United States Department of Agriculture

 Marketing and Regulatory Programs

 Animal and Plant Health Inspection Service

 Veterinary Services

 VS Guidance 13410.1

1

Disease Testing and Sampling Requirements for Imported Live Poultry and Birds (and 

Hatching Eggs of Poultry, Birds, and Ratites) during Entry Quarantines

1. Purpose and Background

This guidance outlines sampling requirements for imported live poultry, birds, and hatching

eggs of poultry, birds, and ratites during import entry quarantine at facilities approved by the 

Animal and Plant Health Inspection Service (APHIS) Veterinary Services (VS) division 

(hereinafter “VS”). The entry quarantine period for applicable poultry or hatching eggs is at 

least 30 days, and may be extended in case of infection or illness found during the 

quarantine. During the quarantine period, facilities will submit samples to the National 

Veterinary Services Laboratories (NVSL) for testing by virus isolation for avian influenza and 

Newcastle disease.

1 The NVSL must find the samples free of these viruses before the birds 

or eggs can be released from quarantine.

The National Animal Import Center Director or the Director of an Animal Import Center may 

approve the collection of additional specimens to be tested for other avian pathogens and 

may also ask the NVSL to use other testing protocols in addition to virus isolation. Requests 

for additional tests should only be made in the event of a disease outbreak during which 

quarantined birds manifest clinical signs other than those of avian influenza or Newcastle 

disease. Importers should consult the NVSL on specimen collection procedures for tests not 

covered by this guidance. Additional test requests will incur associated testing fees, for 

which the importer is responsible.

This guidance document represents the Agency’s position on this topic. It does not create or 

confer any rights for or on any person and does not bind the U.S. Department of Agriculture 

(USDA) or the public. The information it contains may be made available to the public. While 

this document provides guidance for users outside VS, VS employees may not deviate from 

the directions provided herein without appropriate justification and supervisory concurrence.

2. Document Status

A. Review date: March 31, 2020.

B. This document updates and replaces Veterinary Services Memorandum No 591.22, 

which is rescinded. The content of this document reflects program changes and 

enhances implementation.

3. Reason for Reissuance

Not applicable.

 1 1) The NVSL charges a user fee directly to the importer separate from quarantine fees. Current fee rates 

can be found on the NVSL diagnostic testing Web site. 
 United States Department of Agriculture

 Marketing and Regulatory Programs

 Animal and Plant Health Inspection Service

 Veterinary Services

 VS Guidance 13410.1

2

4. Authority and References

A. Authorities (Code of Federal Regulations (CFR)):

• 7 CFR 371.4

• 9 CFR 93 subparts A and B

B. References

• VS Guidance 13401.2, “APHIS Approval and Oversight of Privately Owned Avian

Quarantine Facilities in the United States”

• VSG 5510.1, “Procedures for Processing Cooperative Service Agreements for 

Privately Owned Quarantine Facilities for Commercial Birds, Poultry and any Avian 

Eggs for Hatching,” (11/04).

• VS Notice 10-07, Diagnostic Submissions to the National Veterinary Services 

Laboratory is listed on the VS Share point for Notices.

• VS Form 10-4, “Specimen Submission,” (8/09).

• NVSL diagnostic testing user fees.

5. Audience 

VS employees, other Federal and State agencies, and members of the public.

6. Guidance

Note: VS personnel includes personnel VS contracts to perform quarantine oversight 

functions; they must physically collect all samples for disease testing. This function may not 

be delegated to the facility’s representatives.

A. Instructions for Specimen Collection from Eggs Imported for Hatching

1) VS generally does not require sampling eggs before hatching. If VS determines that 

hatchlings show signs of illness, the NVSL may be consulted for guidance on

specimen collection/submission procedures of any unhatched eggs.

2) VS personnel must maintain a daily log (VS Form 17-12 or equivalent, as approved 

by a VS representative). Any mortalities, hatchings, etc., must be accounted for 

using this form or an approved equivalent. See VS Guidance 13401.2 for 

recordkeeping requirements for poultry or hatching eggs during a quarantine period.

3) Eggs that have not hatched at the end of the hatching cycle, and which VS

determines do not need to be sent to the NVSL, must be destroyed via incineration 

or other sterilization method as allowed by local ordinances.
 United States Department of Agriculture

 Marketing and Regulatory Programs

 Animal and Plant Health Inspection Service

 Veterinary Services

 VS Guidance 13410.1

3

B. Instructions for Specimen Collection from Poultry Imported Live or Hatched from 

Imported Eggs

(Note: see Section C for swab sampling and pooling techniques.)

