2010 Johne’s Disease Fecal Proficiency Panel    

General Summary

October 4, 2010

Overview

A total of 61 laboratories participated in the 2010 Johne’s Disease Fecal Proficiency Panel (5

Canadian, 3 European Union, 1 New Zealand and 52 USA laboratories). In the USA, laboratories must

order separate panels and demonstrate proficiency for each method they wish to use for the Johne’s

Disease National Program. Overall, the number of laboratories requesting proficiency panels for PCR and

pooled testing increased from 2009. Concurrently, there was a sharp decline in the number of

laboratories requesting individual panels for both 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.  Therefore, results provided in

Table 1 include these retests. The TREK liquid media culture system was the only method where all

laboratories passed the individual panel. This year, no laboratory used the Bactec 460 liquid media

culture system.

Table 1. Summary results of the 2010 Johne’s Disease Fecal Proficiency Panel. In order to pass

results must meet the criteria listed in the 2006 Uniform Program Standards for the Voluntary

Bovine Johne's Disease Control Program.

# passed 

(%)

# not 

passing 

(%)

# passed 

on retest 

(%)

# not 

passing 

on retest 

(%)

# not 

returned 

(%)

Total 

Shipped

Total shipped 

in 2009 

(%change)

Individual Panel

Direct PCR (all) 47 (81%) 6 (10%) 3 (5%) 1 (2%) 1 (2%) 58 52 (+12%)

Tetracore 25 (86%) 3 (10%)* 1(3%)A 29 27 (+7%)

Applied Biosystems 11 (79%) 1 (7%)C 1 (7%)B 1 (7%)B 14 6 (+133%)

In House / Other 11 (78%) 2 (14%) 1 (7%) 14 17 (‐18%)

Liquid Systems (all) 29 (88%) 3 (9%) 1 (3%) 33 43 (‐23%)

MGIT 960 6 (43%) 3 (29%) 9 14 (‐36%)

TREK 23 (100%) 23 27 (‐15%)

HEY Solid Media (all) 21 (81%) 2 (8%) 1 (4%) 2 (8%) 26 36 (‐28%)

Individual Panel Total 106 (81%) 18 (14%) 7 (5%) 117 131 (‐11%)

Pooling Panel

Direct PCR (all) 27 (79%) 4 (12%) 2 (6%) 1 (3%) 34 26 (+31%)

MGIT 960 5 (83%) 1 (17%) 6 3 (+100%)

TREK 13 (93%) 1 (7%) 14 16 (‐13%)

HEY 5 (83%) 1 (17%) 6 6 (0%)

Pooled Panel Total 50 (83%) 7 (12%) 2 (3%) 1 (2%) 60 51 (+15%)

CThis laboratory did not request a retest.

*All 3 laboratories requested a retest, 1 retested with TetracoreA the other 2 retested with Applied 

BiosystemsB

General Summary of the 2010 Johne’s Disease Fecal Proficiency Panel Page | 1  
   

Individual Panel Description  

Each individual panel consisted of 26 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.  All 117 individual panels contained the same set of samples. Panels were assembled

in groups, each with a different key (See Table 6 at the end of this report for the key).  Table 2 shows the

categorical (positive/negative) summary performance for each identification method by animal ID.  Two

cows (0901, 09‐07865) were shedding at such low levels less than 70% of samples were identified as

positive by participating laboratories; consequently these  samples were removed for official grading

purposes.  Cow 10‐01627 was shedding a ‘bison strain’ of Mycobacterium avium subsp. paratuberculosis

(MAP), as determined by IS1311 restriction endonuclease assay (Whittington et. al, Mol Cell Probe,

2001). Consistent with previous literature, laboratories had difficulty isolating this strain using HEY

media (5 out of 48 positive), yet this cow was shedding at a high level based on PCR and days to positive

using  routine liquid culture protocols..  NVSL has isolated this strain from bison originating from

Montana and Oregon, elk from Oregon, and now cattle from Idaho.  

