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SAM 303.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 303

Supplemental Assay Method for the Titration of Distemper Virus in 

Embryonated Chicken Eggs

Date: October 30, 2014

Number: SAM 303.05

Supersedes: SAM 303.04, June 30, 2011

Standard Requirements: 9 CFR 113.302 and 113.306

Contact: Alethea 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, Evaluation, and Licensing

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.
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Supplemental Assay Method for the Titration of Distemper Virus in Embryonated Chicken Eggs

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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 Test Vaccine

4. Performance of the Test

5. Interpretation of the Test Results

6. Report of Test Results

7. References 

8. Summary of Revisions
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Supplemental Assay Method for the Titration of Distemper Virus in Embryonated Chicken Eggs

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1. Introduction

This Supplemental Assay Method (SAM) describes an in vivo method for assaying the viral 

content of mink distemper virus (MDV) vaccines and that of chicken-embryo-adapted canine

distemper virus (CDV) Positive Control. The method uses embryonated chicken eggs as the test 

system. The distemper viruses (DV) endpoint is determined by quantification of viral plaques on 

the chorioallantoic membrane (CAM) of inoculated chicken embryos.

2. Materials

2.1 Equipment/instrumentation

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

below.

2.1.1 Egg incubator, 36°± 2°C, humidified (Jamesway Model No. 252, Midwest 

Incubators Sales and Service)

2.1.2 Cabinet, laboratory biosafety level-2

2.1.3 Water bath, 36°± 2°C

2.1.4 Vortex mixer (Vortex-2 Genie, G-560, Scientific Industries Inc.)

2.1.5 Colony counter, darkfield Quebec (Model 3330, Reichert Scientific 

Instruments)

2.1.6 Syringe, 2-mL

2.1.7 Micropipettor, 200-μL, and tips

2.2 Reagents/supplies

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

reagents and supplies must be sterile.

2.2.1 CDV Positive Control, Lederle strain of CDV

2.2.2 Embryonated chicken eggs, 7 days, from specific pathogen free hens, in 

accordance with title 9, Code of Federal Regulations (9 CFR)
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2.2.3 Diluent

1. Nutrient broth, 8.0 g 

2. Deionized water (DI), 1000 mL

3. Mix until dissolved.

4. Sterilize at 15 ± 1 psi, 121°± 5°C for 30 ± 5 minutes. 

5. Store at 2°- 7°C.

2.2.4 70% Ethanol Solution 

1. 718 mL ethanol, 95%

2. 282 mL DI

2.2.5 2% Iodine Solution 

1. 2.0 g iodine

2. 100 mL 70% Ethanol Solution

2.2.6 Egg-candling light

2.2.7 Etcher/engraver, electric

2.2.8 Needles, 22-gauge x 1-inch and 20-gauge x 1-inch

2.2.9 Syringe, 1-mL tuberculin

2.2.10 Polystyrene tubes, 12 x 75-mm

2.2.11 Graduated cylinders, 25-mL, 50-mL, 100-mL, and 250-mL, sterile

2.2.12 Suction bulb, rubber

2.2.13 Forceps, curved-tip and blunt thumb

2.2.14 Petri dishes, 100 x 15-mm
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2.2.15 Pipette-aid

2.2.16 Duco®cement

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel shall have training in the propagation of animal viruses and the quantitation of 

virus infectivity in embryonated chicken eggs.

3.2 Preparation of equipment/instrumentation

On the day of test initiation, set the 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 inoculation, using the egg-candling light, check each 

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

Appropriately dispose of eggs which do not meet these criteria. Candle a 

sufficient number of eggs for the Test Vaccine, the CDV Positive Control, 

the Diluent Control, and the Uninoculated Control eggs. 

2. Label 5 eggs for each dilution of the Test Vaccine and CDV Positive

Control. Also label 5 additional eggs as Diluent Controls and 5 others as 

Uninoculated Controls. The Diluent Control eggs will be inoculated with 

the diluent only. 

The Uninoculated Controls will not be inoculated (their CAMs will not be 

dropped). Place labeled eggs in double-layer cardboard flats.

3. Using the egg-candling light, hold the egg with the large end up and 

slowly turn the egg to locate the embryo. (If the embryo is hard to discern, 

give the egg a gentle, twisting shake to locate it.) Locate the edge of the 

air sac on the side opposite the embryo, and mark it with a pencil. The first 

drill site will be above this marked line by the center of the air sac. The 

second drill site will be half way between the edge of the air sac and the 

tip of the narrow end of the egg, on a site clear of large blood vessels. 

