UNCONTROLLED COPY

SAM 320.04

 Page 1 of 11

United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 320

Supplemental Assay Method for Titration of Chlamydophila felis (formerly 

Feline Chlamydia psittaci) in Embryonated Chicken Eggs

Date: November 20, 2014

Number: SAM 320.04

Supersedes: SAM 320.03, June 30, 2011

Standard Requirement: 9 CFR 113.71

Contact: Alethea M. Fry, (515) 337-7200

Sandra K. Conrad

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 06Jan15

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 09Jan15

Byron E. Rippke, Director

Policy, Licensing, and Evaluation

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 09Jan15

Rebecca L.W. Hyde, Section Leader

Quality Management

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.
Center for Veterinary Biologics SAM 320.04

Testing Protocol Page 2 of 11

Supplemental Assay Method for Titration of Chlamydophila felis (formerly Feline Chlamydia psittaci)

in Embryonated Chicken Eggs

UNCONTROLLED COPY

Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents/control procedures

3.4 Preparation of the sample

4. Performance of the Test

5. Interpretation of the Test Results

6. Report of Test Results

7. References 

8. Summary of Revisions
Center for Veterinary Biologics SAM 320.04

Testing Protocol Page 3 of 11

Supplemental Assay Method for Titration of Chlamydophila felis (formerly Feline Chlamydia psittaci)

in Embryonated Chicken Eggs

UNCONTROLLED COPY

1. Introduction

This Supplemental Assay Method (SAM) is a titration method for assaying live chicken-embryoadapted Chlamydophila felis (formerly feline Chlamydia psittaci) vaccines for potency. The 

method uses embryonated chicken eggs as the indicator host system. This assay is used to 

demonstrate the presence of live chlamydia and to determine the titer of a vaccine serial by the 

chicken embryo death pattern 10 days after inoculation.

2. Materials

2.1 Equipment/instrumentation

2.1.1 Egg incubator, 36°± 2°C, humidified

2.1.2 Cabinet, laboratory biosafety level-2 

2.1.3 Water bath, 36°± 2°C 

2.1.4 Vortex mixer 

2.1.5 Micropipettor, 1000-μL, and tips 

2.2 Reagents/supplies

2.2.1 C. felis Positive Control, Cello strain

2.2.2 Embryonated chicken eggs, 6- to 7-day, specific pathogen free, in 

accordance with Title 9, Code of Federal Regulations (9 CFR)

2.2.3 7.5% Sodium Bicarbonate

1. 7.5 g sodium bicarbonate

2. Q.S. to 100 mL with deionized water (DI).

3. Sterilize by autoclaving at 121°± 2°C, 15 psi for 30 ± 10 minutes.

4. Store at 2°- 7°C. 
Center for Veterinary Biologics SAM 320.04

Testing Protocol Page 4 of 11

Supplemental Assay Method for Titration of Chlamydophila felis (formerly Feline Chlamydia psittaci)

in Embryonated Chicken Eggs

UNCONTROLLED COPY

2.2.4 1% Phenol Red

1. 1.0 g phenol red

2. Q.S. to 100 mL with DI.

3. Store at 2°- 7°C. 

2.2.5 Sucrose phosphate buffer of Bovarnick (Chlamydophila Diluent)

1. 74.6 g sucrose (C12H22O11)

2. 0.42 g potassium phosphate, monobasic, anhydrous (KH2PO4)

3. 1.25 g potassium phosphate, dibasic, anhydrous (K2HPO4)

4. 0.92 g monosodium glutamate (C5H8NO4Na)

5. 1.0 mL gentamicin 

6. 1 mL 1% Phenol Red

7. Dissolve ingredients in DI to make 1 L.

8. Filter through a 0.22-μm filter.

9. Adjust pH to 7.0-7.2 with 7.5% Sodium Bicarbonate. 

10. Store at 2°- 7°C.

2.2.6 Ethanol Solution, 70%

1. 718 mL ethanol, 95%

2. 282 mL DI

3. Store at room temperature.

2.2.7 Tincture of Iodine, 2%

1. 2 g iodine
Center for Veterinary Biologics SAM 320.04

Testing Protocol Page 5 of 11

Supplemental Assay Method for Titration of Chlamydophila felis (formerly Feline Chlamydia psittaci)