1) Sampling within 24 hours of arrival or hatching

a. Swab collection: Collect cloacal or oropharyngeal swab samples from all animals

(up to a maximum of 150) that die within 24 hours of arrival at the quarantine 

facility (or within 24 hours of hatching for eggs). The oropharyngeal (OP) site 

should be used for sample collection from chickens, turkeys, and game birds 

(gallinaceous species). For waterfowl use only the cloacal site.

b. Submit samples to the NVSL as described in section D.

2) Sampling during days 2-16 (from dead poultry only)

a. Tissue samples

1. Collect tissue samples from dead poultry on either days 2, 7, and 12 or days 

3, 8, and 13 (every 5th day).

2. Sample all dead birds up to a maximum of 30 dead poultry collected per 

individual collection day (and up to a maximum of 90 animals sampled 

between days 2 and 16).

3. Up to a total of 5 tissue specimens (from no more than 5 different animals of 

the same species) can be pooled into one tube. Do not pool tissue specimens 

from multiple species (for example, do not mix chicken with duck tissue 

specimens).

4. VS should instruct the importer, the quarantine operator, or both to hold all 

mortalities in a cooler in between days designated for VS sampling.

b. Swab samples

1. In addition to tissue samples, collect swab samples from dead poultry, one 

swab per animal, on either days 2, 7, and 12 or days 3, 8, and 13 (every 5th

day).Swab no more than 40 dead poultry per individual collection day.

2. Swabs from five animals (from the same species) should be pooled into one 

broth tube. Submit up to 40 samples (8 broth tubes) per collection day, with a 

maximum submission for the 3 collection days of 24 broth tubes which 

represents a maximum of 120 animals. If there are fewer dead animals, 

sampling numbers will be fewer. These numbers reflect maximums. If there is

much additional mortality, consult the NVSL to determine if sampling should 

be increased.
 United States Department of Agriculture

 Marketing and Regulatory Programs

 Animal and Plant Health Inspection Service

 Veterinary Services

 VS Guidance 13410.1

4

3) Sampling during days 7-14 (live poultry)

a. 75 live poultry should be sampled by swabbing only (one swab per animal) as a 

one-time event during days 7 through 14. (If the quarantined lot contains fewer 

than 75 animals, sample the whole shipment.)

b. Swab the oropharynx of non-waterfowl species (for waterfowl, swab the cloaca).

c. Any clinically ill poultry should be sampled first, with the remainder from 

apparently healthy animals.

d. Sample poultry located in different areas throughout the facility.

e. All species within the facility should be represented in the samples selected, but

do not pool more than one species together in a tube.

f. VS encourages collection between days 12 and 14 whenever possible.

4) If exceptional mortality occurs during days 2 through 16 (or later during the 

quarantine), additional sampling may be advisable. Personnel collecting samples 

should consult with the NVSL regarding unusual mortality events.

C. Tissue and Swab Sampling Protocols

1) General tissue collection from dead poultry

Remove spleen, trachea, lungs, and a portion of the terminal gut. Place the organs 

(or parts thereof, depending on the size of the birds) in 5.5 ml of brain-heart infusion 

(BHI) broth and freeze promptly. Ensure that BHI broth covers all tissue within the 

tube. (Note: Dead poultry should be collected daily (by the importer) and refrigerated

per VS Guidance 13401.2.)

2) General swab collection from dead poultry: 

Sampling must be vigorous to obtain tissue of the cloaca or oropharynx. Use one 

swab per animal. Do not use cotton swabs. The NVSL recommends pooling five 

swabs per tube of broth; however, do not pool swabs collected from different 

species, and do not pool tracheal and cloacal swabs. If VS personnel need further 

instructions on how to collect swabs, they should consult the NVSL “Disease–

Specific Guide to Sample Collection.” Note: The oropharyngeal (OP) site should be 

used for sample collection from chickens, turkeys, and game birds (gallinaceous 

species). Only the cloacal site should be used for waterfowl.

3) VS offices must plan collections to ensure samples arrive at the NVSL during the 

business week (unless otherwise prearranged with the NVSL).
 United States Department of Agriculture

 Marketing and Regulatory Programs

 Animal and Plant Health Inspection Service

 Veterinary Services

 VS Guidance 13410.1

5

Table 1 Summary of Sampling

* Pooled is defined as collected tissues or swabs from no more than 5 different animals of the 

same species.

