Center for Veterinary Biologics SAM632.06

Testing Protocol Page 1 of 10

 

United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 632

Supplemental Assay Method for Potency Testing of Salmonella choleraesuis

Bacterins

Date: June 17, 2022

Number: SAM 632.06 

Supersedes: SAM 632.05, May 24, 2016 

Standard Requirement: 9 CFR 113.122 

Contact Person: Email: Methodsrequest.notification@usda.gov 

Phone: Center for Veterinary Biologics, 515-337-6100 

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.

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Supplemental Assay Method for Potency Testing of Salmonella choleraesuis Bacterins

Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

2.3 Animals

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Selection and handling of test mice

3.3 Preparation of supplies/equipment

3.4 Preparation of reagents 

 

4. Performance of the Test

4.1 Vaccination of test animals 

4.2 Preparation of challenge in a biological safety cabinet

4.3 Timing and administration of challenge 

4.4 Observation of mice after challenge

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 Potency Testing of Salmonella choleraesuis Bacterins

1. Introduction

This Supplemental Assay Method (SAM) describes procedures for potency testing biological 

products containing Salmonella choleraesuis, as prescribed in title 9, Code of Federal 

Regulations (9 CFR), part 113.122. Mice are vaccinated twice, 14 days apart, and challenged 

with a standard dose of virulent S. choleraesuis 7 to 10 days after the second vaccination. 

2. Materials

2.1 Equipment/instrumentation 

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

below. 

2.1.1 Spectronic 20D+ spectrophotometer (Spectronics Instruments)

2.1.2 Sterile inoculating loop 

2.1.3 Bunsen burner or Bacti-Cinerator® (if non-sterile wire loop is used)

2.1.4 Incubator, 35°- 37°C

2.1.5 Micropipettors, 20- to 1000-μL

2.1.6 Test tube mixer, vortex-type 

2.1.7 Crimper for aluminum seals on serum vials

2.1.8 Rotary shaker 

2.1.9 Biological safety cabinet

2.2 Reagents/supplies

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

2.2.1 Appropriately qualified S. choleraesuis challenge culture, available from 

the biologics manufacturer. This reagent is supplied to the Center for Veterinary 

Biologics (CVB), upon request, with a reagent information sheet indicating 

handling and test information. 

2.2.2 Test bacterin(s) containing S. choleraesuis

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Supplemental Assay Method for Potency Testing of Salmonella choleraesuis Bacterins

2.2.3 Appropriately qualified S. choleraesuis reference bacterin, available from 

the biologics manufacturer. This reagent is supplied to the CVB, upon request, 

with a reagent information sheet indicating handling and test information. 

2.2.4 Syringes, 1-mL Tuberculin 

2.2.5 Needles, 26-gauge, 3/8-inch

2.2.6 Glass serum bottles, 10- to 100-mL 

2.2.7 Rubber stoppers, 13 x 20-mm, and aluminum cap for serum bottle 

2.2.8 Screw-cap tubes, 13 x 100-mm and 15 x 125-mm

2.2.9 Screw-cap flask, 1-L

2.2.10 Pipettes, 5- to 25-mL 

2.2.11 Micropipette tips, up to 1000-μL capacity

2.2.12 Media, as specified by the biologics manufacturer.

2.3 Animals

2.3.1 Mice, 16-22 g. Although the 9 CFR does not specify a specific mouse type 

or source, some colonies of mice may be relatively resistant to salmonellosis and 

therefore less suitable for this assay. 

2.3.2 Sixty mice are required for each serial to be tested (20 mice/dilution; 

3 dilutions/serial). Sixty additional mice are required for the reference bacterin. 

Thirty mice are required to determine the LD50 of the challenge inoculum. All 

mice must be from the same source colony and of similar weight and/or age.

3. Preparation for the Test

3.1 Personnel qualifications/training

Technical personnel need a working knowledge of the use of general laboratory 

chemicals, equipment, and glassware; and must have specific training and experience in 

sterile technique, the handling of live bacterial cultures, and the handling of mice.

3.2 Selection and handling of test mice

3.2.1 Mice of either sex may be used, but females are recommended.

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Supplemental Assay Method for Potency Testing of Salmonella choleraesuis Bacterins

3.2.2 All mice must be housed and fed in a similar manner.

3.2.3 Identify each cage of mice by treatment group. 

3.2.4 If any mice die after vaccination but prior to challenge with live 

S. choleraesuis, necropsy these mice to determine cause of death, if the cause of 

death is not outwardly apparent. If the cause of death is unrelated to vaccination, 

file the necropsy report with the test records, and no additional action is needed. 

