General Summary of the 2011 Johne’s Disease Fecal Proficiency Panel Page | 1  

2011 Johne’s Disease Fecal Proficiency Panel    

General Summary

October 18, 2011

Overview

A total of 62 laboratories participated in the 2011 Johne’s Disease Fecal Proficiency Panel (6

Canadian, 3 European Union, 1 New Zealand and 52 USA laboratories). Overall, the number of

laboratories requesting proficiency panels for direct PCR and pooled testing slightly increased from 2010

and held steady or decreased for liquid and solid media culture. 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. If preliminary results indicated that the laboratory had failed, they were given the

opportunity to retake the proficiency panel provided the results could be completed by September 30th,

2011.  The results provided in Table 1 include these retests.  

Table 1. Summary results of the 2011 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.

# passed 

1st attempt 

(%)

# failed 

1st attempt 

(%)

# passed 

2nd attempt 

(%)

# failed 

2nd attempt 

(%)

# labs 

not 

retesting

Total 

Shipped

Total shipped in 

2010 (%change)

Individual Panel

Direct PCR (all) 40 (75%) 13 (25%) 8 (80%) 2 (20%) 3 63 58 (+9%)

Tetracore 17 (68%) 8 (32%) 5(83%) 1 (17%) 2 31 29 (+7%)

Applied Biosystems 16 (89%) 2 (11%) 1 1 19 14 (+26%)

In House / Other 7 (70%) 3 (30%) 2 (67%) 1 (33%) 13 14  (‐8%)

Liquid Systems (all) 29 (94%) 2 (6%) 1 1 32 33 (‐3%)

MGIT 960 9 (100%) 9 9 (0%)

TREK 20 (91%) 2 (9%) 1 1 23 23 (0%)

HEY Solid Media (all) 22 (92%) 2 (8%) 1 25 26 (‐8%)

Individual Panel Total 91 (84%) 17 (16%) 10 2 4 120 117 (+3%)

Pooling Panel

Direct PCR (all) 35 (95%) 2 (5%) 1 1 38 34 (+12%)

MGIT 960 7 (100%) 7 6 (+17%)

TREK 15(94%) 1 (6%) 1 16 14 (+13%)

HEY 4 (100%) 4 6 (‐33%)

Pooled Panel Total 61 (95%) 3 (5%) 1 2 65 60 (+8%)
   

General Summary of the 2011 Johne’s Disease Fecal Proficiency Panel Page | 2  

Individual Panel Description  

Each individual panel consisted of 25 unknown 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. Approximately 4 liters of fecal material were collected rectally

per animal, shipped to NVSL, aliquoted as soon as possible in individual vials, and stored at ‐70°C until

kits were distributed.  Panels were assembled in groups, each with a different key (See Table 7 at the

end of this report for the key).  Table 2 shows the categorical (positive/negative) performance for each

identification method by animal ID. Interestingly the most difficult positive sample was different for the

direct PCR assays than the liquid culture assays. Overall 79/126 (63%) of direct PCR tests identified cow

11‐04682 correctly, where liquid culture identified that sample 61/64 (95%) correctly.  The sample from

cow 10‐08079 proved problematic using liquid culture with 55/64 (86%) tests correctly identifying that

cow as positive, compared to 120/126 (95%) for direct PCR.  Correct classification using solid media

culture was problematic with the samples from both cows.  

Table 2. Composition of the 2011 Johne’s Disease Fecal Proficiency Panel, and the overall categorical summary

results per cow for each method performed by laboratories.  

HEY TREK MGIT Tetracore 

Applied 

Biosystems In House 

Cow ID 242 23 7 29 21 13

10-04921 (OH dairy cow) 2 Critical- Neg 98% 98% 100% 95% 98% 88%

09-02557 (ND beef cow) 2 Critical- Neg 100% 100% 100% 95% 98% 96%

10-04999 (OH beef cow) 2 Critical- Neg 100% 100% 100% 95% 98% 100%

11-04682 (IA dairy cow) 2 Low 63% 93% 100% 64% 64% 58%

10-08079 (IA dairy cow) 2 Low 69% 89% 78% 97% 95% 92%

11-04681 (IA dairy cow) 3 Low 97% 99% 100% 97% 100% 92%

09-07866 (IA dairy cow) 1 High 96% 96% 100% 100% 100% 100%

10-08282 (OH dairy cow) 23 Critical- High 100% 96% 100% 98% 100% 92%

10-08284 (OH dairy cow) 2 Critical- High 100% 98% 100% 98% 98% 100%

11-01804 (IA dairy cow) 2 Critical- High 98% 98% 100% 100% 100% 96%

09-01151 (IA beef cow)4 1 Critical- High 100% 94% 100% 92% 94% 100%

10-08425 (OH dairy cow) 2 Critical- High 96% 98% 100% 100% 100% 100%

10-08283 (OH dairy cow) 3 Mod-High 99% 97% 100% 95% 100% 95%

2

Number of proficiency panels submitted per method.

