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SAM 411.06

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 411

Supplemental Assay Method for the Titration of Newcastle Disease Vaccine, 

Infectious Bronchitis Vaccine, and Combination Newcastle Disease-Infectious 

Bronchitis Vaccine in Chicken Embryos

Date: January 10, 2018

Number: SAM 411.06

Supersedes: SAM 411.05, November 28, 2014

Standard Requirement: 9 CFR 113.327 and 113.329

Contact: Sandra K. Conrad, (515) 337-7200

Debra R. Narwold

Approvals: /s/Alethea M. Fry for Date: 13Feb18

Geetha B. Srinivas, Section Leader

Virology

/s/Paul J. Hauer Date: 26Feb18

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 28Feb18 

 Linda S. Snavely Date

 Quality Management Program Assistant
Center for Veterinary Biologics SAM 411.06

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Supplemental Assay Method for the Titration of Newcastle Disease Vaccine, Infectious Bronchitis Vaccine, 

and Combination Newcastle Disease-Infectious Bronchitis Vaccine in Chicken Embryos

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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 sample

4. Performance of the Test

4.1 Egg inoculation

4.2 Incubation

5. Interpretation of the Test Results

5.1 Controls

5.2 NDV

5.3 IBV

5.4 Calculations

5.5 Retests

5.6 Evaluation of test results

6. Report of Test Results

7. References

8. Summary of Revisions
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Supplemental Assay Method for the Titration of Newcastle Disease Vaccine, Infectious Bronchitis Vaccine, 

and Combination Newcastle Disease-Infectious Bronchitis Vaccine in Chicken Embryos

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1. Introduction

This Supplemental Assay Method (SAM) describes a procedure for titrating Newcastle disease 

virus (NDV) vaccine, infectious bronchitis virus (IBV) vaccine, and combination NDV-IBV

vaccine. The vaccines are reconstituted and inoculated into embryonated chicken eggs in tenfold 

dilutions such that the 50% egg infective doses (EID50) can be calculated directly on a per field 

dose basis by the Reed-Muench method.

2. Materials

2.1 Equipment/instrumentation

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

below.

2.1.1 Centrifuge (Beckman J6-MI, JS-4.2 rotor)

2.1.2 Humidified, rotating egg incubator (Midwest Incubators, Model 252)

2.1.3 Vortex mixer (Thermolyne Maxi Mix II, Model No. M37615)

2.1.4 Pipette (Rainin Pipetman, P1000)

2.1.5 Cool-lite tester (Val-A)

2.1.6 Egg candling light on stand (Speed King)

2.1.7 Etcher electric engraver (Vibro-graver Acme Burgess Inc.)

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 Cotton swabs/cotton balls

2.2.2 Serological pipettes (Falcon)

2.2.3 Specific-pathogen-free (SPF) chick embryos, 9- to 11-day-old

2.2.4 Pipette tips (Rainin Clean-Pak disposable microliter pipette tips RT-200)

2.2.5 Syringe, 1-cc tuberculin, single use (Becton, Dickinson and Company)
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Supplemental Assay Method for the Titration of Newcastle Disease Vaccine, Infectious Bronchitis Vaccine, 

and Combination Newcastle Disease-Infectious Bronchitis Vaccine in Chicken Embryos

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2.2.6 Hypodermic needle, 18-gauge x 1 1/2-inch (Becton, Dickinson and 

Company)

2.2.7 Hypodermic needle, 25-gauge x 5/8-inch (Becton, Dickinson and 

Company)

2.2.8 Glass test tubes, 16 x 125-mm with Morten closures

2.2.9 Glass test tubes, 13 x 100-mm with Morten closures

2.2.10 Duco cement

2.2.11 Pipette tips (Rainin 0-100, 0-200, 100-1000 or equivalent)

2.2.12 Solutions

1. Tryptose phosphate broth (TPB)

TPB 29.5 g

q.s. with distilled or deionized water (DW) 1000.0 mL

Sterilize by autoclaving.