Table 2. Composition of the 2010 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 9 29 14 14

247 (ND beef cow) 2 Critical- Neg 98% 100% 100% 98% 96% 96%

10-01088 (OH beef cow) 2 Critical- Neg 100% 100% 100% 98% 93% 93%

10-02775 (OH beef cow) 2 Critical- Neg 100% 100% 100% 98% 89% 93%

0901 (IA beef cow) 2 Low 50% 59% 28% 26% 25% 46%

09-07865 (IA dairy cow) 2 Low 6% 67% 0% 69% 68% 54%

420 (IA dairy cow) 2 Low 88% 98% 67% 90% 100% 86%

311 (NY dairy cow) 1 High 100% 100% 100% 93% 100% 100%

318 (NY dairy cow) 1 Critical- High 100% 100% 100% 97% 100% 100%

09-07866 (IA dairy cow) 2 High 100% 100% 72% 98% 100% 100%

10-01627 (ID dairy cow)3 2 High 10% 98% 100% 100% 100% 96%

460 (IA dairy cow) 2 Critical- High 100% 100% 78% 97% 100% 100%

09-01151 (IA beef cow)4 2 High 100% 100% 100% 78% 100% 75%

477 (IA dairy cow) 2 Critical- High 98% 100% 94% 97% 100% 100%

233 (IA dairy cow) 2 Critical- High 100% 100% 100% 98% 100% 100%

2

Number of proficiency panels submitted per method.

Liquid Media Direct PCR

% Samples Correctly Classified

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 infected with a 'bison strain' of MAP. 

Inhibition of the PCR reaction is always a potential cause for false negative results, especially

when conducting PCR directly from fecal material. While there may have been sporadic inhibition in past

proficiency panels, this year was the first year systemic inhibition problems were reported for multiple

General Summary of the 2010 Johne’s Disease Fecal Proficiency Panel Page | 2  
   

laboratories. Cow 09‐01151 was a clinical beef cow identified as a heavy shedder in all of the culture

methods; yet 12 laboratories using either a commercial (Tetracore) or in house DNA extraction method

classified at least one sample from this cow as negative. None of these laboratories were using an

internal control which would have detected PCR inhibition.  The 2 commercially available PCR methods

for Johne’s disease in the United States either haven an internal control automatically provided (Applied

Biosystems) or one is available upon request (Tetracore).  An internal control should be added to eac

sample, preferably before DNA extraction. Positive results for the internal control then confirm th

validity of the PCR reaction. Because this identified a widespread problem, NVSL requested that

laboratories misclassifying these samples rerun the samples incorporating the internal control and

further permanently alter their protocols to include this step. Therefore, NVSL strongly recomme

that all dire

h

e

nds

ct PCR methods use an internal control in each test well, regardless of source of PCR

reagent

ly

ify

ing

s.  

According to the 2006 Johne’s Disease Uniform Methods and Rules, laboratories must correct

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

less than 30% of the remaining non‐critical samples. Because 4 samples were removed for official

grading, laboratories were allowed to misclassify up to 3 of the remaining noncritical samples, includ

the samples with the ‘bison strain’ of MAP.  Even though this put laboratories using solid media at a

slight disadvantage, no laboratory failed as a direct result of the ‘bison’ MAP samples.  Table 3 list

specific reasons laboratories failed to pass the proficiency panel for each method.  Misclassifying

negative samples as positive continues to be the most common

s the

reason for failing a proficiency test.  

Reports of contamination overgrowth were low and sporadic.   

ies

tical samples).  They also were required to correctly classify at least

Table 3. Reasons laboratories failed the 2010 Johne’s Disease Fecal Proficiency Panel. Laborator

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

shedding samples as positive (cri

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 0 2100

Missed 5 or more low/ moderate 

shedders (lack of sensitivity) 2 0000

Misclassified a high shedding sample 

as negative 0 0001

A critical sample was contaminated NA NA NA 0 1 0

Multiple reasons cited above 1 0 1 0 1 1

Total failed kits 3 (10%) 2 (14%) 2 (14%) 0 3 (33%) 2 (8%)

0

0

1

Total kitstested 29 14 14 23 9 24

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

panel.  For completeness, all samples were included in this analysis. Similar to last year, TREK media

performed very well, with 65% (15/23) of laboratories correctly identifying 100% (25/25) or 96% (24

of all samples in the proficiency panel. Direct PCR was the next best performing method, with 18%

per

/25)

General Summary of the 2010 Johne’s Disease Fecal Proficiency Panel Page | 3  
   

(10/57) of all laboratories misclassifying 1 or fewer samples per panel.  The 4 very low shedding sample

and the bison strain samples significantly affected the performance of solid

s

media, as over half of the

laboratories using this method misclassified 5 or more samples per panel.  

mance of the TREK media for this sample remained

unchanged at 98% correct for both 2009 and 2010.  

the

uid media, solid media and MGIT 960 liquid media) and direct PCR. A panel

consisted of 25 fecal samples.  

Five of nine laboratories using the MGIT 960 media system misclassified five or more samples.

