General Summary of the 2009 Johne’s Disease Fecal Proficiency Panel Page | 1

2009 Johne’s Disease Fecal Proficiency Panel 

General Summary

October 6, 2009

Overview

A total of 64 laboratories participated in the 2009 Johne’s Disease Fecal Proficiency Panel (6 

Canadian, 3 European Union and 55 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. Table 1 details the number of individual and pooled panels shipped and the pass/fail status for 

each method. Laboratories were allowed to order multiple panels for each method and were notified of 

their preliminary pass/fail status upon submission of their results. They were given the opportunity to 

retake a failed proficiency panel and nearly all chose to do so. Results in Table 1 include retests.

Table 1. Summary results* of the 2009 Johne’s Disease Fecal 

Proficiency Panel. 

# passed 

(%)

# not 

passing (%)

# not 

returned 

(%)

Total 

Shipped

Individual Panel

Direct PCR (all) 40 (77%) 10 (19%) 2 (4%) 52

Tetracore 24 (89%) 3 (11%) 27

Applied Biosystems 6 (100%) 0 6

In House / Other 10 (59%) 7 (41%) 17

Liquid Systems (all) 34 (79%) 5 (12%) 4 (9%) 43

BACTEC 460 2 (100%) 0 2

MGIT 960 6 (43%) 4 (29%) 4 (29%) 14

TREK 26 (96%) 1 (4%) 27

HEY Solid Media (all) 32 (89%) 3 (8%) 1 (3%) 36

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

Pooling Panel

Direct PCR 25 (96%) 0 1 (4%) 26

Liquid 20 (100%) 0 0 20

HEY 6 (100%) 0 0 6

Pooled Panel Total 51 (98%) 0 1 (2%) 52

* 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.

Individual Panel Description 

Each individual panel consisted of 26 samples with one sample identified as a 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 per 

animal and aliquoted as soon as possible in individual vials then stored at -70°C. All 131 individual 

panels contained the same set of samples. Panels were assembled in groups, each with a different key 
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(See Table 6 at the end of this report for the key). Table 2 shows the categorical (positive/negative)

summary performance of each method by cow ID. The culture/PCR error rates were similar with the 

exception of the low shedding samples from cows 3000 and 86 where liquid media outperformed both 

direct PCR and solid culture. Despite similar levels of bacteria recovered using solid or liquid media from 

cow 3000 and 86 (liquid culture average days to positive was 36.4 and 37.0 respectively), direct PCR 

failed to detect over 50% of samples from cow 86.

Table 2. Composition of the 2009 Johne’s Disease Fecal Proficiency Panel, and the overall categorical summary 

results per cow for each method performed by laboratories. 

Cow ID Direct PCR Solid Liquid 

ST10 2 Apr-08 GA Neg 0 96% 98% 97%

247 2 Oct-08 ND Neg 0 98% 100% 96%

492903 2 Apr-08 MT Neg 0 98% 100% 97%

4929223 1 Apr-08 MT Neg 0 96% 100% 97%

3000 1 Apr-08 IA Low 1.5 72% 69% 87%

86 2 Oct-08 IA Low 2 47% 73% 88%

14 2 Apr-08 NY Low 3 96% 92% 91%

420 2 Apr-08 IA Low 4 92% 89% 95%

311 2 Apr-08 NY High 325 99% 98% 100%

339 2 Apr-08 NY High 1000 99% 94% 97%

455 2 Apr-08 IA High 1775 99% 98% 99%

5 2 Apr-08 IA High 3350 98% 97% 99%

4464 2 Apr-08 IA High 5625 100% 100% 100%

392 2 Apr-08 IA High 7275 100% 97% 99%

1 Colony counts were determined by NVSL, averaging results from 3 cultures for each cow. For high shedders 

the inoculum was diluted 10-x until colony counts were under 100 per tube. 

3 Sample was spiked with Mycobacterium fortuitum. 

2 Samples were classified as positive or negative for MAP by the laboratories. If the sample was represented 

twice in the panel, it was counted twice. For example Cow ST10 had 2 samples in each panel and 50 panels 

were submitted using direct PCR (2x50 = 100).

4 One of the two samples from this cow was identified as the positive control. 

% Samples Correctly Classified2 # Vials / 

Panel

Date 

Collected

State of 

Origin

Shedding 

Status

 Avg. CFU/ 

Tube1 

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

classify all high shedding samples as positive, all negative samples as negative and misidentify 4 or fewer 

(<30%) non-critical samples. Table 3 lists the specific reasons laboratories failed to pass the proficiency 

panel for each method. Misclassifying negative samples as positive continues to be the most common 

reason for failing a proficiency test. Reports of contamination overgrowth were low and sporadic. 

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

TREK media had the highest number of panels, 22/27 (81%) that correctly classified all 25 samples 

followed by solid media 13/33 (39%); MGIT 3/10 (30%); and direct PCR 9/49 (18%). Both solid media and 

direct PCR tended to indentify low shedding samples as negative suggesting those samples were near 

the detection limit for these methods. 
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Table 3. Reasons laboratories failed the 2009 Johne’s Disease Fecal Proficiency Panel. 