in Embryonated Chicken Eggs

UNCONTROLLED COPY

2. 100 mL 70% Ethanol Solution

3. Store at room temperature. 

2.2.8 Egg-candling light 

2.2.9 Etcher/engraver, electric, or egg drill

2.2.10 Needles, 22-gauge x 1 1/2-inch and 18-gauge x 1 1/2-inch

2.2.11 Syringe, 1-mL tuberculin

2.2.12 Duco cement

2.2.13 Polystyrene tubes, 17 x 100-mm 

2.2.14 Pipette-aid

2.2.15 Pipettes

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel shall have experience in the propagation and maintenance of chlamydophila 

agents and inoculation techniques of embryonated chicken eggs. Personnel shall be 

proficient with chlamydophila titration techniques using embryonated chicken eggs.

3.2 Preparation of equipment/instrumentation

On the day of test initiation, set a water bath at 36°± 2°C.

3.3 Preparation of reagents/control procedures

3.3.1 Preparation of eggs

1. On the day of test initiation, using an egg-candling light, check each 

egg for viability, proper embryo growth, and integrity of the egg shell. 

Appropriately dispose of eggs which do not meet these criteria.

2. Label each egg by writing a number with a pencil just below the base 

line of the air sac. Numbered eggs are placed in double layer cardboard 

flats.
Center for Veterinary Biologics SAM 320.04

Testing Protocol Page 6 of 11

Supplemental Assay Method for Titration of Chlamydophila felis (formerly Feline Chlamydia psittaci)

in Embryonated Chicken Eggs

UNCONTROLLED COPY

3. Disinfect the air sac end by swabbing with 2% Tincture of Iodine. 

Allow to air dry.

4. Drill a small hole through the disinfected egg shell using an electric 

etcher/engraver or egg drill.

5. Retain 5 uninoculated viable embryonated chicken eggs to serve as 

controls and to monitor nonspecific embryo death.

3.3.2 Preparation of C. felis Positive Control

1. On the day of test initiation, rapidly thaw a vial of Chlamydophila 

Positive Control in a 36°± 2°C water bath.

2. With a 10-mL pipette, dispense 4.5 mL Chlamydophila Diluent into 

each of an appropriate number of 17 x 100-mm polystyrene tubes. Label 

each tube, bracketing the expected Chlamydophila Positive Control

endpoint titer specified in the Center for Veterinary Biologics (CVB) 

Reagent Data Sheet (e.g., label 7 tubes from 10-1through 10-7, 

respectively).

3. With a micropipettor, transfer 500 μL of the Chlamydophila Positive 

Control to the tube labeled 10-1; mix by vortexing.

4. Using a new pipette tip, transfer 500 μL from the 10-1-labeled tube 

(Section 3.3.2[3]) to the 10-2tube; mix by vortexing. 

5. Repeat Section 3.3.2(4) for each of the subsequent dilutions until the 

tenfold dilution series is completed.

3.4 Preparation of the sample

3.4.1 The initial test of a Test Vaccine will be with a single vial (a single sample 

from 1 vial). On the day of inoculation, using a sterile 1.0-mL syringe and an 

18-gauge x 1 1/2-inch needle, rehydrate a vial of the Test Vaccine with the 

provided diluent by transferring 1.0 mL for a 1-mL-dose vaccine, 0.5 mL for a 

1/2-mL-dose vaccine, etc., into the vial containing the lyophilized Test Vaccine; 

mix by vortexing. Incubate for 15 ± 5 minutes at room temperature. 

3.4.2 Multifraction Test Vaccines containing feline rhinotracheitis virus (FRV), 

feline calicivirus (FCV), or feline panleukopenia virus (FPV) are prepared the 

same as chlamydophila single-fraction Test Vaccines. (FRV, FCV, and FPV do 

not replicate in embryonated chicken eggs.)
Center for Veterinary Biologics SAM 320.04

Testing Protocol Page 7 of 11

Supplemental Assay Method for Titration of Chlamydophila felis (formerly Feline Chlamydia psittaci)

in Embryonated Chicken Eggs

UNCONTROLLED COPY

3.4.3 Using the same method described for diluting the Chlamydophila Positive

Control in Section 3.3.2(2) through Section 3.3.2(5), prepare an appropriate 

number of tenfold serial dilutions of the reconstituted Test Vaccine to bracket its 

expected endpoint titer specified in the Animal and Plant Health Inspection 

Service (APHIS) filed Outline of Production.