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. 

4

Sample identified as having PCR inhibition (see text for details)

# 

Vials/

Panel

Shedding 

Status1

3

This cow was represented in the kit twice, except for kits 101-125 where she also replaced 09-01151.

In 2010, samples from a clinical beef cow, 09‐01151, caused significant inhibition in several

laboratories using Tetracore and some in house assays. Over 15 laboratories would have failed the

proficiency panel if those would have been considered critical samples. Consequently, NVSL chose not to

fail laboratories, and instead strongly recommend an internal control be incorporated into the routine

testing of clinical samples. This year NVSL had enough vials from 09‐01151 to include one sample in kit #

1‐100. Only 2 laboratories using Tetracore, and one using Applied Biosystems (who used an in house

confirmatory test because of specificity concerns) failed to correctly identify that animal as positive. This

is significant progress.   
   

General Summary of the 2011 Johne’s Disease Fecal Proficiency Panel Page | 3  

According to the 2010 Johne’s Disease Uniform Methods and Rules, laboratories must correctly

classify all critical high shedding samples as positive, all negative samples as negative and misidentify

less than 30% of the remaining non‐critical samples.  Table 3 lists the specific reasons laboratories failed

to pass the proficiency panel for each method.  

Table 3. Reasons laboratories failed the 2011 Johne’s Disease Fecal Proficiency Panel. Laboratories were

required to correctly identify all the negative samples as negative and all the critical high shedding samples as

positive (critical samples).  They also were required to correctly classify at least 70% of the remaining samples.  

Direct PCR 

(Tetracore)

Direct 

PCR (AB)

Direct PCR 

(In house)

TREK 

liquid 

media

MGIT 

liquid 

media

HEY solid 

media

Misclassified a negative sample as 

positive

6 1 2 1 01

Missed 4 or more low/ moderate 

shedders (lack of sensitivity) 1 0 1 1 01

Misclassified a high shedding sample 

as negative 1 1 0 0 01

A critical sample was contaminated NA NA NA 0 0 0

Multiple reasons cited above 1 0 1 0 0 0

Total failed kits 9 (29%) 2 (11%) 4 (31%) 2 (9%) 0 2 (8%)

Total kits tested 31 19 13 23 9 25

A disturbing trend continued from previous years, and that is an increasing number of

laboratories using direct PCR misclassified negative samples as positive.  While the 2011 non‐infected

cows had not been used in the check test previously, the herds have been.  The major difference in this

panel from previous years is the extreme number of organisms contained in 6 samples. Three of the

cows (2 samples/cow) represented in the panel this year were shedding over 7,000 CFU per tube,

compared to 1 animal in previous years.  This emphasizes the importance of having strict procedures to

prevent cross contamination and amplicon contamination.  Table 4 examines the number of negative

samples reported with Ct values by PCR method.  Interestingly, 28/47 (60%) of laboratories that had

undetermined Ct values for all negative samples correctly classified all of the positive samples. In

contrast, only 5/16 (31%) of the laboratories that reported Ct values in some of the negative samples

were able to correctly classify all the positive samples.   There were a total of 9 laboratories that failed

the initial PT (see table 3) by calling negative samples positive. Six of those laboratories chose to retake

the PT, of those only 2 identified the same cow as positive, which was expected by random chance

alone.  

Table 4. The number of samples from non‐infected cows reported with Ct

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

method.  

Tetracore 

(58 tested)

AB 

(42 tested)

In house 

(26 tested)

10-04921 (OH dairy cow) 6 1 3

10-04999 (OH beef cow) 3 1 1

09-02557 (ND beef cow) 5 1 2
   

General Summary of the 2011 Johne’s Disease Fecal Proficiency Panel Page | 4  

Figure 1 compares the performance of each method by the number of samples misclassified per

panel.  TREK continues to have the highest percentage of laboratories that correctly classify all the

samples at 83% (19/23). This year, MGIT is a close second with 67% (6/9), followed by direct PCR with

46% (29/63). Only 3 of 25 kits submitted using solid media were able to correctly classify all of the

samples.  

