2 Mod-High 99% 100% 100% 100% 98% 100% 100%

15-00471D (FL)4 2 Critical- High 100% 100% 100% 100% 100% 100% 100%

17-03210 (FL) 2 Critical- High 100% 100% 100% 100% 100% 100% 100%

17-02487 (FL) 2 Critical- High 99% 100% 100% 100% 100% 97% 100%

17-03143 (FL) 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. 
   

General Summary of the 2019 Johne’s Disease Fecal Proficiency Panel Page | 3  

Samples from 6 animals were used in previous years and their performance was compared.

Table 3 shows the respective year panels’ categorical (positive/negative) performance for each

identification method by animal ID. Note that animal 17‐03492(WI) was valid in 2017 and 2018 but was

invalid in 2019.

Table 3. Comparison between six animals used in the 2016, 2017, 2018, and 2019 Johne’s Disease Fecal Proficiency

Panels with the overall categorical summary results per cow for each method performed by laboratories.  

   

All Kits HEY TREK MGIT Thermo F. Tetracore In-House

2016 1021 10 13 3 26 27 18

2017 95 9 12 5 28 23 18

2018 87 7 11 4 26 21 18

Cow ID 2019 83 6 9 3 28 18 19

17-03492 (WI) 2017 2 Low-Mod 44% 72% 92% 30% 25% 39% 39%

17-03492 (WI) 2018 2 Low 33% 71% 73% 75% 21% 17% 20%

17-03492 (WI) 2019 2 Low 33% 58% 78% 67% 21% 19% 29%

16-01645 (IA) 2016 2 Mod-High 99% 100% 97% 100% 98% 100% 97%

16-01645 (IA) 2017 2 Mod-High 99% 100% 92% 100% 100% 100% 100%

16-01645 (IA) 2018 2 Mod-High 99% 100% 100% 100% 96% 100% 100%

16-01645 (IA) 2019 2 Mod-High 99% 100% 100% 100% 96% 100% 100%

15-00471D (FL) 2016 2 High 100% 100% 97% 100% 100% 100% 100%

15-00471D (FL) 2017 2 Critical- High 99% 100% 100% 100% 100% 98% 100%

15-00471D (FL) 2018 2 Critical- High 100% 100% 100% 100% 100% 100% 100%

15-00471D (FL) 2019 2 Critical- High 100% 100% 100% 100% 100% 100% 100%

17-03210 (FL) 2018 2 Critical- High 100% 100% 100% 100% 100% 100% 100%

17-03210 (FL) 2019 2 Critical- High 100% 100% 100% 100% 100% 100% 100%

17-02487 (FL) 2017 2 Critical- High 100% 100% 100% 100% 100% 100% 100%

17-02487 (FL) 2018 3 Critical- High 99% 100% 91% 100% 100% 100% 100%

17-02487 (FL) 2019 2 Critical- High 99% 100% 100% 100% 100% 97% 100%

17-03143 (FL) 2018 2 Critical- High 100% 100% 100% 100% 100% 100% 100%

17-03143 (FL) 2019 2 Critical- High 100% 100% 100% 100% 100% 100% 100%

1

Number of proficiency panels submitted per method.

Percent of Samples Correctly Classified

Liquid Media Direct PCR

Panel 

Year

# Vials 

/Panel

Shedding 

Status
   

General Summary of the 2019 Johne’s Disease Fecal Proficiency Panel Page | 4  

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

Interestingly, the reported values for animal 17‐03143 (FL) differ between methods with the averaged

values most similar to the high‐shedding animals for liquid culture and direct PCR.  However, it is more

similar to a low‐shedding animal using solid culture.  These results are similar to those found in 2018 for

this animal and 2014 and 2016 with animal 12‐00956 (KS), which had a mixed infection of both cattle

and ‘bison’ strains of Mycobacterium avium subsp. paratuberculosis (MAP).  Reviewing whole genome

sequence data from our laboratory indicates animal 17‐03143 (FL) likely also has a mixed infection with

both cattle and ‘bison’ strains of MAP, which is consistent with the results shown in Table 4 since ‘bison’

MAP strains do not grow well, if at all, on HEY media. For those laboratories conducting strain

differentiation assays, the ‘bison’ MAP strain should be the dominant strain recovered from the liquid

culture systems and the cattle strain from the HEY media.  

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

HEY1 TREK MGIT Thermo F. Tetracore In-House

Colonies 

per Tube

Days to 

Positive

Days to 

Positive Ct Ct Ct

Cow ID 6 9 3 28 18 19

17-03492 (WI) Low 0.7 35 47 37.3 37.6 35.6

16-01645 (IA) Moderate 23.3 25 29 31.5 29.5 29.8

18-05419A (NE) Moderate 18.3 24 15 30.7 28.2 28.6

18-05419B (NE) Mod-High 25.8 28 27 29.8 28.4 28.3

18-05419C (NE) Mod-High 36.4 23 17 29.1 27.2 27.5

18-05419D (NE) Mod-High 28.1 22 9 28.7 26.5 26.5

15-00471D (FL) Critical- High 29.4 20 22 26.4 25.4 24.6

17-03210 (FL) Critical- High TNTC 16 17 23.6 22.5 21.9

17-02487 (FL) Critical- High TNTC 17 15 23.1 22.0 21.4

17-03143 (FL) Critical- High 2.5 15 13 21.7 19.7 19.7

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 especially skews the values down for the solid culture of high-shedding animals.
   

