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SAM 209.06

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 209

Supplemental Assay Method for Potency Testing Products Containing 

Clostridium haemolyticum Antigen

Date: December 13, 2017

Number: SAM 209.06

Supersedes: SAM 209.05, April 1, 2014

Standard Requirement: 9 CFR, Part 113.107

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

Approvals:

/s/Larry R. Ludemann Date: 19Dec17

Larry R. Ludemann, Section Leader

Bacteriology

/s/Paul J. Hauer Date: 27Dec17

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 28Dec17 

 Linda S. Snavely Date

 Quality Management Program Assistant
Center for Veterinary Biologics SAM 209.06

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Supplemental Assay Method for Potency Testing Products Containing Clostridium haemolyticum Antigen

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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

2.3 Test animals

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Selection and handling of test animals

3.3 Preparation of supplies

3.4 Preparation of reagents

4. Performance of the Test

4.1 Vaccination of test animals

4.2 Timing of challenge

4.3 Preparation of challenge

4.4 Observation of guinea pigs following challenge

5. Interpretation of Test Results

6. Report of Test Results

7. References 

8. Summary of Revisions
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Supplemental Assay Method for Potency Testing Products Containing Clostridium haemolyticum Antigen

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1. Introduction

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

products containing Clostridium haemolyticum antigen as prescribed in title 9, Code of Federal

Regulations (9 CFR), section 113.107. For products that require 2 vaccinations, guinea pigs are 

vaccinated twice, 21 to 23 days apart, and challenged with a standard dose of virulent 

C. haemolyticum spores 14 to 15 days following the second vaccination. For products that 

require a single dose, guinea pigs are vaccinated and challenged with a standard dose of virulent 

C. haemolyticum spores 35 to 38 days following vaccination. This is a 2-stage test in which the 

second stage is applied only when exactly 2 vaccinated guinea pigs die in the first stage.

2. Materials

2.1 Equipment/instrumentation

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

below.

 Mixer, vortex-type

2.2 Reagents/supplies

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

unless specified as required for the test.

2.2.1 C. haemolyticum challenge culture, IRP 526 (06) (This culture must be 

obtained from the United States Department of Agriculture, Animal and Plant 

Health Inspection Service, Veterinary Services, Center for Veterinary Biologics

(CVB), Ames, IA 50010.)

2.2.2 Sodium chloride, reagent grade

2.2.3 Calcium chloride dihydrate, reagent grade

2.2.4 Syringes, needle-locking, 3-mL, 5-mL, and 10-mL

2.2.5 Needles, 23-gauge x 1-inch

2.2.6 Pipettes, 2-mL, 10-mL, and 25-mL

2.2.7 Glass screw-cap tubes, 16 x 125-mm
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2.2.8 Polypropylene screw-top conical tubes, 50-mL

2.2.9 Glass serum bottles, 50-mL

2.2.10 Rubber stoppers, 13 x 20-mm, and aluminum caps for serum bottles

2.2.11 Water, deionized or distilled, or water of equivalent purity

2.3 Test animals

Guinea pigs, 300-500 g. (Eight guinea pigs are required for each serial to be tested. Five 

additional guinea pigs are required as controls for each 1-4 serials tested. All guinea pigs 

used for a test series must be from the same source and housed and fed in the same 

manner.)

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. In addition, personnel need specific training and 

experience in the handling of live bacterial cultures and the handling of guinea pigs.

3.2 Selection and handling of test animals

3.2.1 Select guinea pigs that are healthy and free of external parasites and have 

an unblemished hair coat.

3.2.2 Guinea pigs of either sex or any color may be used.

3.2.3 Examine guinea pigs the day they are received. House appropriately 

allowing adequate space. (Do not mix sexes in any 1 cage.)

3.2.4 Instruct the animal caretakers not to clean the cages following challenge.

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

incinerate the guinea pigs and contaminated bedding and to sanitize the

contaminated cages.

3.3 Preparation of supplies

3.3.1 Sterilize all glassware before use.
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3.3.2 Make sure all disposable supplies (pipettes, syringes, needles, rubber 

stoppers, etc.) are sterile.

3.4 Preparation of reagents

3.4.1 C. haemolyticum challenge culture

The challenge culture, C. haemolyticum IRP 526 (06), is suspended in 50% 

glycerol. Each vial contains approximately 0.8 mL. Store vials of spore 

suspension at -50° to -90°C.

3.4.2 Calcium chloride solution

Dissolve 13.5 g of calcium chloride dihydrate (CaCl2-2H2O) in 180 mL of 

distilled water. Autoclave at ≥121°C for 25-30 minutes following manufacturer’s 

recommendations. Cool and aseptically dispense 27 mL into a 50-mL serum 

bottle. 

Note: Prepare the CaCl2 solution on the day of the challenge. Keep the 

solution on ice until used.

3.4.3 NaCl solution, 0.85%

Dissolve 1.7 g of NaCl in 200 mL of distilled water. Autoclave at ≥121°C for 25-

30 minutes following manufacturer’s recommendations. Cool and dispense 

aseptically 4.5-mL and 9-mL quantities into 16 x 125-mm tubes. 

Note: Prepare the NaCl solution on the day of the challenge. Keep the 

solution on ice while making dilutions.