4. Swab the 2 drill sites with 2% Iodine Solution and allow to air dry.
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5. With the etcher/engraver, gently drill a hole through the shell and inner 

membrane at the mark for the first drill site (on the air sac end). 

6. Turn the egg horizontally on the egg flat and carefully drill a small 

hole through the egg shell at the second drill site, but not through the 

inner shell membrane. 

7. Using the egg-candling light for guidance, drop the CAM by a gentle 

suction with a rubber bulb placed over the first drilled hole on the air sac 

end. This causes the CAM to pull away from the shell under the second 

drilled hole, creating a new artificial air sac for the CAM inoculation. 

8. After dropping the CAMs, return eggs to the egg flat in a horizontal 

position and place them in the 36°± 2°C egg incubator until time of 

inoculation, 2 ± 2 hours.

3.3.2 Diluent for the Diluent Control Eggs

Dispense 2 mL of diluent into a polystyrene tube.

3.3.3 Preparation of CDV Positive Control

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

Control in the water bath.

2. Dispense 1.8 mL of Diluent into sufficient 12 x 75-mm polystyrene 

tubes to bracket the expected endpoint according to the CVB Reagent 

Data Sheet; appropriately label each tube (e.g., 5 tubes, labeled 10-1

through 10-5, respectively). 

3. With a 200-μL micropipettor, transfer 200 μL of the CDV Positive 

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

4. Using a new pipette tip, transfer 200 μL from the 10-1labeled tube 

(Step 3) to the 10-2tube; mix by vortexing. 

5. Repeat Step 4 for each of the remaining tubes, transferring 200 μL to 

the next dilution tube until the tenfold dilution series is completed.
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3.4 Preparation of the Test Vaccine

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 test inoculation:

1. For a single dose of Test Vaccine, using a sterile 1.0-mL syringe and a 

20-gauge x 1-inch needle, rehydrate a vial of the Test Vaccine by 

transferring 1.0 mL for 1-mL-dose vaccines, 0.5 mL for 1/2-mL-dose 

vaccines, etc., of the provided diluent into the vial containing the 

lyophilized Test Vaccine; mix by vortexing. 

2. For a multiple-dose Test Vaccine, remove the seal and stopper from 

the lyophilized Test Vaccine and the provided diluent. Measure the 

provided diluent into a sterile graduated cylinder according to the number 

of doses indicated on the manufacturer’s instructions (e.g., for a 250-dose 

container of 1 mL per dose, measure 250 mL of diluent). Rehydrate the 

Test Vaccine by aseptically pouring the measured diluent into the 

lyophilized Test Vaccine bottle; mix by vortexing.

3.4.2 Incubate for 15 ± 5 minutes at room temperature. 

3.4.3 Using the same method described for diluting the CDV Positive Control in 

Section 3.3.3, Steps 2 through 5, prepare an appropriate number of tenfold serial 

dilutions of the reconstituted Test Vaccine to bracket its expected titration 

endpoint titer specified in the Animal and Plant Health Inspection Service 

(APHIS) filed Outline of Production or special outline.

4. Performance of the Test

4.1 Starting with the highest dilution of the Test Vaccine, mix the dilution tubes by 

vortexing, and aspirate 0.5 mL with a 1-mL syringe and 22-gauge x 1-inch needle. 

Holding the syringe at a 45° angle, insert the tip of the needle through the opening of the 

dropped CAM and deliver 100 μL of the Test Vaccine dilution into each of 5 

eggs/dilution. This allows the inoculum to be delivered onto the CAM without causing 

damage by the needle.

4.2 With the same syringe, and after vortexing, draw up an equal volume of the next 

lower Test Vaccine dilution and inoculate the dropped CAMs of 5 more eggs. 

4.3 Inoculate the remaining dilutions of the Test Vaccine as described in Section 4.1. 

Separate syringes are not necessary between each dilution in a series if inoculating from 

the most dilute to the most concentrated within that series (e.g., 10-3through 10-1).
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4.4 In a similar manner, inoculate 5 eggs/dilution of the CDV Positive Control. 

4.5 Inoculate each of the 5 Diluent Control eggs with 100 μl of the Diluent. To 

prevent cross-contamination, it is recommended to inoculate these eggs first.

4.6 Seal the inoculation opening and the suction hole of all eggs with Duco®cement 

and allow to air dry 5 ± 2 minutes.

4.7 Incubate the inoculated eggs and Diluent Controls at 36°± 2°C in the horizontal 

position on the cardboard egg flat for 6 days.