2. Penicillin/Streptomycin (pen/strep)

penicillin g 500 units/mL

streptomycin 2 mg/mL

q.s. with DW 1000 mL

3. Sterile DW

Sterilize by autoclaving.

4. 70% alcohol

ethyl alcohol 70.0 mL

q.s. with DW 30.0 mL

5. Iodine 2% in alcohol

iodine 2.0 g

ethyl alcohol (70%) 100.0 mL
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Supplemental Assay Method for the Titration of Newcastle Disease Vaccine, Infectious Bronchitis Vaccine, 

and Combination Newcastle Disease-Infectious Bronchitis Vaccine in Chicken Embryos

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3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have experience or training in this protocol. This includes knowledge of 

aseptic biological laboratory techniques and preparation, proper handling, and disposal of 

biological agents, reagents, tissue culture samples, and chemicals. Personnel must also 

have knowledge of safe operating procedures and policies and training in the operation of 

the necessary laboratory equipment listed in Section 2.1.

3.2 Preparation of equipment/instrumentation

Operate all equipment/instrumentation according to manufacturers’ instructions and 

monitor in compliance with current corresponding standard operating procedures or 

equivalent.

3.3 Preparation of reagents/control procedures

Prepare reference viruses in the same manner as sample preparation.

3.4 Preparation of the sample

3.4.1 NDV vaccine

1. Reconstitute NDV vaccine, frozen or lyophilized, in 4°C tryptose 

phosphate broth (TPB) to a total volume as listed in Table 1. Vigorously 

mix for a minimum of 30 seconds, and hold in an ice bath for 15 minutes

to allow for virus disaggregation. Mix again and further dilute the 

reconstituted vaccine in TPB to obtain the final concentration (1 dose per 

0.1 mL) as shown in Table 1.

2. Make tenfold dilutions of the final virus concentration, 10-1through 

10-8, by serially mixing 0.5 mL of the virus with 4.5 mL of TPB plus 

antibiotics (500-u penicillin and 2.0-mg streptomycin per mL TPB). Use a 

separate pipette or pipette tip for each transfer. Mix each dilution well 

before proceeding to the next dilution, and keep all dilutions on ice.

3.4.2 IBV vaccine

Prepare the same as for the NDV vaccine (Section 3.4.1), making tenfold 

dilutions through 10-6.
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Supplemental Assay Method for the Titration of Newcastle Disease Vaccine, Infectious Bronchitis Vaccine, 

and Combination Newcastle Disease-Infectious Bronchitis Vaccine in Chicken Embryos

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3.4.3 Combination NDV-IBV vaccine

Prepare the same as for the NDV vaccine. For titration of the Newcastle disease 

virus fraction, the dilutions are inoculated without further treatment. For titration 

of the infectious bronchitis virus fraction, neutralize the NDV by adding 0.8 mL

of each dilution, 10-2through 10-6, to an equal volume (0.8 mL) of anti-NDV 

serum. Mix well, and incubate in an ice bath for 30 minutes prior to inoculation.

Table 1

Number of 

doses in 

1 vial

Reconstituted

Volume of Vaccine

Additional dilution to reach

 1 dose/0.1mL inoculation

 (or 0.2mL of product + antiserum)

 100

 500

 1,000

 2,000

 2,500

 5,000

 10,000

 15,000

 20,000

 25,000

 10.0 mL

 50.0 mL

 30.0 mL

 60.0 mL

 50.0 mL

 100.0 mL

 100.0 mL

 100.0 mL

 100.0 mL

 100.0 mL

none

none

1.5 mL vaccine plus 3.5 mL diluent

1.5 mL vaccine plus 3.5 mL diluent

2.0 mL vaccine plus 8.0 mL diluent

2.0 mL vaccine plus 8.0 mL diluent

1.0 mL vaccine plus 9.0 mL diluent

1.0 mL vaccine plus 14.0 mL diluent 

1.0 mL vaccine plus 19.0 mL diluent 

1.0 mL vaccine plus 24.0 mL diluents

4. Performance of the Test

4.1 Egg inoculation

Prior to reconstituting the vaccine, prepare and label the appropriate number of eggs for 

allantoic cavity inoculation.