4. Performance of the Test

4.1 Vaccination of test animals

4.1.1 Check the label on each container of product to be tested for identity and 

recommended field dose. (The guinea pig dose is 1/5 of the recommended calf 

dose.)

4.1.2 Thoroughly mix the product by striking the container against the palm of 

the hand at least 25 times before the syringes are filled. Use 5-mL or 10-mL

syringes, fitted with 23-gauge x 1-inch needles.
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4.1.3 Vaccinate 8 guinea pigs with each serial of product. Administer the 

vaccine subcutaneously in the ventral thoracic area. Administer the first 

vaccination on the RIGHT side of the guinea pig.

4.1.4 For 2-dose products, administer the second vaccination 21 to 23 days after 

the first. Administer the second vaccination subcutaneously in the ventral thoracic 

area on the LEFT side of the guinea pig.

4.2 Timing of challenge

4.2.1 Challenge 8 guinea pigs per serial of product 14 to 15 days after the 

second vaccination for 2-dose products or 35 to 38 days after vaccination for 

single-dose products.

4.2.2 Challenge the nonvaccinated control guinea pigs at the same time. The 5 

nonvaccinated guinea pigs may serve as controls for up to 4 serials of product. 

4.3 Preparation of challenge

4.3.1 Thaw a vial of challenge culture, IRP 526 (06), at room temperature and 

use immediately.

4.3.2 Mix the contents thoroughly by shaking the vial vigorously.

4.3.3 Use a 2-mL pipette to remove 0.7 mL of spore suspension from the vial. 

Pipette 0.5 mL of spore suspension into a glass tube containing 4.5 mL of cold, 

sterile 0.85% NaCl solution (1:10 dilution). Further dilute the spore suspension by 

adding 1.0 mL of well mixed 1:10 dilution to 9.0 mL of cold, sterile 0.85% NaCl 

solution (1:100 dilution).

Note: The C. haemolyticum spore suspension is viscid and must be pipetted 

with care to insure delivery of the complete volume.

4.3.4 Use a 2-mL pipette to remove 2 mL of well-mixed spore suspension 

diluted 1:100. Transfer 1 mL into a 50-mL serum vial containing 39 mL of cold, 

sterile calcium chloride solution (final concentration of calcium chloride solution 

equals approximately 5%). Cap and seal the bottle. Mix this 1:4000 challenge 

suspension thoroughly and place on ice. Allow the challenge suspension to remain 

on ice approximately 10 minutes before inoculating the first guinea pig.

4.3.5 Mix the challenge thoroughly before each syringe is filled. Use 3-mL

syringes fitted with 23-gauge x 1-inch needles to inoculate the guinea pigs. 

Inoculate each guinea pig intramuscularly in the thigh of the right or left hind leg 

with 0.5 mL of challenge material. The challenge dilution contains approximately 

100 guinea pig lethal dose 50 (LD50) per 0.5 mL.
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Note: Each 40-mL volume of the 1:4000 challenge dilution may be used to 

challenge up to 40 vaccinates and 5 nonvaccinated controls. This constitutes 

a challenge unit. Additional 40-mL volumes of challenge must be prepared if 

additional challenge units are required.

4.3.6 Complete the inoculation of the guinea pigs in each challenge unit within 

approximately 30 minutes from the time the challenge dilution is prepared.

4.4 Observation of guinea pigs following challenge

Observe the guinea pigs for 72 hours following challenge and record deaths. Also 

examine the test animals for sores, edema, and general physical condition. Record 

these observations on the daily record. The test is concluded at the end of the

72 hours.

Note: 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 Test Results

The test is interpreted as prescribed in 9 CFR 113.107. For a valid test, at least 80% of the 

controls shall die within the 3-day postchallenge observation period. 

If this requirement is met, the results of the potency test shall be evaluated according to the 

following table:

Stage Number of 

vaccinates

Cumulative 

number of 

vaccinates

Cumulative total number 

of deaths for a satisfactory 

test

Cumulative total 

number of deaths for 

an unsatisfactory test

1 8 8 1 or less 3 or more

2 8 16 4 or less 5 or more

The second stage is required only when exactly 2 animals die in the first stage. The second stage 

test is performed in a manner identical to the first stage test.

6. Report of Test Results

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

7.1 Title 9, Code of Federal Regulations, section 113.107, U.S. Government Printing 

Office, Washington, DC.

7.2 History of reagents: C. haemolyticum, IRP 89 (strain 7170), used to prepare IRP 

526 (06), was obtained from Montana State University, Bozeman, Montana, in 1962. The 

number of passages is unknown.

8. Summary of Revisions

Version .06

 Updated 3.4.3 to reflect correct sodium chloride dispense volumes.

 Updated 7.2 to correct lot number.

 The Director was updated on the cover page.

Version .05

 The Bacteriology Section Leader was updated.

 Minor word changes for clarification of procedures.

Version .04

 The Contact information has been updated. 

 The IRP number has been updated throughout the document from 477 to 526 (06).

Version .03

 The document number has been changed from BBSAM0209 to SAM 209.

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:

 IRP 315 has been changed to IRP 477 throughout the document.
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 The acceptable guinea pig weight has been changed to match the regulation (9 CFR 

113.107).

 The humane endpoint wording has been slightly revised for consistency.

 The contact person has been changed to Janet M. Wilson.