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SAM 611.06

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 611

Supplemental Assay Method for Potency Testing of 

Erysipelas Bacterins in Mice

Date: December 15, 2017

Number: SAM 611.06

Supersedes: SAM 611.05, June 18, 2014

Standard Requirement: 9 CFR 113.119

Contact: Janet M. Wilson, (515) 337-7245

Approvals:

/s/Larry R. Ludemann Date: 20Dec17

Larry R. Ludemann, Section Leader

Bacteriology

/s/Paul J. Hauer Date: 22Dec17

Paul J. Hauer, Director

Policy, Evaluation, and Licensing

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.

Entered into CVB Quality Management System

by: /s/Linda S. Snavely 26Dec17 

Linda S. Snavely Date

Quality Management Program Assistant
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Supplemental Assay Method for Potency Testing of Erysipelas Bacterins in Mice

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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 Postinoculation plate count in a biological safety cabinet

4.5 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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1. Introduction

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

products containing Erysipelothrix rhusiopathiae, as prescribed in the title 9, Code of Federal 

Regulations (9 CFR), part 113.119. Mice are vaccinated and then challenged with a standard 

dose of virulent E. rhusiopathiae 14 to 21 days after vaccination. 

2. Materials

2.1 Equipment/instrumentation 

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

below.

2.1.1 Spectrophotometer, Spectronic 20 D+ (Spectronic Instruments) 

2.1.2 Sterile inoculating loop

2.1.3 Bunsen burner (if nonsterile wire loop is used)

2.1.4 Incubator, 35°- 37°C

2.1.5 Crimper for aluminum caps on serum vials

2.1.6 Biological safety cabinet

2.2 Reagents/supplies

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

2.2.1 E. rhusiopathiae strain E1-6P challenge culture. This culture is available 

from the Center for Veterinary Biologics (CVB). Refer to the current reagent data 

sheet for additional information.

2.2.2 Test bacterin(s) containing E. rhusiopathiae

2.2.3 E. rhusiopathiae reference bacterin, available from the CVB. Refer to the 

current reagent data sheet for additional information.

2.2.4 Syringes, 1-mL tuberculin 

2.2.5 Needles, 26-gauge x 3/8-inch
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2.2.6 Glass serum bottle, 20- to 100-mL

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

2.2.8 Glass screw-cap tubes, 13 x 100-mm

2.2.9 Pipettes, 5-mL and 25-mL

2.2.10 Erysipelas medium broth

2.2.11 Bovine blood agar plates, 5%

2.2.12 Peptone buffer, 1%

2.2.13 Saline, 0.85%

2.3 Animals

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

type or sex, the CVB uses CF-1 female mice. 

2.3.2 Eighty mice are required for each serial to be tested (20 mice/dilution; 4 

dilutions/serial). Eighty 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. 

Note: Although 9 CFR regulations require only 3 dilutions/serial, the CVB 

tests an additional dilution to increase the probability of bracketing the PD50

dilution. 

3. Preparation for the Test

3.1 Personnel qualifications/training

Technical personnel must have working knowledge of the use of general laboratory 

chemicals, equipment, and glassware and 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.

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

E. rhusiopathiae, 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 CVBInspection 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 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 E. rhusiopathiae reference bacterin. Refer to the current reagent data sheet 

for use dilutions.

3.4.2 Test bacterin(s) containing E. rhusiopathiae. For each test bacterin, make 

threefold dilutions in the appropriate diluent (see Section 4.1.3) immediately prior 

to use. Use dilutions identical to the dilutions used for the Reference bacterin (see 

Section 3.4.1). Place each of the dilutions in separate sterile serum bottles. 

3.4.3 E. rhusiopathiae challenge culture. Refer to the current reagent data sheet 

for preparation and storage information.

3.4.4 Saline, 0.85% - National Centers for Animal Health (NCAH) Media 

#30201

Sodium chloride 8.5 g

Deionized water q.s. to 1000 mL

Autoclave 20 to 40 minutes at ≥ 121°C. Store at 20°- 25°C for no longer than 1 

year.
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3.4.5 Peptone buffer, 1%--NCAH Media #10522

Peptone 10 g

Sodium phosphate, dibasic 12.01 g

Potassium phosphate, monobasic 2.09 g

Deionized water q.s. to 1000 mL

Adjust pH to 7.4 ± 0.1. Autoclave 20 to 40 minutes at ≥ 121°C. Cool before 

using. Store at 20°- 25°C for no longer than 6 months.

3.4.6 Bovine blood agar, 5%--NCAH Media #10006

Blood agar base powder 40 g

Deionized water q.s. to 950 mL

Autoclave 20 to 40 minutes at ≥ 121°C. Cool to 45°- 47°C. 

Add:

Defibrinated bovine blood 50 mL

Pour into sterile petri dishes. Cool to 20°- 25°C. Store at 2°- 7°C for no longer 

than 6 months.