If death is attributable to the test bacterin, report the death immediately to the 

CVB-Inspection and Compliance, which may request further safety testing of the 

bacterin.

3.2.5 When the test is concluded, instruct the animal caretakers to euthanize and 

incinerate the mice and to sanitize the contaminated rooms. 

3.3 Preparation of supplies/equipment

3.3.1 Use only sterile bacteriological supplies. 

3.3.2 Operate and maintain all equipment according to manufacturers’ 

recommendations and applicable standard operating procedures. 

3.4 Preparation of reagents

3.4.1 S. choleraesuis reference bacterin, provided by the biologics 

manufacturer. Refer to the current reagent information sheet for details. 

 

3.4.2 S. choleraesuis challenge culture, provided by the biologics manufacturer. 

Refer to the current reagent information sheet for details.

4. Performance of the Test

4.1 Vaccination of test animals

4.1.1 Check the label on each product and Section VI of the current Outline of 

Production to confirm identity and dose volume. 

4.1.2 Test each test bacterin and the reference bacterin at 3 fivefold dilutions. 

Typically, test the bacterins at 1:5, 1:25, and 1:125 dilutions. Refer to the current 

reagent information sheet for any starting dilutions of the reference bacterin. It is 

permissible to make fivefold dilutions other than those described as long as the 

reference and test bacterins are tested at the same dilutions. For viscous bacterins, 

it is advisable to start at 1:2 or 1:3 and make fivefold dilutions from this starting 

point to increase injectability of the product at the low dilution.

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Supplemental Assay Method for Potency Testing of Salmonella choleraesuis Bacterins

4.1.3 Thoroughly mix product by inverting end-to-end at least 10 times. Make 

the appropriate fivefold dilutions of the reference bacterin as specified on the 

reagent information sheet. Make identical fivefold dilutions of the test bacterin(s) 

in the diluent approved in the specific outline of production for that product. 

(Some oil-adjuvanted products require oil-based diluents.) Place each dilution in a 

separate sterile injection vial. Prepare dilutions immediately prior to use; do not 

store in diluted form. 

 

4.1.4 Vaccinate separate groups of 20 mice with each of the 3 test bacterin 

dilutions and 3 reference bacterin dilutions. For reference bacterin groups, inject 

each mouse intraperitoneally with the dose supplied on the reagent information 

sheet. Inject test bacterins intraperitoneally at a dose volume that corresponds to 

1/20 of the lowest dose recommended on the product label or Section VI of the 

current Outline of Production. This volume must not be < 0.1 mL. 

Note: It is permissible to vaccinate a few extra mice in each group to 

compensate for any potential deaths that may occur prior to challenge. 

However, if extra mice are vaccinated, all surviving at the time of challenge 

must be challenged with live S. choleraesuis and included in data 

calculations.

4.1.5 Revaccinate the mice in a similar manner 14 days after the first 

vaccination.

4.1.6 Retain 30 non-vaccinated mice to determine LD50 of the challenge.

4.2 Preparation of challenge in a biological safety cabinet

4.2.1 Prepare the challenge and LD50 determination dilutions according to the 

firm-supplied information. 

4.2.2 Place the vials of challenge inoculum and additional dilution tubes on ice. 

Keep on ice throughout the challenge. 

4.3 Timing and administration of challenge

4.3.1 Challenge all vaccinates 7 to 10 days after the second vaccination. 

4.3.2 Challenge non-vaccinated LD50 controls at the same time as the 

vaccinates.

4.3.3 Inoculate each vaccinated mouse with 0.25 mL of challenge inoculum (see 

Section 4.2.6) intraperitoneally, using a 1-mL Tuberculin syringe and 26-gauge, 

3/8-inch needle. 

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Supplemental Assay Method for Potency Testing of Salmonella choleraesuis Bacterins

4.3.4 Inoculate separate groups of 10 non-vaccinated control mice 

intraperitoneally with 0.25 mL of each of the LD50 dilutions. 

4.4 Observation of mice after challenge

4.4.1 Observe the mice up to twice daily for 14 days after challenge. Record 

deaths and euthanize any moribund mice as recommended by the Institutional 

Animal Care and Use Committee. 

4.4.2 If deaths occurring after challenge are suspected to be due to causes other 

than salmonellosis, necropsy such mice to determine the cause of death. If cause 

of death is unrelated to vaccination and/or challenge, do not include data from 

these mice in the total deaths for the test.

5. Interpretation of the Test Results

Interpret the test as prescribed in 9 CFR 113.122. 

