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SAM 123.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 123

Supplemental Assay Method for Pseudorabies Virus Challenge Test in Swine

Date: October 21, 2014

Number: SAM 123.05

Supersedes: SAM 123.04, September 9, 2011

Standard Requirement: 9 CFR, Parts 113.213(c)(2)(vi) and 113.318(b)(3)

Contact: Alethea M. Fry, (515) 337-7200

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 22Dec14

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 29Dec14

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 29 Dec14

Rebecca L.W. Hyde, Section Leader

Quality Management

Center for Veterinary Biologics

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable.
Center for Veterinary Biologics SAM 123.05

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Supplemental Assay Method for Pseudorabies Virus Challenge Test in Swine

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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents/control procedures

3.4 Preparation of the sample

4. Performance of the Test

5. Interpretation of the Test Results

6. Report of Test Results

7. References

8. Summary of Revisions
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Supplemental Assay Method for Pseudorabies Virus Challenge Test in Swine

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1. Introduction

This Supplemental Assay Method (SAM) describes an in vivo method which employs challenge 

of swine to determine the potency of killed Pseudorabies virus (PRV) vaccines or the 

immunogenicity of PRV Master Seed viruses as specified in title 9, Code of Federal Regulations

(9 CFR).

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 Digital thermometer

2.1.2 Centrifuge with rotor

2.1.3 Water bath

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below. All 

reagents and supplies must be sterile.

2.2.1 PRV-susceptible swine at the minimum age recommended for vaccination 

and shown to be negative for PRV serum neutralizing (SN) antibodies by the 

current version of SAM 117, Supplemental Assay Method for Titration of 

Pseudorabies Virus Neutralizing Antibody (Constant Virus – Varying Serum 

Method. Swine shall be considered susceptible if there is no neutralization at a 

final serum dilution of 1:2. Other tests of equal sensitivity acceptable to the 

Animal and Plant Health Inspection Service (APHIS) may be used.

Numbers of animals required for testing:

1. 9 CFR 113.213: 5 vaccinated swine (VS) + 5 controls (CONT)

2. 9 CFR 113.318(b)(4): 5 VS + 5 CONT

3. 9 CFR 113.(b)(1)-(3): 20 VS + 5 CONT

2.2.2 PRV Challenge, Becker strain only with permission from State 

Veterinarian approving the procedure.
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2.2.3 Minimum Essential Medium (MEM) (National Centers for Animal Health

(NCAH) Media #20030)

1. 9.61 g MEM with Earles salts without bicarbonate

2. 1.1 g sodium bicarbonate (NaCHO3)

3. Q.S. to 1000 mL with deionized water (DI), adjust pH to 6.8-6.9 with 

2N hydrochloric acid (HCl)

4. Sterilize through a 0.22-μm filter.

5. Store at 2°- 7°C.

2.2.4 Serum separation tubes with 20-gauge x 1 1/2-inch Vacutainer® needles

2.2.5 Polystyrene tubes, 12 x 75-mm

2.2.6 Syringes, 3- and 10-mL and needles, 20 gauge x 1 1/2-inch

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have training and experience in evaluating swine for clinical signs of 

disease due to PRV infection. A current Animal Use Application must be available prior 

to ordering the swine for the test. All constraints in the application must be followed.

3.2 Preparation of equipment/instrumentation

Set the water bath at 36°± 2°C.

3.3 Preparation of reagents/control procedures

Rapidly thaw the PRV Challenge in a 36°± 2°C water bath; dilute in MEM as 

recommended on the Center for Veterinary Biologics (CVB) Reagent Data Sheet.

3.4 Preparation of the sample

3.4.1 No preparation of Test Vaccine/Master Seed is required.
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3.4.2 Preparation of blood samples

1. Allow blood samples to clot in the serum separation tubes at room 

temperature for 20 ± 5 minutes.

2. Separate serum from the clot by centrifuging the tubes at 1000 X g for 

20 ± 5 minutes (2,000 rpm, Model J6B centrifuge with a JS-4.0 rotor).

3. Pour off the serum into labeled 12 x 75-mm polystyrene tubes. 

Maintain each animal’s serum separately.

4. Store serum samples at -20°± 5°C until tested for SN antibodies 

according to the current version of SAM 117.

4. Performance of the Test

4.1 On the day of the first vaccination, bleed, using the Vacutainer®system, from the 

anterior vena cava of all VS and CONT swine for SN antibody susceptibility 

determination. Using a 10-mL syringe and needle, administer 1 dose of Test 

Vaccine/Master Seed as recommended on the label to all VS. Replace the needle between 

swine. Follow label recommendations for interval between vaccinations if 2 doses are to 

be administered to the swine; repeat the vaccination if required. 

Note: CONT swine are not vaccinated. 

4.2 At 14 to 28 days postvaccination after the last vaccination, draw blood samples, 

using the Vacutainer® system, from the anterior vena cava of all VS and CONT swine.