3.4.7 Horse Infusion Broth--NCAH Media #40143

Horse meat (no fat) 454 g

Horse liver 18 g

Deionized water 1000 mL

Grind tissue and dispense in hot water in a cooker. Heat to boiling and simmer for 

approximately 1 hour. Remove from heat and allow to settle at least 2 hours. Skim 

off fat. Strain through cheese cloth and discard meat. Filter broth through No. 2 

Whatman filter paper. Use in Section 3.4.8 or hold for future use at -20°C or 

lower.

3.4.8 Erysipelas Medium (broth)--NCAH Media #10133

Horse Infusion Broth (Section 3.4.7) 1000 mL

Sodium phosphate, dibasic 11 g

Potassium phosphate, monobasic 1 g

Oxgall® (1 g dissolved in 10 mL deionized water) 10 mL

Peptone 20 g

Gelatin, granulated 5 g
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Heat to just below boiling to dissolve the gelatin. Cool to 55°- 57°C. Adjust pH to 

7.6 ± 0.1.

Add:

Dextrose 5 g

Horse serum (not heat inactivated) 100 mL

Filter while still warm through a sterile, disposable 0.2-μm filter. Adjust final pH 

to 7.7 ± 0.1 with filtered acid or base. Store at 2°- 7°C for no longer than 6 

months.

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 for identity and dose volume.

4.1.2 Test each test bacterin and the reference bacterin at a minimum of 4

threefold dilutions. 

4.1.3 Thoroughly mix product by inverting end-to-end. Make the appropriate 

threefold dilutions of the reference bacterin in saline. Make identical threefold 

dilutions of the test bacterin(s) in saline or the diluent approved in the specific 

Outline of Production for that product. (Some oil-adjuvanted products require oilbased diluents.) Place each dilution in a separate sterile serum bottle. 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 test bacterin 

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

each mouse with 0.2 mL subcutaneously. Inject test bacterins subcutaneously at a 

dose volume that corresponds to 1/10 of the smallest dose recommended on the 

product label or Section VI of the current Outline of Production. This volume 

must not be < 0.1 mL.

4.1.5 Retain 30 nonvaccinated mice to determine LD50 of the challenge.

4.2 Preparation of challenge in a biological safety cabinet

4.2.1 Reconstitute a vial of challenge in 1.5 mL 1% peptone buffer. 

4.2.2 Inoculate 100 mL of erysipelas medium broth with the entire contents of a 

vial of reconstituted culture.
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4.2.3 Incubate the inoculated broth at 35°- 37°C for 18 to 24 hours.

4.2.4 Perform a Gram stain using standard methods. If the Gram stain shows a 

pure culture of Gram-positive rods, continue with the challenge procedure. If the 

culture is not pure, begin the preparation of the challenge again (Section 4.2.1).

4.2.5 Dilute overnight culture as necessary in sterile erysipelas challenge culture 

broth to 74-78% T at 600 nm, using a Spectronic 20D+ spectrophotometer. Use 

sterile erysipelas medium broth as a blank for the spectrophotometer. 

4.2.6 Prepare a 10-5dilution of the standardized culture in sterile erysipelas 

challenge medium broth. This is the inoculum used to challenge the vaccinated 

mice. Dispense an aliquot of the challenge liquid into a serum vial and seal it with 

a rubber stopper and aluminum ring. 

4.2.7 Continue preparing additional tenfold dilutions (10-6to 10-9) of the 

standardized culture for postinoculation plate counts and challenge LD50

determination. Dispense an aliquot of each LD50 dilution (10-7 to 10-9) in a 

separate serum vial and seal.

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

Keep on ice through challenge procedure and until culture is added to plates for 

postinoculation plate counts.

4.3 Timing and administration of challenge

4.3.1 Challenge all vaccinated mice 14 to 21 days after the vaccination. 

Inoculate each vaccinated mouse with 0.2 mL of challenge inoculum (see Section 

4.2.6) subcutaneously, using a 1-mL tuberculin syringe and 26-gauge x 3/8-inch

needle.

4.3.2 Inoculate the nonvaccinated control mice (separate groups of 10 mice 

each) immediately following the challenge of vaccinated mice. Each mouse is 

inoculated subcutaneously with 0.2 mL of the appropriate LD50 dilution (10-7to 

10-9prepared in Section 4.2.7) using a 1-mL tuberculin syringe and 26-gauge x 

3/8-inch needle.

4.4 Postinoculation plate count in a biological safety cabinet

4.4.1 After the mice are challenged, perform a colony count on blood agar 

plates using the tubes retained for this purpose.

4.4.2 All bacterial suspensions must be mixed well prior to placing an aliquot 

on an agar plate. Plate each dilution (10-5to 10-7from Sections 4.2.6 and 4.2.7) in 

triplicate using 0.1 mL on bovine blood agar. Inoculum must be spread evenly on 
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the surface of the agar plates and not allowed to pool around the edges. Complete 

all plate inoculations within 1 hour of challenge.