5.1 Calculate the LD50 (theoretical dose/dilution at which the challenge would be 

lethal to 50% of the control mice) of the challenge inoculum using the Reed-Muench or 

Spearman-Kärber method of estimation. A valid test must have an LD50 between 10 and 

1,000. 

5.2 Calculate the PD50 of the reference bacterin and each test bacterin (theoretical 

dose/dilution at which the bacterin would protect 50% of the mice) using the ReedMuench or Spearman-Kärber method of estimation. 

5.3 At least 2 dilutions of the reference must protect > 0% and < 100% of the mice for a 

valid test. The lowest dilution of the reference shall protect > 50% of the mice. The 

highest dilution of the reference shall protect < 50% of the mice. 

5.4 If the PD50 of the reference cannot be calculated because the lowest dilution tested 

protects < 50% of the mice, the serial may be retested, provided the following: 

5.4.1 If the serial is not retested, it is unsatisfactory.

5.4.2 If the protection provided by the lowest dilution of the reference bacterin

exceeds that provided by the lowest dilution of the test serial by at least 6 mice, 

the test serial is unsatisfactory without additional testing. 

5.4.3 If the total number of mice protected by the reference bacterin (sum of 

survivors in all dilution groups) exceeds the total number protected by the test 

serial by 8 mice or more, the test serial is unsatisfactory without additional 

testing.

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Supplemental Assay Method for Potency Testing of Salmonella choleraesuis Bacterins

5.5 If the PD50 of the test serial in a valid test cannot be calculated because the highest 

dilution protected more than 50% of the mice, the serial is satisfactory without further 

testing.

5.6 Divide the PD50 of each test serial by the PD50 of the reference to calculate the 

relative potency (RP) for each serial.

5.7 If the RP of the test serial(s) is ≥ 0.5, the serial is satisfactory. 

 

5.8 If the RP of the test serial(s) is < 0.5, the serial is unsatisfactory. 

5.9 A test serial with an RP < 0.5 may be retested by conducting 2 independent 

replicate tests in a manner identical to the initial test. Calculate the results of the retests in 

the following manner: 

 

5.9.1 Average the RP values of the retests.

 

5.9.2 If the average RP of the retests is < 0.5, the serial is unsatisfactory. 

 

5.9.3 If the average RP of the retests is ≥ 0.5 AND the RP obtained in the 

original test is ≤1/3 the average (RP) of the retests, the test bacterin is satisfactory. 

Consider the initial test to be the result of test system error.

5.9.4 If the average of the retests is ≥ 0.5 BUT the RP of the original test is 

> 1/3 of the average RP of the retests, calculate a new average RP using the RP 

values obtained in all tests (original plus retests). If the new average RP is ≥ 0.5, 

the test bacterin is satisfactory. If the new average RP is < 0.5, the test bacterin is 

unsatisfactory. 

6. Report of Test Results

Report results of the test(s) as described by standard operating procedures. 

7. References

7.1 Title 9, Code of Federal Regulations, part 113.122, U.S. Government Printing 

Office, Washington, DC. 

7.2 Reed LJ, Muench H, 1938. A simple method of estimating 50% endpoints. Am J 

Hygiene, 27:493-497. 

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Supplemental Assay Method for Potency Testing of Salmonella choleraesuis Bacterins

8. Summary of Revisions 

Version .05 

• Updated coversheet and contact information.

Version .05 

• The Section Leader and Director information has been updated.

• The CVB no longer provides the challenge reagent. The SAM was updated to reflect 

this change.

Version .04

• The CVB no longer provides the qualified reference bacterin. The SAM was updated 

to reflect this change.

Version .03

• The Contact information has been updated. 

• 2.1.3/4.5.1: These sections have been updated to reflect current practices.

Version .02 

This document was revised to clarify 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: 

• 2.1 A sterile inoculating loop and biological safety cabinet have been added to the 

list of equipment.

• 2.1.1 Information regarding the spectrophotometer currently in use has been 

provided. 

• 2.2.8 Screw-cap tubes, 15 x 125-mm, have been added. 

• 2.2.15 A screw-cap flask has been added. 

• 3.4.7 A recipe for bovine blood agar plates has been added. 

• 4.2.6 The second gram stain done on the challenge material has been removed. 

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• Bovine blood agar plates have been added as an additional media for plate counts. 

• Information regarding autoclave parameters has been added through Section 3. 

• References to the current reagent data sheet have been added throughout the 

document. 

• The use of a biological safety cabinet has been added throughout the document. 

• References to internal documents have been replaced with summary information. 

• The contact has been changed to Janet Wilson. 

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