Is your herd Mycoplasma hyopneumoniae negative?

A. Sponheim1,2; E. Fano1; D. Polson1; K. Doolittle1, C. Fitzgerald3, M. Pieters2

1Boehringer Ingelheim Vetmedica, Inc., St. Joseph, USA; 2University of Minnesota, St. Paul, USA, 3Iowa State University, Ames, USA

Overview

• Take Home Message• Background Information• Objective• What is stochastic modeling?• Materials and methods• Applications• Conclusions

Take Home Message

• Sampling guidelines developed specifically for Mhp

• Number of animals to sample based on risk willing to assume

Background Information

• Mhp continues to be an important issue• Many farms have undergone elimination• Diagnostics are challenging• Novel sample types suggest higher DxSe

– Sample procedure comparison for PCR (Fablet, 2010; Pieters and Rovira, 2013; Sievers, 2015)

– Effect of pooling on DxSe (Sievers, 2015)• sample size + pooling = herd detection rate

AND saves $$

Approach

• Two phases– Pooling for DxSe

• In vitro phase– Sampling guideline development

• Stochastic modeling

Phase 1

• To determine the effect of pooling laryngeal swab samples on Mhp detection in low prevalence populations

Materials & Methods

• Artificial samples− Mhp strain AP 414 (20 Ct) − Oral fluids from Mhp negative farm− PBS for dilution

• Life Technologies VetMAX-Plus PCR reagents• 540 samples tested• Pooled samples

– 3:1– 5:1

Materials & Methods

• Distribution – Low (26 Ct) – Medium (31 Ct) – High (36 Ct)

Materials & Methods

– Pooling: 1 known positive sample + 2 or 4 negative samples– *Power calculation: 2 proportions, Minitab 17 (α probability 0.05,

baseline 0.5, power 0.8, proportion 0.3, expected difference 20%, N 90)

Individual positive samples

5:1 pool 3:1 poolIndividual negative samples

Prevalence scenarioLow Ct value (26) N=90

6 PCRs 90 PCRs* 90 PCRs*

Medium Ct value (31) N=90

6 PCRs 90 PCRs* 90 PCRs*

High Ct value (36) N=90

6 PCRs 90 PCRs* 90 PCRs*

Mycoplasma hyopneumoniae PCR testing method

6 PCRs

Materials & Methods

• PCR plate organization

1 2 3 4 5 6 7 8 9 10 11 12

1 C- PL3 01

PL3 02

PL3 03

PL3 04

PL3 05

PL3 06

PL3 07

PL3 08

PL3 09

PL3 10

PL3 11

2 C+ PL3 12

PL3 13

PL3 14

PL3 15

PM301

PM302

PM303

PM304

PM305

PM306

PM307

3 L1+ PM308

PM309

PM310

PM311

PM312

PM313

PM314

PM315

PH3 01

PH3 02

PH3 03

4 M1+ PH3 04

PH3 05

PH3 06

PH3 07

PH3 08

PH3 09

PH3 10

PH3 11

PH3 12

PH3 13

PH3 14

5 H1+ PH3 15

PL5 01

PL5 02

PL5 03

PL5 04

PL5 05

PL5 06

PL5 07

PL5 08

PL5 09

PL5 10

6 C1- PL5 11

PL5 12

PL5 13

PL5 14

PL5 15

PM501

PM502

PM503

PM504

PM505

PM506

7 PM507

PM508

PM509

PM510

PM511

PM512

PM513

PM514

PM515

PH5 01

PH5 02

PH5 03

8 PH5 04

PH5 05

PH5 06

PH5 07

PH5 08

PH5 09

PH5 10

PH5 11

PH5 12

PH5 13

PH5 14

PH5 15

Results

Phase 2

• To determine ante-mortem laryngeal swab sampling protocols for detection of Mhp by PCR at low prevalence levels

Why important?

• No specific guidelines available for Mhp testing

• Sampling guidelines depend on DxSeand DxSp of testing procedure, herd size, detection probability, and expected prevalence when determining number of animals & number of times to sample

What is stochastic modeling?

Slide courtesy of CJ Fitzgerald

Materials & Methods

Materials & Methods

• Stochastic model– 100 iterations

Stochastic model developed by Dale Polson

ApplicationsNumber of sample collections needed for a population size of ≥2,500 at varying Mycoplasma hyopneumoniae group prevalences by number of

laryngeal swab samples collected and pooled by 3.

Based on 99% LCL of Se (79.1%)of artificial laryngeal swab pools of 3:1 where 1 of 3 samples are high Ct value and population size is ≥2,500. All replicates were required to have a detection probability of 95% or higher.

N Individuals 30 60 90 120N 3:1 pools 10 20 30 40

Percent Prevalence1% 15 8 6 42% 8 4 3 33% 6 3 2 24% 4 2 2 25% 3 2 2 2

ApplicationsNumber of sample collections needed for a population size of ≥ 2,500 at varying Mycoplasma hyopneumoniae group prevalences by number of

laryngeal swab samples collected and pooled by 5.

Based on 99% LCL of Se (58.5%)of artificial laryngeal swab pools of 5:1 where 1 of 5 samples are high Ct value and population size is ≥2,500. All replicates were required to have a detection probability of 95% or higher.

N Individuals 30 60 90 120N 5:1 pools 6 12 18 24

Percent Prevalence1% 21 11 7 62% 11 5 4 33% 7 4 3 24% 5 3 2 25% 4 3 2 2

Applications• Is your farm negative?

– Incoming gilts from Isolation or GDU– Sentinel replacements to validate the negative

status of sow herd– Due to wean piglets to validate lack of vertical

transmission

Limitations

• One strain of Mhp• One sample type• Intra-laboratory variability• Sampling interval

Next steps

• Laryngeal sample collections completed +30 days apart

• Field validation• Tables available for specific scenarios

– nessx317@umn.edu– amanda.sponheim@boehringer-

ingelheim.com

Conclusions

• Sampling guidelines developed specifically for Mhp

• Number of animals to sample based on risk willing to assume

Acknowledgements

• Christensen Farms • Dr. Laura Dalquist• Dr. Mike Eisenmenger• Dr. Tom Wetzell• Jill Johnson

• Alyssa Anderson• University of Minnesota

SROC breeding herd farm staff

• Boehringer Ingelheim Vetmedica, Inc.