These laboratories were not able to identify any of the low shedding samples from cow 09‐07865, and

only 5/18 of cow 0901. Laboratories using this media system also had difficulty with cow 420. The same

collection from this cow was used in the 2009 proficiency panel.  Laboratories using the MGIT system in

2009 misclassified this cow 3/20 times (85% correct); whereas this year laboratories misclassified this

cow 6/18 times (67% correct). In contrast, the perfor

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

three culture methods (TREK liq

30%

35%

4%

13% 13%

4%

0% 0% 0%

8%

38%

54%

0% 0%

22%

0%

22%

56%

7%

11%

28% 26%

16%

12%

0%

10%

20%

30%

40%

50%

60%

100%  correct Misclassified 1 Misclassified 2 Misclassified 3 Misclassified 4 Misclassified ≥ 5

% proficiency panels

TREK

Solid Media

MGIT

DirPCR

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 4 lists the contents of each pool. Dependi

key (see

ng on the

Table 7 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 two pools that contained

heavy shedding samples. Laboratories were allowed to misclassify one of the two pooled samples

containing only low shedding samples.   

General Summary of the 2010 Johne’s Disease Fecal Proficiency Panel Page | 4  
   

Table 4. Composition of the 2010 Johne’s Disease Fecal Pooling Proficiency Panel

Cow ID

Avg. CFU/ 

tube*

1 High, 4 Negative samples 460 400

1 High, 4 Negative samples 455 1775

1 Low, 4 Negative samples 420 4

1 Low, 4 Negative samples 14 3

5 Negative samples

Positive sample(s) 

description

*Refers to the positive sample, not the pooled sample

Table 5 further describes the performance of each method used in the pooled proficiency test.  

Even though this pooling panel had 2 pools consisting of 4 negative samples and 1 low shedding sample,

only 1 laboratory using solid media misclassified both pools. TREK media detected the highest

percentage of positive pools.  For direct PCR, 2 laboratories identified a pool with the low shedding

samples as suspect or inconclusive.  For the purposes of Table 5, these pools were not considered

misclassified.  

Table 5. Performance of each method used in the Johne’s Disease 2010

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

panel.  All laboratories achieved a passing score.  

Direct

PCR MGIT TREK

Solid

media

Identi fied the negative pool as positive 1010

A high shedding pool was identi fied as

negative

3100

Both low shedding pools were identi fied as

negative

0001

One low shedding pool was misidenti fied as

negative

7303

All 5 pools were identi fied correctly 22 2 13 2

Total 33 6 14 6

No.panels*

Panels

that

failed

Panels

that

passed

Individual detailed results and statistics for each panel will be provided to individual laboratories

before October 20, 2010. Certificates of approval will be mailed in November, 2010. 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 the2010 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 2010 Johne’s Disease Fecal Proficiency Panel Page | 5  
   

General Summary of the 2010 Johne’s Disease Fecal Proficiency Panel Page | 6  

Any questions or comments can be directed to:

Beth Harris, M.S., Ph.D.        Suelee Robbe‐Austerman, DVM, PhD

USDA/APHIS/NVSL        USDA/APHIS/NVSL

Head, Mycobacteria /Brucella Section     VMO, Mycobacteria/Brucella Section

Office: 515.337.7362         Office: 515.337.7837

Beth.N.Harris@aphis.usda.gov       Suelee.Robbe‐Austerman@aphis.usda.gov

Table 6.  2010 Johne’s Disease Individual Fecal Proficiency Panel key by kit number. Samples are coded by color according to

shedding status as follows: Negative, Low – eliminated from official grading, Noncritical positive samples, Critical – high shedding

samples, Positive control.

Vial # 1-20 21-40 41-60 61-80 81-100 101-120

1 09-07865 (IA dairy cow) 10-01088 (OH beef cow) 10-01627 (ID dairy cow) 311 (NY dairy cow) 09-01151 (IA beef cow) 247 (ND beef cow)

2 10-01627 (ID dairy cow) 10-01627 (ID dairy cow) 10-01088 (OH beef cow) 10-01627 (ID dairy cow) 10-01627 (ID dairy cow) 09-01151 (IA beef cow)

3 10-01088 (OH beef cow) 10-01627 (ID dairy cow) 311 (NY dairy cow) 10-01088 (OH beef cow) 10-01088 (OH beef cow) 10-01627 (ID dairy cow)

4 311 (NY dairy cow) 311 (NY dairy cow) 247 (ND beef cow) 318 (NY dairy cow) 247 (ND beef cow) 10-01088 (OH beef cow)

5 247 (ND beef cow) 247 (ND beef cow) 09-07865 (IA dairy cow) 247 (ND beef cow) 09-07865 (IA dairy cow) 247 (ND beef cow)

6 09-01151 (IA beef cow) 09-01151 (IA beef cow) 09-01151 (IA beef cow) 09-07865 (IA dairy cow) 10-01088 (OH beef cow) 09-07865 (IA dairy cow)