Time Submission 

Type

Sample types

Tissue or Swabs

Max. # 

poultry 

sampled

Maximum #

pooled* samples 

sent to NVSL

Notes

Arrival-24 

hours Dead poultry Swabs from cloacal or oropharyngeal sites 150 30

Days 2-16

(sample 

every 5th 

day)

Dead poultry

Tissue:

Sample no more than 

30 dead animals per 5-

day period

90 18

If less than 30 

dead per 

sampling 

period sample 

all

Swabs:

Sample no more than 

40 dead animals per 5 

day period

120 24

Days 7-14

(one time 

sampling)

Live poultry Swabs:

cloacal or 

oropharyngeal

75 15

Sample the 

whole 

shipment, if

less than 75

animals

Total 435 87 pooled 

samples
 United States Department of Agriculture

 Marketing and Regulatory Programs

 Animal and Plant Health Inspection Service

 Veterinary Services

 VS Guidance 13410.1

6

D. Packing, Shipping, and Identification of Specimens

1) Pack samples in dry ice (or with multiple frozen gel packs) in a standard TC-34 

specimen mailer or other replacement form approved by the NVSL. Tighten 

specimen tube caps securely to avoid specimen leakage in transit.

2) To prevent contamination of outer shipping specimen containers, store them outside 

the quarantine area in the facility. 

3) For this guidance, a submission consists of all of the specimens collected on a 

given date. Identify samples with consecutive numbers. Ensure the sample 

identification number on the sample label matches the numbers listed on VS Form 

10-4.

4) All blocks on VS Form 10-4, “Specimen Submission,” should be filled out as 

completely as possible. Record all information related to the given group of birds on 

VS Form 10-4 or current equivalent. The following items are of particular importance: 

a. Block 9: Examinations Requested -- Newcastle disease and avian influenza by 

virus isolation.

b. Block 15: Referral Number assigned by the submitter; serves as the submitter’s 

own reference.

c. Block 21: Identification – assigned by the submitter. Should be consecutive 

numbers, and must correspond with the number placed on the tube.

d. Block 22: Additional Data: Import permit number, expected release date or any 

other optional information to provide the NVSL about the specimens. The last 

submission from the quarantined lot should include the statement, "LAST 

SUBMISSION FROM THIS QUARANTINE." 

5) Specimens should be frozen as soon after collection as possible. Place tubes with 

submission forms (VS Form 10-4 or current equivalent) in a sealed plastic bag.

Disinfect the bag with a 2 percent solution of One Stroke Environ, Virkon S, or an 

APHIS-approved disinfectant, before removing it from the quarantine facility. Place 

the sealed and disinfected bag in the shipping container that has been held outside 

the bird holding area. Pack submissions from one quarantine station only in the 

shipping container. Shipment will be made by the most expedient courier service

available and consistent with current NVSL protocols. Current shipping requirements 

and mailing addresses can be found on the NVSL Web site.
 United States Department of Agriculture

 Marketing and Regulatory Programs

 Animal and Plant Health Inspection Service

 Veterinary Services

 VS Guidance 13410.1

7

E. Reporting Laboratory Results for Imported Poultry

1) The NVSL will generate a laboratory report for each submission received.

2) VS personnel responsible for quarantine oversight will:

a. Be solely responsible for furnishing laboratory results to the importer and to the 

quarantine facility. Only final and conclusive laboratory results should be 

released to the importer.

b. Report positive laboratory results of Newcastle disease and/or avian influenza to

the importer via a written report on receipt of the laboratory report. The written 

report will list the location of the quarantine, the importer’s name and contact 

information, the species involved, the date of the collection, and the date of viral

isolation by the NVSL.

c. Notify the supervising director by phone and email of any positive laboratory 

results for Newcastle disease or avian influenza.

d. Inform the importer when virus isolates, other than Newcastle disease or avian 

influenza virus, are made; and/or whether embryo (hatching egg) deaths that 

may occur during quarantine require additional laboratory work and additional 

user fee charges.

e. Inform the importer when additional laboratory work will result in holding the 

poultry beyond the minimum 30-day quarantine period.

f. Furnish copies of laboratory reports to the importer on request. 

3) The supervising director will notify the VS National Import Export Services Director of 

Live Animals, Riverdale, MD, by phone and email of any positive laboratory results

for Newcastle disease or avian influenza.

F. