Center for Veterinary Biologics SAM 607.05

Testing Protocol Page 1 of 11

United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 607

Supplemental Assay Method for Potency Testing of Fowl Cholera 

(Pasteurella multocida) Bacterins, Type 1

Date: June 17, 2022 

Number: SAM 607.05 

Supersedes: SAM 607.04, May 27, 2016 

Standard Requirement: 9 CFR 113.117 

Contact: 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 Fowl Cholera (Pasteurella multocida) Bacterins, Type 1

Table of Contents

8. 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 birds

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 Post-inoculation plate count in a biological safety cabinet 

4.5 Observation of chickens after 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 of Fowl Cholera (Pasteurella multocida) Bacterins, Type 1

1. Introduction

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

products containing avian Pasteurella multocida type 1, as prescribed in Title 9, Code of Federal

Regulations (9 CFR), part 113.117. Chickens are vaccinated twice, 21 days apart, and challenged 

with a standard dose of virulent P. multocida, type 1, 14 days after the second vaccination. This 

is a 2-stage test in which the second stage is applied when 7 or 8 vaccinated chickens die in the 

first stage.

2. Materials

2.1 Equipment/instrumentation 

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

below. 

2.1.1 Spectrophotometer, Spectronic 20D+ (Spectronic 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 Crimper for aluminum seals on serum vials

2.1.7 Test tube mixer, vortex-type 

2.1.8 Biological safety cabinet

2.2 Reagents/supplies

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

2.2.1 P. multocida, type 1, strain X-73. 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). Refer to the 

current reagent data sheet for details.

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Supplemental Assay Method for Potency Testing of Fowl Cholera (Pasteurella multocida) Bacterins, Type 1

2.2.2 Test bacterin(s) containing P. multocida, type 1 

2.2.3 Syringes, Luer-lock, 3-mL or 5-mL 

2.2.4 Needles, 18-gauge x 1 1⁄2-inch

2.2.5 Glass serum bottle, 20- to 100-mL 

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

2.2.7 Glass screw-top tubes, 13 x 100-mm, with caps

2.2.8 Pipettes, 5-mL, 10-mL, 25-mL 

2.2.9 Micropipette tips, up to 1000-μL capacity

2.2.10 Bovine blood agar plates 

2.2.11 Tryptose broth 

2.2.12 Sterile cotton swabs

2.2.13 Poultry leg bands (size 9) or livestock spray paint, 1 color per treatment 

group, for animal identification

2.3 Animals

Chickens, leghorn type, at least 12 weeks of age. Twenty chickens are required for each 

serial to be tested. Ten additional chickens are required as controls. All birds must be 

from the same source and hatch. The birds must be from flocks with no history of fowl 

cholera. Birds must not be previously vaccinated with any products containing P. 

multocida. 

3. Preparation for the Test

3.1 Personnel qualifications/training

Technical personnel need working knowledge of the use of general laboratory chemicals, 

equipment, and glassware; and need to have specific training and experience in sterile 

technique, the handling of live bacterial cultures, and the handling of poultry. 

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Supplemental Assay Method for Potency Testing of Fowl Cholera (Pasteurella multocida) Bacterins, Type 1

3.2 Selection and handling of test birds

3.2.1 Chickens of either sex may be used. 

3.2.2 House and feed all chickens in a similar manner.

3.2.3 It is permissible to house vaccinates and controls in the same enclosure, 

provided that space allocation is sufficient to meet requirements set forth by the 

CVB/National Veterinary Services Laboratories (NVSL) Animal Care and Use 

Committee.

3.2.4 Positively identify each bird by treatment group. Identification may be by 

means of leg bands or livestock body paint. 

8. If leg bands are used, band each leg in case 1 band is lost. 

2. If body paint is used, freshen it at least every 3 weeks. 

3.2.5 If any chickens die after vaccination for suspected vaccine related causes, 

but prior to challenge with live P. multocida, these birds must be necropsied to 

determine cause of death. If cause of death is unrelated to vaccination, the 

pathologist’s report is filed with the test records and no additional action is taken. 

If death is attributable to the test bacterin, the death must be reported immediately 

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

the bacterin.

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

incinerate the birds and to sanitize contaminated rooms. 

3.3 Preparation of supplies/equipment

3.3.1 Sterilize all glassware before use. 

3.3.2 Use only sterile bacteriological supplies (pipettes, syringes, needles, 

rubber stoppers, saline, etc.). 

3.3.3 All equipment must be operated according to manufacturers’ 

recommendations and applicable standard operating procedures. 

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Supplemental Assay Method for Potency Testing of Fowl Cholera (Pasteurella multocida) Bacterins, Type 1

3.4 Preparation of reagents

3.4.1 P. multocida, type 1 (Lyon and Little classification), strain X-73 challenge 

culture. Refer to the current reagent data sheet for details on storage and 

preparation. 

3.4.2 Tryptose broth – National Centers for Animal Health (NCAH) Media 

#10404 

Tryptose broth powder 26 g 

Deionized water q.s. 1 L 

Autoclave 15 minutes at ≥ 121°C. Cool before using. Store at 20°- 25°C for no 

more than 6 months. 

3.4.3 Bovine blood agar – NCAH Media #10006 

Blood agar base powder 40 g 

Deionized water q.s. 950 mL 

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

Add: 

Defibrinated bovine blood 50 mL 

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

more than 6 months. 

4. Performance of the Test

4.1 Vaccination of test animals

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

of Production to confirm identity, recommended field dose, and route of injection. 

4.1.2 Thoroughly mix product by inverting end-to-end at least 10 times before 

the syringes are filled. Use 3- or 5-mL syringes, fitted with 18-gauge x 1 1⁄2-inch

needles.

4.1.3 Vaccinate separate groups of no more than 21 chickens with each of the 

test bacterins. Use the dose volume and injection route recommended on the 

product label for each bacterin. Unless otherwise specified on the product label 

and/or Section VI of the current Outline of Production, subcutaneous injections 

are given in the unfeathered, loose skin on the back of the lower neck.

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Supplemental Assay Method for Potency Testing of Fowl Cholera (Pasteurella multocida) Bacterins, Type 1

4.1.4 Revaccinate the chickens in a similar manner 21 days after the first 

vaccination.

4.1.5 Retain no more than 11 chickens as nonvaccinated controls. 

4.2 Preparation of challenge in a biological safety cabinet

4.2.1 Reconstitute a vial of challenge culture in 1 mL tryptose broth. 

4.2.2 Inoculate 2 blood agar plates with 100 μL of reconstituted culture and 

streak for isolation. 

4.2.3 Incubate the inoculated blood agar plates at 35°- 37°C for 16 to 19 hours. 

4.2.4 Use plates that have pure growth by visual inspection to prepare the 

challenge inoculum. 

8.3.2 Scrape several bacterial colonies from the surface of the blood agar 

plates using a sterile cotton swab and suspend in tryptose broth in a 

13 x 100-mm tube. Add bacterial growth until the suspension 

measures 71-75%T at 630 nm using a Spectronic 20D+

spectrophotometer or equivalent. Use sterile tryptose broth in a 13 

x 100-mm tube as a blank for the spectrophotometer. 

4.2.6 Prepare a 10-5 dilution of the standardized culture in tryptose broth. This is 

the inoculum used to challenge the chickens. Dispense challenge liquid into a 

serum vial and seal with a rubber stopper and aluminum ring. 

4.2.7 Prepare 10-6, 10-7, 10-8 dilutions for postinoculation plate counts or 

alternatively, save an aliquot of the challenge dilution in a separate vial and

prepare these additional dilutions later (see Section 4.4). 

4.2.8 Place vial(s) of challenge inoculum and additional dilution tubes on ice. 

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

postinoculation plate count. 

4.3 Timing and administration of challenge

4.3.1 Challenge 20 vaccinates per serial of product not less than 14 to 18 days 

after the second vaccination. Euthanize any additional vaccinated birds at this 

time.

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Supplemental Assay Method for Potency Testing of Fowl Cholera (Pasteurella multocida) Bacterins, Type 1

4.3.2 Challenge nonvaccinated controls at the same time as the vaccinates. 

Euthanize any additional control birds at this time. 