General Summary of the 2019 Johne’s Disease Fecal Proficiency Panel Page | 5  

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

that were misclassified (Figure 1) using all 25 samples. In this analysis 67% of laboratories using the TREK

system correctly classified all the samples. For the laboratories using solid media 33% correctly classified

all the samples. The performance of the three direct PCR methods was similar to last year with slightly

more laboratories correctly calling all the samples, but many still had difficulty with samples from animal

17‐03492 (WI). Twenty‐one percent of laboratories using In‐house direct PCR methods correctly

classified all the samples.  

Figure 1. Percentage of 2019 Johne’s disease fecal proficiency panels by number of samples misclassified for the

three culture (TREK liquid media, solid 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 2019 Johne’s Disease Fecal Proficiency Panel Page | 6  

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 misidentify

70% (~3 samples) or fewer of the remaining, valid, non‐critical samples. Table 5 lists the specific 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 2019 Johne’s Disease Fecal Proficiency Panel.  

Because direct PCR is now the most common organism detection assay offered, the

performance of that assay across laboratories becomes more important. Variation in reported cycle

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

reported Ct for the 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. The diluted

samples, 18‐05419A (NE), 18‐05419A (NE), 18‐05419A (NE), 18‐05419A (NE), 15‐00471D (FL), all

performed similar to natural shedding animals.

Figure 2. Average, and 1 standard deviation, reported Ct of 2019 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.

2019 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

1 21 1

Missed 4 or more low / moderate 

shedders (lack of sensitivity) 1

Misclassified a high shedding sample 

as negative

Multiple reasons cited above 1

Total failed kits 2 (11%) 3 (11%) 1 (5%) 0 (0%) 1 (33%) 0 (0%)

Total kits tested 18 28 19 9 3 6
   

General Summary of the 2019 Johne’s Disease Fecal Proficiency Panel Page | 7  

False positive results with either direct fecal PCR or confirmatory culture 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

evenly distributed amongst the three negative animals that were used in this year’s panel when

considering the number of vials included. Interestingly, laboratories using In‐House methods accounted

for 6 of 8 Ct values from animal 18‐01899 (IA). There were a total of 9 laboratories that reported Ct

values on at least one negative sample. Of those 9 laboratories, 5 failed the PT (see Table 5) by calling a

negative sample positive and is a reduction from last year’s panel (12 of 15 failed).  Although fewer

laboratories reported Ct values for negative samples, it continues to be an issue. Almost half the

laboratories reporting Ct values called them negative.

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  

Twenty five 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 of panels. To pass, laboratories were required to

correctly classify the negative pool and the two pools that contained a high‐shedding animal (17-03210 

& 17-02487). Laboratories were allowed to pass even if they misclassified one of the other pools (17‐

04003 or 17‐04070).

Table 7. Composition of the 2019 Johne’s Disease Fecal Pooling

Proficiency Panel.

Tetracore Thermo F. In-House Total

18-01897 (IA) 1 1

18-01898 (IA) 2 1 3

18-01899 (IA) 3 6 9

Num. panels 

reporting Ct 2379

Cow ID

Avg. 

CFU/ tube*

1 High, 4 Negative samples 17-03210 ~2500

1 High, 4 Negative samples 17-02487 ~1000

1 Moderate, 4 Negative samples 17-04003 30

1 Moderate, 4 Negative samples 18-05419A 18

5 Negative samples

Positive sample(s) 

description

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

General Summary of the 2019 Johne’s Disease Fecal Proficiency Panel Page | 8  

Table 8 describes the performance of each method used to test the pool. It is commendable that

all laboratories using solid culture passed.  All but one laboratory passed the pooled panel using direct

PCR and liquid culture methods.  Three laboratories using liquid culture misclassified the negative pool

and another misclassified multiple pools.  

Table 9. Performance of each method used in the Johne’s Disease 2018 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 2019 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 1

Identi fied a high ‐shedding pool as negative 1

Two non‐critical pools were identi fied as negative

Failed due to multiple criteria

One non‐critical pool was misidenti fied as negative

All 5 pools were identi fied correctly 49 11 3

Total Failed Pooled Kits 1 (2%) 1 (8%) 0 (0%)

Total 50 12 3

No. panels

Panels

that

failed

Panels

that

passed

2019
   

General Summary of the 2019 Johne’s Disease Fecal Proficiency Panel Page | 9  

Appendix 1. 2019 Johne’s Disease Individual Fecal Proficiency Panel key by kit number. Samples are coded

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

shedding samples. Sample 26 was the positive control.

