UNCONTROLLED COPY

SAM 200.07

Page 1 of 9

United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 200

Supplemental Assay Method for Potency Testing Products Containing 

Clostridium chauvoei Antigen

Date: December 13, 2017

Number: SAM 200.07

Supersedes: SAM 200.06, February 15, 2017

Standard Requirement: 9 CFR 113.106

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 200.07

Testing Protocol Page 2 of 9

Supplemental Assay Method for Potency Testing Products Containing Clostridium chauvoei Antigen

UNCONTROLLED COPY

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 and administration of challenge

4.4 Observation of guinea pigs following challenge

5. Interpretation of the Test Results

6. Report of Test Results

7. References 

8. Summary of Revisions 
Center for Veterinary Biologics SAM 200.07

Testing Protocol Page 3 of 9

Supplemental Assay Method for Potency Testing Products Containing Clostridium chauvoei Antigen

UNCONTROLLED COPY

1. Introduction

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

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

Regulations (9 CFR), section 113.106. 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. chauvoei 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. chauvoei 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.

2.2.1 Clostridium chauvoei challenge culture, IRP 631. (This culture should be 

obtained from the United States Department of Agriculture, Veterinary Services, 

Center for Veterinary Biologics (CVB).)

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, 5-mL, and 25-mL

2.2.7 Glass dilution bottles, 160-mL

2.2.8 Glass serum bottle, 50-mL
Center for Veterinary Biologics SAM 200.07

Testing Protocol Page 4 of 9

Supplemental Assay Method for Potency Testing Products Containing Clostridium chauvoei Antigen

UNCONTROLLED COPY

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

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

2.3 Test animals

Guinea pigs, 300-500 grams. (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 should 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 should have working knowledge of the use of general laboratory 

chemicals, equipment, and glassware and have specific training and experience in the 

handling of live bacterial cultures and the handling of laboratory animals.

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.

3.3.2 Make sure all supplies (pipettes, syringes, needles, rubber stoppers, etc.) 

are sterile.
Center for Veterinary Biologics SAM 200.07

Testing Protocol Page 5 of 9

Supplemental Assay Method for Potency Testing Products Containing Clostridium chauvoei Antigen

UNCONTROLLED COPY

3.4 Preparation of reagents

3.4.1 Clostridium chauvoei challenge culture

The Clostridium chauvoei challenge culture, IRP 631, is suspended in 50% 

glycerol. Each vial contains approximately 1.5 mL. Store vials of spore 

suspension lower than -60°C.

3.4.2 Calcium chloride solution

Dissolve 27 grams of calcium chloride dihydrate (CaCl2∙2H2O) in 360 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 grams of Sodium Chloride (NaCl) in 200 mL of distilled water. 

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

recommendations. Cool and dispense aseptically 99 mL into a 160-mL glass 

dilution bottle and a 36 mL quantity into a 50-mL glass serum bottle.

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.

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.
Center for Veterinary Biologics SAM 200.07

Testing Protocol Page 6 of 9

Supplemental Assay Method for Potency Testing Products Containing Clostridium chauvoei Antigen

UNCONTROLLED COPY

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 and administration of challenge

4.3.1 Thaw a vial of culture suspension 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 1.2 mL of spore suspension from the vial. 

Transfer 1 mL of spore suspension into a glass dilution bottle containing 99 mL of 

cold, sterile 0.85% NaCl solution (1:100 dilution).

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

care to ensure delivery of the complete volume.

4.3.4 Use a 2-mL pipette to remove 1.2 mL of well-mixed 1:100 dilution of 

spore suspension. Transfer 1.0 mL of the diluted spore suspension into a glass

dilution bottle containing 36 mL of cold, sterile 0.85% NaCl solution (1:3700 

dilution).

4.3.5 Use a 5-mL pipette to remove 5.0 mL of well-mixed 1:3700 dilution of 

spore suspension. Transfer 3 mL into a 50-mL serum vial containing 27 mL of 

cold, sterile calcium chloride solution (final concentration of calcium chloride 

solution equals approximately 5%). Cap and seal the bottle. Mix this 1:37,000 

challenge suspension thoroughly and place it on ice. Allow the challenge

suspension to remain on ice approximately 10 minutes before inoculating the first 

guinea pig.

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

needle-locking 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.
Center for Veterinary Biologics SAM 200.07

Testing Protocol Page 7 of 9

Supplemental Assay Method for Potency Testing Products Containing Clostridium chauvoei Antigen

UNCONTROLLED COPY

Note: Each 30-mL volume of the 1:37,000 challenge dilution may be used to 

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

a challenge unit. Additional 30-mL volumes of challenge must be prepared if

additional challenge units are required.

4.3.7 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

The guinea pigs are observed for 72 hours following challenge. Deaths are recorded. The 

test animals are also examined for sores, edema, and general physical condition. These 

observations are also noted 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 the Test Results

The test is interpreted as prescribed in 9 CFR 113.106. For a valid test, at least 80 percent 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.
Center for Veterinary Biologics SAM 200.07

Testing Protocol Page 8 of 9

Supplemental Assay Method for Potency Testing Products Containing Clostridium chauvoei Antigen

UNCONTROLLED COPY

7. References

7.1 Title 9, Code of Federal Regulations, section 113.106, U.S. Government Printing 

Office, Washington, DC.

7.2 History of Clostridium chauvoei IRP 631: This spore suspension was prepared 

October 16, 2015. It replaces IRP 509 (04) and was prepared from C. chauvoei IRP 206, 

which was prepared from C. chauvoei Lot F, obtained from American Cyanamid in 1962. 

8. Summary of Revisions

Version .07

 Update 3.4.3 to reflect correct sodium chloride dispense volumes.

Version. 06

 Challenge reagent lot information updated.

Version .05

 Bacteriology Section Leader updated.

 Minor word changes for clarification of procedures.

Version .04

 The Contact information has been updated.

Version .03

 The document number has been changed from BBSAM0200 to SAM 200.

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:

 2.2.7 16 x 125-mm screw-capped test tubes have been changed to 160-ml glass

dilution bottles.
Center for Veterinary Biologics SAM 200.07

Testing Protocol Page 9 of 9

Supplemental Assay Method for Potency Testing Products Containing Clostridium chauvoei Antigen

UNCONTROLLED COPY

 2.3.1 The acceptable guinea pig weight has been changed to match the regulation (9 

CFR 113.106).

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

 IRP 434 has been changed to IRP 509(04) throughout the document.

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