Figure1.  Percentage of 2010 Johne’s disease fecal proficiency panels by number of samples misclassified for the three culture

methods (TREK liquid media, solid media and MGIT 960 liquid media) and direct PCR. A panel consisted of 25 fecal samples.  

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 5 lists the contents of each pool. Depending on the

key (see Table 8 at the end of this report) the vial numbers associated with each pool varied.  

Laboratories were required to correctly classify the negative pool and the one pool that contained the

very high shedding animal (10‐08425). Laboratories were allowed to misclassify one of the other three

pooled samples.   
   

General Summary of the 2011 Johne’s Disease Fecal Proficiency Panel Page | 5  

Table 5. Composition of the 2011 Johne’s Disease Fecal Pooling

Proficiency Panel

Cow ID

Avg. 

CFU/ 

tube*

1 Very High, 4 Negative samples 10-08425 >10,000

1 High, 4 Negative samples 08-04828 480

1 high (bison strain), 4 Negative samples 10-01627 N/A

1 mod/high, 4 Negative samples 10-08283 75

5 Negative samples

Positive sample(s) 

description

*Refers to the positive sample, not the pooled sample

Table 6 further describes the performance of each method used in the pooled proficiency test.  

For direct PCR, 2 laboratories failed because they both misclassified the pools containing 10‐01627

(bison stain), and 10‐08283. For the 6 passing laboratories that misclassified one sample, 4 missed the

lowest shedding pool containing 10‐08283, and 2 missed the pool containing the bison strain (10‐1627).

The 1 laboratory that failed using TREK misclassified the pools containing 10‐08283 and 08‐04828. As

expected the 4 laboratories that used solid media misclassified the pool containing the sample with the

bison strain (10‐01627).  

Table 6. Performance of each method used in the Johne’s Disease 2011 Fecal Pooling

Proficiency Panel. A total of 5 pooled samples were in each panel.   

Direct

PCR MGIT TREK

Solid

media

Identi fied the negative pool as positive 0000

A high shedding pool was identi fied as

negative

0000

Two non‐critical pools were identi fied as

negative

2010

One non‐critical pool was misidenti fied as

negative

7004

All 5 pools were identi fied correctly 29 7 15 0

Total 38 7 16 4

No. panels

Panels

that

failed

Panels

that

passed

A current listing of all the approved laboratories is available in the NVLS web site:  

http://www.aphis.usda.gov/animal_health/lab_info_services/approved_labs.shtml .   

Remaining sample vials from the2011 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:

http://www.aphis.usda.gov/animal_health/lab_info_services/reagents.shtml.  
   

General Summary of the 2011 Johne’s Disease Fecal Proficiency Panel Page | 6  

Table 7.  2011 Johne’s Disease Individual Fecal Proficiency Panel key by kit number. Samples are coded by color according to

shedding status as follows: Negative, Noncritical positive samples, Critical – high shedding samples. Sample 26 was the positive

control.

Vial # 1-25 26-50 51-75 76-100 101-125

1 09-02557 (ND beef cow) 10-08079 (IA dairy cow) 10-04921 (OH dairy cow) 09-01151 (IA beef cow) 10-08282 (OH dairy cow)

2 10-04921 (OH dairy cow) 10-04921 (OH dairy cow) 10-08079 (IA dairy cow) 10-04921 (OH dairy cow) 10-04921 (OH dairy cow)

3 10-08079 (IA dairy cow) 10-04921 (OH dairy cow) 09-01151 (IA beef cow) 10-08079 (IA dairy cow) 10-08079 (IA dairy cow)

4 09-01151 (IA beef cow) 09-01151 (IA beef cow) 10-08284 (OH dairy cow) 11-04681 (IA dairy cow) 10-08284 (OH dairy cow)

5 10-08284 (OH dairy cow) 10-08284 (OH dairy cow) 09-02557 (ND beef cow) 10-08284 (OH dairy cow) 09-02557 (ND beef cow)

6 10-08282 (OH dairy cow) 10-08282 (OH dairy cow) 10-08282 (OH dairy cow) 09-02557 (ND beef cow) 10-08079 (IA dairy cow)

7 09-02557 (ND beef cow) 09-02557 (ND beef cow) 10-08079 (IA dairy cow) 10-08079 (IA dairy cow) 11-04681 (IA dairy cow)