Appendix 2. 2019 Johne’s Disease Pooled Fecal Proficiency Panel key by kit number.

Vial # 1-25 26-50 51-75 76-100 100-105

1 18‐05419A (NE) 18‐05419D (NE) 18‐05419B (NE) 18‐05419C (NE) 18‐05419A (NE)

2 18‐05419B (NE) 17‐02487 (FL) 17‐03210 (FL) 18‐05419D (NE) 18‐05419C (NE)

3 15‐00471D (FL) 17‐03786 (IA) 17‐03787 (IA) 18‐05419D (NE) 17‐02487 (FL)

4 17‐03787 (IA) 17‐04259 (IA) 18‐05419A (NE) 17‐03143 (FL) 17‐03786 (IA)

5 17‐03143 (FL) 17‐03210 (FL) 17‐02487 (FL) 17‐04259 (IA) 17‐03787 (IA)

6 17‐03492 (WI) 17‐03787 (IA) 17‐03786 (IA) 16‐01645 (IA) 17-03143 (FL)

7 18‐05419B (NE) 15‐00471D (FL) 17‐03210 (FL) 18‐05419B (NE) 17‐03787 (IA)

8 18‐05419A (NE) 17‐03787 (IA) 17‐04259 (IA) 17‐02487 (FL) 17‐03210 (FL)

9 18‐05419D (NE) 17‐03210 (FL) 17‐03143 (FL) 17‐03786 (IA) 17‐03492 (WI)

10 17‐02487 (FL) 18-05419B (NE) 16‐01645 (IA) 17‐03492 (WI) 18‐05419D (NE)

11 17‐04259 (IA) 16‐01645 (IA) 18‐05419C (NE) 15‐00471D (FL) 17‐03143 (FL)

12 18-05419C (NE) 17‐03143 (FL) 15‐00471D (FL) 17‐03787 (IA) 18‐05419B (NE)

13 17‐03210 (FL) 17‐03492 (WI) 17‐03786 (IA) 17‐03210 (FL) 16‐01645 (IA)

14 17‐03786 (IA) 18‐05419D (NE) 17‐04259 (IA) 17-04259 (IA) 18‐05419A (NE)

15 15‐00471D (FL) 18‐05419A (NE) 17‐03492 (WI) 17‐03143 (FL) 18‐05419D (NE)

16 17‐04259 (IA) 18‐05419B (NE) 15‐00471D (FL) 17‐04259 (IA) 15‐00471D (FL)

17 18‐05419D (NE) 17‐03143 (FL) 17-04259 (IA) 18‐05419A (NE) 17‐04259 (IA)

18 16‐01645 (IA) 17‐03492 (WI) 18‐05419D (NE) 17‐02487 (FL) 17‐03210 (FL)

19 17‐02487 (FL) 18‐05419C (NE) 18‐05419A (NE) 17‐03786 (IA) 17‐03492 (WI)

20 17‐03786 (IA) 15‐00471D (FL) 17‐02487 (FL) 17‐03210 (FL) 18‐05419B (NE)

21 17‐04259 (IA) 17‐04259 (IA) 17‐03492 (WI) 17‐03787 (IA) 17‐02487 (FL)

22 17‐03143 (FL) 18‐05419A (NE) 18‐05419D (NE) 18‐05419B (NE) 17‐03786 (IA)

23 17‐03787 (IA) 17‐02487 (FL) 18‐05419B (NE) 15‐00471D (FL) 17‐04259 (IA)

24 17‐03210 (FL) 17‐03786 (IA) 17‐03143 (FL) 17‐03492 (WI) 15‐00471D (FL)

25 17‐03492 (WI) 17‐04259 (IA) 17‐03787 (IA) 18‐05419A (NE) 17‐04259 (IA)

26 16‐01645 (IA) 16‐01645 (IA) 16‐01645 (IA) 16‐01645 (IA) 16‐01645 (IA)

Pool Description

Kit#     

1‐20

Kit#     

21‐40

Kit#     

41‐60

Kit#     

61‐70

5 Negative samples 5 2 4 1

1 mod (18-05419A), 4 Negative samples 4 3 1 5

1 mod-high (17-04003), 4 Negative samples 2 4 3 2

1 high (17-02487), 4 Negative samples 1 5 2 3

1 high (17-03210), 4 Negative samples 3 1 5 4

Pool Sample Number
   

General Summary of the 2019 Johne’s Disease Fecal Proficiency Panel Page | 10  

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

515.337.7388.  

Report was prepared by:

Kevin D. Stokes, PhD

USDA/APHIS/DB/NVSL

Mycobacteria & Brucella Section

Kevin.D.Stokes@USDA.GOV