4.3 For a Test Vaccine, if at least 4 of the 5 VS have not developed titers of at least 

1:8 and the remaining VS have not developed a titer of 1:4, the VS and CONT swine may 

be tested by challenging as stated below. For an MSV, all swine are challenged.

4.4 Using a 3-mL syringe without a needle, administer intranasally, 2 mL of the 

diluted PRV Challenge per swine (1 mL/nostril), during inhalation to both VS and CONT 

swine. Swine should be held vertically with head up and not sedated or anesthetized for 

the challenge.

4.5 For -1 and 0 days prechallenge exposure, determine and record rectal 

temperatures.

4.6 Observe swine daily for 14 days postchallenge in the morning before and during 

feeding. Note and record all clinical observations. After clinical observations are 

recorded, determine rectal temperatures and record daily for 7 days postchallenge.
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4.7 Clinical observations

4.7.1 For 9 CFR 113.318(b)(3)(i) CONT swine, severe central nervous system 

(CNS) signs include, but are not limited to: Falling, difficulty rising, inability to 

rise, head tilt, head pressing, paralysis, tremors, spasms, convulsions, paddling, 

opisthotonos, circling, and coma.

4.7.2 For 9 CFR 113.213(c)(2)(vi) CONT swine, CNS signs include all of the 

signs listed previously: Plus pruritus, teeth grinding, empty chewing, persistent or 

unusual vocalization, disorientation, ataxia, stumbling, loss of postural control, 

and proprioceptive placing deficits.

4.7.3 Clinical signs of PRV infection in VS include, but are not limited to: All 

of the CNS signs listed previously (Sections 4.7.1 and 4.7.2), plus anorexia on 2 

or more consecutive DPC, fever of μ 106°F (μ 41.1°C) on any 2 or more DPC, 

stunting, weakness, vomiting, diarrhea, constipation on any 2 or more DPC, 

blindness or other ocular disease, persistent sneezing, persistent or deep coughing, 

labored breathing, and pneumonia.

4.7.4 For the purposes of this testing, the following clinical signs of PRV 

infection will not be included in the evaluations, due to their ambiguity: Transient 

inappetence, depression, shivering, occasional sneezing, occasional upper 

respiratory cough, reduction in rate of weight gain, and fever which is transient or 

< 106°F (< 41.1°C).

4.7.5 Moribund animals exhibiting clinical signs consistent with the expected 

disease pathogenesis that are unable to rise or move under their own power may 

be humanely euthanized and considered as deaths as outlined in 9 CFR 117.4.

5. Interpretation of the Test Results

5.1 For swine challenge-exposure no point system or weighted scoring system is 

allowed.

5.2 Validity requirements

5.2.1 9 CFR 113.318 (b)(3)(i): If at least 4 of the 5 CONT swine do not develop 

severe CNS signs or die, the test is inconclusive and may be repeated. For each 

swine, this development will be considered the observations on 1 or more days as 

described in Section 4.7.1.
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5.2.2 9 CFR 113.213 (c)(2)(vi): If at least 4 of the 5 CONT swine do not 

develop CNS signs or die, the test is inconclusive and may be repeated. For each 

swine, this development will be considered the observations on 1 or more days as 

described in Section 4.7.2.

5.3 Test Vaccine evaluation criteria

5.3.1 9 CFR 113.213: If 2 or more of the VS develop clinical signs of PRV 

infection or die, the Test Vaccine is unsatisfactory.

5.3.2 9 CFR 113.318(b): If 2 or more of the VS develop clinical signs of PRV 

infection or die, the Master Seed is unsatisfactory. 

6. Report of Test Results

Record all test results on a test record.

7. References

7.1 Title 9, Code of Federal Regulations, parts 113.213 and 113.318, U.S. 

Government Printing Office, Washington, DC.

7.2 Vannier P. Experimental infection of fattening pigs with Pseudorabies

(Aujeszky's disease) virus: efficacy of attenuated live and inactivated virus vaccines in 

pigs with or without passive immunity. Am J Vet Res 1986; 46:1478-1502.

8. Summary of Revisions

Version .05

 The Contact information has been updated; however, the Virology Section has elected 

to keep the same next review date for the document.

Version .04

 The phrase "available from the Center for Veterinary Biologics/CVB" has been 

removed from the document as these reagents are no longer supplied by the CVB.

Version .03

 The Contact information has been updated.
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Version .02

This document was revised to clarify the practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact 

the outcome of the test, the following changes were made to the document:

 The document number has been changed from MVSAM123 to SAM 123.

 The Contact has been changed from Larry Ludemann and Steve Hanson to Joseph 

Hermann and Peg Patterson.

 2.2.2: A statement regarding virus permissions has been added.

 2.2.3: The amount of sodium bicarbonate (NaHCO3) has been changed from 2.2 g to 

1.1 g.

 4.7.5: The 9 CFR 117.4 statement has been added.

 The phrase “Reference and Reagent Data Sheet” has been changed to “Reagent Data 

Sheet” throughout the document.

 The refrigeration temperatures have been changed from 4°± 2°C to 2°- 7°C.