Laboratories were required to correctly identify all the negative samples as negative 

and all the high shedding samples as positive (critical samples). They also were 

required to correctly classify at least 70% of all 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 2 0 3 1 1 0

Missed 5 or more low/ moderate 

shedders (lack of sensitivity) 1 0 1 0 1 1

Misclassified a high shedding 

sample as negative

0 0 1 0 0 1

A critical sample was 

contaminated NA NA NA 0 0 1

Multiple reasons cited above 0 0 2 0 2 0

Total failed kits 3 (11%) 0 7 (41%) 1 (4%) 4 (40%) 3 (9%)

Total kits tested 27 6 17 27 10 35

Figure1. Percentage of 2009 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. 

81%

11%

4%

0%

4%

39%

27%

18%

9%

6%

30%

20%

0%

10%

40%

18%

29%

22%

18%

12%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

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

% proficiency panels

TREK

Solid Media

MGIT

DirPCR

Pooling Panel Description 

Twenty five samples were provided with instructions to pool 5 samples together, for a total of 5 

pooled samples. Table 4 lists the contents of each pool. Depending on the key (see table 7 at the end of 

this report) the vial numbers associated with each pool varied. Laboratories were required to correctly 
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classify the negative pool and the two pools that contained heavy shedders. Laboratories were allowed 

to misclassify one of the two pooled samples containing only medium or low shedding samples. All 

laboratories submitting results in 2009 achieved a passing score. 

Table 4. Composition of the 2009 Johne’s Disease Fecal Pooling 

Proficiency Panel.

Pool Description Cow ID 

Avg. CFU/ 

tube* 

1 High, 4 Negative samples 392 7275

1 Moderately High, 4 Negative samples 311 325

2 Moderate, 3 Negative samples 18 14

2 Low, 3 Negative samples 86 2

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. 

While all laboratories passed, liquid media continues to detect the highest number of positive pools. 

The only pool that was misclassified was the pool with 2 low shedding (cow 86) and 3 negative samples. 

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

Disease 2009 Fecal Pooling Proficiency Panel. A total of 5 pooled 

samples were in each panel. All laboratories achieved a passing 

score. 

100% correctly 

classified

Misclassified 1 

sample Total

Liquid Media 19 (95%) 1 (5%) 20

TREK 16 0 16

MGIT 2 1 3

Bactec 1 0 1

Solid Media 5 (83%) 1 (17%) 6

Direct PCR 15 (60%) 10 (40%) 25

Tetracore 12 5 17

Applied Biosystems 1 3 4

In House 2 2 4

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

around October 20, 2009. Certificates of approval will be mailed in November, 2009. 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 the2009 Proficiency Panel have been made available to 

laboratories for validation or research purposes. Available samples can be viewed in the reagents 
General Summary of the 2009 Johne’s Disease Fecal Proficiency Panel Page | 5

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. 

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: 515.663.7362 Office: 515.663.7837

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

Table 6. 2009 Johne’s Disease Individual Fecal Proficiency Panel key by kit number

Vial # 1-20 21-40 41-60 61-80 81-100 101-120 121-140

1 14 420 86 247 339 ST10 492922*

2 339 455 ST10 311 492903 420 392

3 247 86 446 ST10 14 492922* 455

4 311 ST10 14 420 339 392 ST10

5 ST10 446 339 455 247 455 420

6 455 14 247 86 311 247 392

7 247 5 311 ST10 ST10 311 492903

8 311 446 ST10 446 420 420 339

9 339 420 455 14 492922* 392 247

10 492903 392 247 5 392 492903 311

11 3000 492903 311 339 455 339 420

12 5 3000 339 420 247 492903 492903

13 86 5 492903 392 311 14 311

14 492922* 86 3000 492903 420 339 5

15 392 492922* 5 3000 392 247 14

16 14 392 86 5 492903 311 339

17 5 455 492922* 86 3000 5 247

18 446 247 392 492922* 5 446 ST10

19 420 311 14 392 86 455 3000

20 392 339 5 455 446 86 5

21 492903 492903 446 247 14 ST10 86

22 420 14 420 311 5 3000 446

23 455 339 392 339 ST10 5 14

24 86 247 492903 492903 455 86 86

25 ST10 311 420 14 86 14 455

26 446 ST10 455 446 446 446 446

* Sample was spiked with Mycobacterium fortuitum . 

The bolded 446 sample was identified as the positive control

Table 7. 2009 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, 4 Negative samples 1 4 5 2

1 Moderately High, 4 Negative samples 3 3 4 1

2 Moderate, 3 Negative samples 5 2 3 4

2 Low, 3 Negative samples 2 5 1 3

5 Negative samples 4 1 2 5

Pool Sample Number