4. Performance of the Test

4.1 Mix the dilution tubes by vortexing, and aspirate 0.5 mL of the highest (most 

dilute) tenfold dilution of the Test Vaccine with a 1-mL syringe and 22-gauge x 1 1/2-

inch needle. Holding the syringe vertically, insert the needle to a depth of 1.25 ± 0.25 

inch. Inoculate 100 μL of the Test Vaccine into the yolk sac of each of 5 eggs/dilution.

4.2 With the same syringe, aspirate an equal volume of the vortexed next lower Test 

Vaccine dilution and inoculate the yolk sacs of 5 more eggs. 

4.3 Inoculate the remaining dilutions. Separate syringes are not necessary between 

dilutions in a dilution series when dispensing from the most dilute to the most 

concentrated within that series but are required between series. 

4.4 In a similar manner, inoculate 5 eggs/dilution of the Chlamydophila Positive

Control (dilutions 10-7through 10-4, Section 3.3.2(2)). 

4.5 Seal the inoculation hole of all eggs with Duco cement and allow to air dry 5 ± 2 

minutes.

4.6 Return all inoculated embryonated chicken eggs to the egg incubator. (At this 

time, inoculated eggs can be incubated in egg flats for the required 11 ± 1 days time 

interval. Use double egg flats to prevent accidental egg rupture during 

candling/handling.)

4.7 On the second day postinoculation, candle the inoculated and uninoculated 

control eggs. Identify, record day of death, and appropriately discard dead embryonated 

chicken eggs. Deaths occurring prior to 3 days postinoculation are regarded as 

nonspecific deaths.

4.8 Candle the embryonated chicken eggs daily through 11 ± 1 days postinoculation. 

Identify, record day of death, and appropriately discard dead embryonated chicken eggs. 

Deaths occurring on 3 days postinoculation through 11 ± 1 days postinoculation are 

regarded as specific and are used to calculate the titer of the Test Vaccine.
Center for Veterinary Biologics SAM 320.04

Testing Protocol Page 8 of 11

Supplemental Assay Method for Titration of Chlamydophila felis (formerly Feline Chlamydia psittaci)

in Embryonated Chicken Eggs

UNCONTROLLED COPY

4.9 Calculate the C. felis endpoints of the Test Vaccine and the Chlamydophila 

Positive Control using the Spearman-Kärber method as commonly modified. The titers 

are expressed as log10 50% chicken embryo lethal dose (CELD50) per mL.

Example:

10-4dilution of Test Vaccine = 5/5 embryos dead

10-5dilution of Test Vaccine = 5/5 embryos dead

10-6dilution of Test Vaccine = 2/5 embryos dead

10-7dilution of Test Vaccine = 0/5 embryos dead

Test Vaccine titer = (X - d/2 + [d • s] where:

X = reciprocal log10 of highest dilution with all chicken embryos dead due to 

chlamydophila infection (5)

d = log10 of tenfold dilution factor (1)

s = sum of proportions of infected dead chick embryos for all dilutions tested 

starting at X:

(5+2+0) = 7 = 1.4

 5 5

Test Vaccine titer = (5 - 1/2 + [1 • 1.4]) = 5.9

Adjust the titer to the recommended Test Vaccine dose as follows:

A. Divide the Test Vaccine Dose by the Inoculation Dose where:

Test Vaccine Dose = manufacturer’s recommended vaccination dose (for this 

Chlamydophila Test Vaccine, the recommended dose is 1 mL).