4.3.3 Inoculate each chicken with 0.5 mL of challenge inoculum (10-5 dilution 

of standardized culture, see Section 4.2.6) intramuscularly in the breast muscle, 

using a 3-mL syringe and 18-gauge x 1 1⁄2-inch needle.

8.3 Postinoculation plate count in a biological safety cabinet

4.4.1 After birds are challenged, prepare 10-6, 10-7, 10-8 dilutions using tryptose 

broth as the diluent. 

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

an agar plate. Plate each dilution in triplicate using 0.1 mL on bovine blood agar. 

Inoculum must be spread evenly on 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 18 to 30 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

4.5 Observation of chickens after challenge

4.5.1 Observe chickens up to twice daily for 14 days after challenge. Record 

deaths and euthanize any moribund birds as recommended by the Institutional 

Animal Care and Use Committee.

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

than fowl cholera, have such chickens necropsied to determine 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.

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Supplemental Assay Method for Potency Testing of Fowl Cholera (Pasteurella multocida) Bacterins, Type 1

5. Interpretation of Test Results

Interpret the test as prescribed in 9 CFR 113.117. 

5.1 For a valid test, at least 8 of 10 control chickens must die during the 14-day 

postchallenge period, and the plate count of the challenge inoculum must be at least 

250 cfu/dose. 

Stage Number of 

vaccinates

Cumulative 

number of 

vaccinates

Cumulative number of dead vaccinates 

for...

Satisfactory serial Unsatisfactory serial

1 20 20 6 or less 9 or more

2 20 40 15 or less 16 or more

5.2 The second stage may be conducted when 7 or 8 vaccinates die in the first stage 

of a valid test. The serial is Unsatisfactory if the test is not repeated. The second-stage 

test is performed in a manner identical to the first-stage test and evaluated according to 

the Table in Section 5.1. 

5.3 If fewer than 8 of 10 control chickens die during the postchallenge period, or if 

the challenge dose was less than 250 CFU, the test is considered invalid due to 

insufficient challenge and is reported as Inconclusive. The test may be repeated without 

prejudice, and the repeat test is considered to be a first-stage test.

6. Report of Test Results

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

7. References

Title 9, Code of Federal Regulations, part 113.117, U.S. Government Printing Office, 

Washington, DC. 

8. Summary of Revisions 

Version .05

• Updated coversheet and contact information. 

Version .04

• The Section Leader and Director information has been updated. 

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Supplemental Assay Method for Potency Testing of Fowl Cholera (Pasteurella multocida) Bacterins, Type 1

Version .03

• The Contact information has been updated. 

• 2.1.3: This section has been updated to reflect current practices.

• 2.2.12/4.2.5: Sterile cotton swabs have been added.

• 4.1.3/4.1.5: Bird group numbers have been updated to reflect 9 CFR 113.117.

• 4.2.2: Volume has been added for clarification.

• 4.3.1/4.3.2: Instructions on euthanizing extra birds have been added. 

• 4.5.1: This section has 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:

• 2.1.1 Parameters for the spectrophotometer have been updated.

• 2.1.2/2.1.3 Information pertaining to the inoculating loop has been updated.

• 2.1.8 A biological safety cabinet has been added to the equipment needed to conduct 

this test.

• 2.2.4 The size of needles used to conduct the test has been modified. 

• 2.2.12 The size of leg band used has been indicated. 

• 3.4.2 The autoclave operation parameters have been changed.

• 4.1.1 Section VI of the Outline of Production has been added as additional source of 

information regarding the product to be used to vaccinate the animals. 

• 4.2.5 The %T range for the currently used spectrophotometer has been updated. 

• 4.3.1 This Section has been altered to repeat the 9 CFR parameters for the test.

• Plate count dilution details have been added. 

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Supplemental Assay Method for Potency Testing of Fowl Cholera (Pasteurella multocida) Bacterins, Type 1

• Information regarding the challenge culture has been modified to indicate the current 

reagent data sheet throughout the document. 

• References to internal CVB SOPs have been replaced with summarized information 

throughout the document. 

• Details regarding the location of the subcutaneous injection have been added. 

• The contact person has been changed to Janet Wilson. 

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