4.8 Candle inoculated eggs daily. Those chicken embryos dying within the first 24 

hours after inoculation are considered to be nonspecific and are discarded. 

4.9 Harvest and examine the CAMs.

4.9.1 Using curved-tip forceps, open the egg over the area opposite the 

inoculation site. Remove and discard the embryo, yolk sac, and fluids, leaving the 

CAM attached to its shell.

4.9.2 Detach the CAM with blunt-thumb forceps. Place all 5 CAMs from 1 

dilution into a Petri dish. CAMs from Diluent Controls are placed in a separate 

Petri dish. 

4.9.3 With the aid of a darkfield Quebec bacterial colony counter, examine 

CAMs for typical 1- to 4-mm diameter DV plaques, characterized by 

circumscribed white to grayish-white raised areas. 

1. CAMs containing 1 or more DV plaques are considered to be DV 

positive. 

2. Record results as the number of plaque-positive CAMs versus the total 

number of CAMs examined for each dilution of a Test Vaccine and the 

CDV Positive Control.

4.9.4 Calculate the DV endpoints of the Test Vaccine and the CDV Positive

Control using the method of Spearman-Kärber as modified by Finney. The titers 

are expressed as log10 50% chicken embryo infective dose (CEID50). 
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Example:

10-1dilution of Test Vaccine = 5/5 CAMs plaque positive

10-2dilution of Test Vaccine = 3/4 CAMs plaque positive

10-3dilution of Test Vaccine = 1/5 CAMs plaque positive

10-4dilution of Test Vaccine = 0/5 CAMs plaque positive

Test Vaccine titer = (X - d/2 + [d ● S]) where:

X = log10 of the reciprocal of the highest dilution with all CAMs plaque positive (1)

d = log10 of tenfold dilution factor (1)

S = sum of proportion of CAMs plaque positive for all dilutions tested:

5 3 1 0 20 + 15 + 4 39

5 + 4 + 5 + 5 = 20 = 20 = 1.95 

Test Vaccine titer = (1 - 1/2) + (1 ● 1.95) = 2.45

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

A. divide the Test Vaccine Dose by the Inoculation Dose

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

MDV vaccine, the recommended dose is 1 mL)

Inoculation Dose = amount of diluted Test Vaccine inoculated onto each CAM 

of the embryonated chicken eggs (for this test MDV vaccine, the inoculation dose 

is 0.1 mL)

1 mL dose = 10

 0.1 mL

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

below:

Log of 10 = 1

Test Vaccine titer = 2.45 + 1 = 3.45

Therefore the titer of the MDV Test Vaccine is 103.5 CEID50/mL.
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5. Interpretation of the Test Results

5.1 Valid assay

5.1.1 The calculated titer of the CDV 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.

5.1.2 The lowest inoculated dilution of the CDV Positive Control must induce 

plaques in 100% of the CAMs (5/5). If an endpoint is not reached (1 or more 

CAMs are plaque positive at the highest dilution), the titer is expressed as “greater 

than or equal to” the calculated titer. If an endpoint is critical to testing, the 

highest (most dilute) must exhibit no CDV plaques (0/5). 

5.1.3 The Diluent and Uninoculated Controls must not exhibit any typical DV 

plaques as described above.

5.1.4 At least 4 of the 5 chicken embryos inoculated with a given test dilution 

should remain alive throughout the 6-day incubation period. 

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

and may be retested without prejudice.

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

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

SATISFACTORY.

5.4 In a valid test, if the titer of the Test Vaccine is less than the titer specified in the 

APHIS filed Outline of Production, the Test Vaccine is retested according to 9CFR, Part 

113.8.b.

6. Report of Test Results

Report results as CEID50 per dose of Test Vaccine. 

7. References

7.1 Title 9, Code of Federal Regulations, part 113.306, U.S. Government Printing 

Office, Washington, DC.

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

Comstock Publishing Associates, Ithaca, NY, 1978, pg 731.
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7.3 Finney, DJ, Statistical methods in biological assay. Griffin, London. 3rd edition, 

1978, pp. 394-401.

8. Summary of Revisions

Version .05

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

to keep the same next review date for the document.

Version .04

 The Contact information has been updated.

Version .03

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

document.

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:

 The “Introduction” has been rewritten to provide clarity.

 4.9.4 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. This 

reflects the parameters established and monitored by the Rees system. 

 “Test Serial” was changed to “Test Vaccine” throughout the document.

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

throughout the document.

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

individual items.