Inoculation Dose = amount of diluted Test Vaccine with which each chicken embryo is 

inoculated (for this Chlamydophila Test Vaccine, the inoculation dose is 0.1 mL)

 1 mL dose = 10

0.1 mL inoculum

B. Calculate the log10 of value in A and add it to the Test Vaccine titer as illustrated 

below:

Log of 10 = 1

Test Vaccine titer 5.9 + 1 = 6.9

Therefore, the Test Vaccine Titer = 106.9 CELD50/mL.
Center for Veterinary Biologics SAM 320.04

Testing Protocol Page 9 of 11

Supplemental Assay Method for Titration of Chlamydophila felis (formerly Feline Chlamydia psittaci)

in Embryonated Chicken Eggs

UNCONTROLLED COPY

5. Interpretation of the Test Results

5.1 For a valid assay

5.1.1 All 5 of the uninoculated embryonated chicken eggs must be viable at the

end of the test.

5.1.2 At least 4 eggs/dilution must be viable 3 days postinoculation for each 

Test Vaccine and the Chlamydophila Positive Control. 

5.1.3 The calculated CELD50 titer of the Chlamydophila Positive Control must 

fall within plus or minus 2 standard deviations (± 2 SD) of its mean titer, as 

established by a minimum of 10 previous titrations.

5.1.4 If the validity requirements are not met, then the assay is considered a NO 

TEST and can be retested without prejudice.

5.2 In a valid test, if the titer of the Test Vaccine is equal to or greater than the titer 

specified in an APHIS filed Outline of Production, the Test Vaccine is considered 

SATISFACTORY.

5.3 In a valid test, if the titer of the Test Vaccine is lower than the titer specified in an 

APHIS filed Outline of Production, the Test Vaccine shall be retested in accordance with 

9 CFR 113.8(b).

6. Report of Test Results

Report results as CELD50 per dose.

7. References

7.1 Title 9, Code of Federal Regulations, part 113.71, U.S. Government Printing 

Office, Washington, DC. 

7.2 Cottral, GE (Ed.). Manual of standardized methods for veterinary microbiology. 

Comstock Publishing Associates, Ithaca, New York, 1978, pg. 731.

7.3 Finney, DJ. Statistical method in biological assay, 3rd edition. Griffin, London, 

1978, pg. 508.
Center for Veterinary Biologics SAM 320.04

Testing Protocol Page 10 of 11

Supplemental Assay Method for Titration of Chlamydophila felis (formerly Feline Chlamydia psittaci)

in Embryonated Chicken Eggs

UNCONTROLLED COPY

7.4 Richmond JY, McKinney RW (Eds.). Biosafety in microbiological and 

biomedical laboratories. U.S. Department of Health and Human Services, U.S. 

Government Printing Office, Washington, DC, 1993, pg. 177.

7.5 Storz, J. Chlamydia and chlamydia-induced diseases. Charles C. Thomas 

Publishing Company, Springfield, Illinois, 1971, pg. 358.

8. Summary of Revisions

Version .04

 The Contact information has been updated; however, the Virology Section has elected 

to keep the same next review date for the document.

Version .03

 The Contact information has been updated.

Version .02

This document was rewritten 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:

 The document number has been changed from MVSAM0320 to SAM 320.

 The Contact has been changed from Ione Stoll to Victor Becerra and Sandra Conrad.

 1.2: “Key Words” has been deleted.

 4.9: Additional steps have been added to clarify the titer calculations by the 

Spearman-Kärber formula.

 The refrigeration temperatures have been changed from 4°± 2°C to 2°- 7°C. 

 The term “chicken embryo infective dose (CEID)” has been changed to “chicken 

embryo lethal dose (CELD)” throughout the document.

 Based on the present nomenclature “Feline Chlamydia psittaci” has been changed to 

Chlamydophila felis”; therefore, the Genus “Chlamydia” has been changed to 

“Chlamydophila” and the species “felis” has been introduced.
Center for Veterinary Biologics SAM 320.04

Testing Protocol Page 11 of 11

Supplemental Assay Method for Titration of Chlamydophila felis (formerly Feline Chlamydia psittaci)

in Embryonated Chicken Eggs

UNCONTROLLED COPY

 The term “Reference” has been changed to “Positive Control” throughout the 

document.

 “Reference and Reagent Data Sheet” has been changed to “Reagent Data Sheet” 

throughout the document.

 “Test Serial” has been changed to “Test Vaccine” through the document.

 The footnotes have been deleted with any pertinent references now noted next to the 

individual items.