4.1.1 NDV inoculation 

Inoculate the 10-4through 10-8virus dilutions of the NDV or NDV-IBV vaccine. 

Use 5, 9- to 11-day-old embryonated chick eggs per dilution (25 eggs total), and 

inoculate 0.1 mL of the appropriate dilution per embryo.

4.1.2 IBV inoculation

Inoculate the 10-2through 10-6dilutions of the IBV or the NDV-neutralized NDVIBV vaccines. Use 6, 9- to 11-day-old embryonated chick eggs (30 eggs total). 

Inoculate 0.1 mL of the appropriate dilution of the IBV vaccine or 0.2 mL of the 

appropriate dilution of the NDV-neutralized NDV-IBV vaccine per embryo.
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Supplemental Assay Method for the Titration of Newcastle Disease Vaccine, Infectious Bronchitis Vaccine, 

and Combination Newcastle Disease-Infectious Bronchitis Vaccine in Chicken Embryos

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4.2 Incubation

Incubate the eggs for 7 days candling daily with adjustments made for weekends. Deaths 

occurring the first 24 hours shall be considered due to trauma and not used in 

calculations. At least 4 embryos per dilution must be viable at 24 hours postinoculation 

for a valid test.

5. Interpretation of the Test Results

5.1 Controls

Titrate a known positive reference virus with each group of titrations. The titer of the 

positive reference virus must be within the established range for the test results to be 

valid.

5.2 NDV

Record all deaths occurring after 24 hours as positive. On the seventh day 

postinoculation, open all remaining eggs and examine the embryos. All obviously stunted 

embryos are considered positive.

5.3 IBV

Record all deaths occurring after 24 hours as positive. On the seventh day 

postinoculation, open all remaining eggs and examine the embryos for IBV lesions. An 

embryo exhibiting 1 or more lesions is considered positive.

1. Massachusetts type

Check for stunting, curling, and clubbed down.

2. Other types

Check for stunting, curling, and clubbed down. Open the embryos and check for 

bile stasis (dark green liver) and kidney urates.

5.4 Calculations

Determine the log 10 EID50 titer using the method of Reed-Muench. This dilution and 

inoculation procedure allow for a direct readout on a per dose basis. Round to 1 decimal.
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Supplemental Assay Method for the Titration of Newcastle Disease Vaccine, Infectious Bronchitis Vaccine, 

and Combination Newcastle Disease-Infectious Bronchitis Vaccine in Chicken Embryos

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5.5 Retests

Conduct retests as required by title 9, Code of Federal Regulations (9 CFR), section 

113.8(b), and requirements of minimum release in firm’s current Outline of Production, 

Part V.

5.6 Evaluation of test results

5.5.1 The 9 CFR 113.8(b) defines the criteria for a satisfactory/unsatisfactory 

serial.

5.5.2 The firm’s requirements of minimum release/stability titers for each 

vaccine are listed in the current Outline of Production, Part V, for the specific 

product code.

6. Report of Test Results

Titers are reported out as EID50 per bird dose.

7. References

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

Washington, DC.

7.2 Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty percent 

endpoints. Am. J. Hyg. 27:493-497.

8. Summary of Revisions

Version .06

 Minor changes to dilution nomenclature.

 Table 1 updated.

Version .05

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

to keep the same next review date for the document.
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Supplemental Assay Method for the Titration of Newcastle Disease Vaccine, Infectious Bronchitis Vaccine, 

and Combination Newcastle Disease-Infectious Bronchitis Vaccine in Chicken Embryos

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Version .04

 The Contact information has been updated.

Version .03

 The document number has been changed from VIRSAM0411 to SAM 411.

 The Contact information has been updated.

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:

 2.2.12(2) 15.775 g of penicillin has been changed to 500 units/ml and 100 g of 

streptomycin has been changed to 2 mg/ml.

 2.2.12(3) The “Normal Saline” formula has been deleted.

 3.4.3 Table 1 has been updated to provide more clarification.

 4.2 Clarification has been made to allow for adjustments on weekends.

 7.1 The reference of the 9CFR was added to the list of References.