7 09-07865 (IA dairy cow) 09-07865 (IA dairy cow) 10-01088 (OH beef cow) 10-01088 (OH beef cow) 318 (NY dairy cow) 10-01088 (OH beef cow)

8 10-01627 (ID dairy cow) 247 (ND beef cow) 318 (NY dairy cow) 09-01151 (IA beef cow) 09-07865 (IA dairy cow) 09-07865 (IA dairy cow)

9 10-01088 (OH beef cow) 10-01088 (OH beef cow) 09-01151 (IA beef cow) 09-07865 (IA dairy cow) 10-01627 (ID dairy cow) 10-01627 (ID dairy cow)

10 318 (NY dairy cow) 318 (NY dairy cow) 09-07865 (IA dairy cow) 10-01627 (ID dairy cow) 247 (ND beef cow) 09-01151 (IA beef cow)

11 247 (ND beef cow) 09-01151 (IA beef cow) 10-01627 (ID dairy cow) 247 (ND beef cow) 09-01151 (IA beef cow) 311 (NY dairy cow)

12 09-01151 (IA beef cow) 09-07865 (IA dairy cow) 247 (ND beef cow) 09-01151 (IA beef cow) 311 (NY dairy cow) 318 (NY dairy cow)

13 0901 (IA beef cow) 460 (IA dairy cow) 477 (IA dairy cow) 460 (IA dairy cow) 460 (IA dairy cow) 420 (IA dairy cow)

14 09-07866 (IA dairy cow) 09-07866 (IA dairy cow) 0901 (IA beef cow) 420 (IA dairy cow) 420 (IA dairy cow) 0901 (IA beef cow)

15 477 (IA dairy cow) 477 (IA dairy cow) 460 (IA dairy cow) 0901 (IA beef cow) 0901 (IA beef cow) 460 (IA dairy cow)

16 460 (IA dairy cow) 10-02775 (OH beef cow) 09-07866 (IA dairy cow) 460 (IA dairy cow) 0901 (IA beef cow) 09-07866 (IA dairy cow)

17 460 (IA dairy cow) 233 (IA dairy cow) 477 (IA dairy cow) 09-07866 (IA dairy cow) 460 (IA dairy cow) 477 (IA dairy cow)

18 233 (IA dairy cow) 420 (IA dairy cow) 233 (IA dairy cow) 477 (IA dairy cow) 09-07866 (IA dairy cow) 420 (IA dairy cow)

19 420 (IA dairy cow) 477 (IA dairy cow) 420 (IA dairy cow) 233 (IA dairy cow) 477 (IA dairy cow) 0901 (IA beef cow)

20 477 (IA dairy cow) 420 (IA dairy cow) 420 (IA dairy cow) 420 (IA dairy cow) 233 (IA dairy cow) 09-07866 (IA dairy cow)

21 10-02775 (OH beef cow) 10-02775 (OH beef cow) 10-02775 (OH beef cow) 10-02775 (OH beef cow) 10-02775 (OH beef cow) 10-02775 (OH beef cow)

22 420 (IA dairy cow) 0901 (IA beef cow) 0901 (IA beef cow) 0901 (IA beef cow) 09-07866 (IA dairy cow) 10-02775 (OH beef cow)

23 10-02775 (OH beef cow) 09-07866 (IA dairy cow) 09-07866 (IA dairy cow) 09-07866 (IA dairy cow) 420 (IA dairy cow) 477 (IA dairy cow)

24 0901 (IA beef cow) 0901 (IA beef cow) 10-02775 (OH beef cow) 477 (IA dairy cow) 10-02775 (OH beef cow) 460 (IA dairy cow)

25 09-07866 (IA dairy cow) 460 (IA dairy cow) 460 (IA dairy cow) 10-02775 (OH beef cow) 477 (IA dairy cow) 233 (IA dairy cow)

26 233 (IA dairy cow) 233 (IA dairy cow) 233 (IA dairy cow) 233 (IA dairy cow) 233 (IA dairy cow) 233 (IA dairy cow)

Table 7. 2010 Johne’s Disease Pooled Fecal Proficiency Panel key by kit number

Pool Description

Kit#     

1‐20

Kit#     

21‐40

Kit#     

41‐50

Kit#     

51‐60

1 High (cow 460), 4 Negative samples 4 3 5 1

1 High (cow 455), 4 Negative samples 5 4 2 3

1 Low (cow 420), 4 Negative samples 3 2 1 4

1 Low (cow 14), 4 Negative samples 1 5 4 2

5 Negative samples 2 1 3 5

Pool Sample Number