4.4.3 Incubate the plates aerobically at 35°- 37°C for 48 to 72 hours.

4.4.4 Using the dilution yielding 30-300 colonies per plate, calculate the colony 

forming units (CFU)/challenge dose according to the following formula:

Colony count 

sum

x

1

x

1

x

Challenge 

dilution

x

Challenge 

vol. (mL)



CFU

Number of 

plates

Dilution factor 

plated

Plated volume 

(mL)

1 Dose Dose

4.4.5 Record the plate count (CFU/dose) of the challenge on the test result form 

for informational purposes to track trends and to troubleshoot problem tests. The 

9 CFR does not specify a minimum or maximum CFU/dose for this test.

4.5 Observation of mice after challenge

4.5.1 Observe the mice daily for 10 days after challenge. Record deaths.

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

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

death is unrelated to vaccination and/or challenge, do not include the deaths in the 

total deaths for the test.

5. Interpretation of the Test Results

5.1 Interpret the test as prescribed in 9 CFR 113.119.

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

be lethal to 50% of the control mice) of the challenge inoculum using the ReedMuench or Spearman-Kärber method of estimation. For a test to be valid, the 

challenge inoculum must contain at least 100 LD50/0.2 mL dose.

5.1.2 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 Reed-Muench or Spearman-Kärber method of estimation.

5.1.3 If the PD50 of the reference bacterin cannot be calculated because the 

lowest dilution protects  50% of the mice or the highest dilution protect  50% 

of the mice the test is invalid. The reference also must protect  0% and  100% 

of the mice at 2 or more dilutions in a valid test. At least two dilutions of the 

reference shall protect > 0% and two dilutions shall protect  100% of the mice. 
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The lowest dilution of the reference shall protect > 50% of the mice. The highest 

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

5.1.4 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:

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

2. If the protection provided by the lowest dilution of the reference 

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

mice, the test serial is unsatisfactory without additional testing.

3. If the total number of mice protected by the reference (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.

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

the highest dilution protected  50% of the mice, the serial is satisfactory without 

further testing.

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

the relative potency (RP) for each serial.

5.1.7 If the RP of the test bacterin is ≥ 0.6, the test bacterin is satisfactory.

5.1.8 If the RP of the test serial(s) is < 0.6, the test serial is unsatisfactory. 

5.1.9 A test serial with an RP < 0.6 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:

1. Average the RP values of the retests. 

2. If the average RP of the retests is < 0.6, the serial is unsatisfactory. 

3. If the average RP of the retests is ≥ 0.6 AND the RP obtained in the 

original test is ≤ 1/3 of the average RP of the retests, the test bacterin is 

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

4. If the average of the retests is ≥0.6 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 
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RP is ≥ 0.6, the test bacterin is satisfactory. If the new average RP is 

< 0.6, 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 113.119, 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.

7.3 Cottral, G.E., (Ed.), 1978. Manual of standardized methods for veterinary 

microbiology. Comstock Publishing Associates, Ithaca, NY. pg. 731. 

7.4 Finney, DJ, 1978, Statistical method in biological assay. Griffin, London. 3rd 

edition, pp. 394-401. 

8. Summary of Revisions

Version .06

 Updated cover page information.

Version .05

 The Bacteriology Section Leader was updated.

 Minor word changes for clarification of procedures.

Version .04

 The Contact information has been updated.

 3.4: Autoclave time ranges have been added.

 References to NVSL have been updated to NCAH throughout the document.
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Version .03

 4.2/4.3: Clarification changes have been made to these sections.

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 biological safety cabinet has been added to the equipment needed to conduct 

this test.

 2.2.1 The strain designation has been corrected.

 2.2.4 The tuberculin designation of the 1 mL syringe has been added.

 2.2.5 The larger needle size has been deleted.

 2.2.10 The media designation has been updated and made consistent throughout the 

remainder of the document.

 3.4.7 and 3.4.8 The media recipe has been further clarified.

 4.1.1 Section VI of the current Outline of Production has been added as an additional 

source of information

 4.1.5 Section VI of the current Outline of Production has been added to indicate the 

route of vaccination.

 4.2.2 The amount of media has been updated.

 4.2.7 The dilution scheme relative to the standardized culture has been added for 

clarity.

 4.2.8 This Section has been added to allow the option of preparing plate count 

dilutions at the same time as the dilutions of the challenge inoculums.

 5.1 The option of using the Reed-Muench or Spearman-Kärber method of estimation 

has been added.

 Information regarding the challenge culture and reference bacterin has been modified 

to indicate the current reagent data sheet throughout the document.
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 References to internal CVB SOPs have been replaced with summarized information 

throughout the document.

 The contact person has been changed to Janet Wilson.