8 10-04921 (OH dairy cow) 10-08284 (OH dairy cow) 11-04681 (IA dairy cow) 10-08282 (OH dairy cow) 09-02557 (ND beef cow)

9 10-08079 (IA dairy cow) 10-08079 (IA dairy cow) 10-08282 (OH dairy cow) 09-02557 (ND beef cow) 10-04921 (OH dairy cow)

10 11-04681 (IA dairy cow) 11-04681 (IA dairy cow) 09-02557 (ND beef cow) 10-04921 (OH dairy cow) 10-08284 (OH dairy cow)

11 10-08284 (OH dairy cow) 10-08282 (OH dairy cow) 10-04921 (OH dairy cow) 10-08284 (OH dairy cow) 10-08282 (OH dairy cow)

12 10-08282 (OH dairy cow) 09-02557 (ND beef cow) 10-08284 (OH dairy cow) 10-08282 (OH dairy cow) 10-08282 (OH dairy cow)

13 11-04682 (IA dairy cow) 11-04681 (IA dairy cow) 10-08425 (OH dairy cow) 11-04681 (IA dairy cow) 11-04681 (IA dairy cow)

14 10-04999 (OH beef cow) 10-04999 (OH beef cow) 11-04682 (IA dairy cow) 11-01804 (IA dairy cow) 11-01804 (IA dairy cow)

15 10-08425 (OH dairy cow) 10-08425 (OH dairy cow) 11-04681 (IA dairy cow) 11-04682 (IA dairy cow) 11-04682 (IA dairy cow)

16 11-04681 (IA dairy cow) 10-08283 (OH dairy cow) 10-04999 (OH beef cow) 11-04681 (IA dairy cow) 11-04682 (IA dairy cow)

17 11-04681 (IA dairy cow) 09-07866 (IA dairy cow) 10-08425 (OH dairy cow) 10-04999 (OH beef cow) 11-04681 (IA dairy cow)

18 09-07866 (IA dairy cow) 11-01804 (IA dairy cow) 09-07866 (IA dairy cow) 10-08425 (OH dairy cow) 10-04999 (OH beef cow)

19 11-01804 (IA dairy cow) 10-08425 (OH dairy cow) 11-01804 (IA dairy cow) 09-07866 (IA dairy cow) 10-08425 (OH dairy cow)

20 10-08425 (OH dairy cow) 11-01804 (IA dairy cow) 11-01804 (IA dairy cow) 11-01804 (IA dairy cow) 09-07866 (IA dairy cow)

21 10-08283 (OH dairy cow) 10-08283 (OH dairy cow) 10-08283 (OH dairy cow) 10-08283 (OH dairy cow) 10-08283 (OH dairy cow)

22 11-01804 (IA dairy cow) 11-04682 (IA dairy cow) 11-04682 (IA dairy cow) 11-04682 (IA dairy cow) 10-04999 (OH beef cow)

23 10-08283 (OH dairy cow) 10-04999 (OH beef cow) 10-04999 (OH beef cow) 10-04999 (OH beef cow) 11-01804 (IA dairy cow)

24 11-04682 (IA dairy cow) 11-04682 (IA dairy cow) 10-08283 (OH dairy cow) 10-08425 (OH dairy cow) 10-08283 (OH dairy cow)

25 10-04999 (OH beef cow) 11-04681 (IA dairy cow) 11-04681 (IA dairy cow) 10-08283 (OH dairy cow) 10-08425 (OH dairy cow)

26 10-08283 (OH dairy cow) 10-08283 (OH dairy cow) 10-08283 (OH dairy cow) 10-08283 (OH dairy cow) 10-08283 (OH dairy cow)

Table 8. 2011 Johne’s Disease Pooled Fecal Proficiency Panel key by kit number

Pool Description

Kit#     

1‐25

Kit#     

25‐50

Kit#     

51‐75

5 Negative samples 5 1 2

1 mod/high (10-08283), 4 Negative samples 2 3 4

1 High-bison strain (10-01627), 4 Negative samples 4 5 1

1 High (08-4828), 4 Negative samples 3 4 5

1 Very High (10-08425), 4 Negative samples 1 2 3

Pool Sample Number

Any questions or comments can be directed to the Diagnostic Bacteriology Laboratory at 515.337.7388

or 515.337.7568.  

Report was prepared by:

Suelee Robbe‐Austerman, DVM, PhD

USDA/APHIS/NVSL

Head, Mycobacteria /Brucella Section

Suelee.Robbe‐Austerman@aphis.usda.gov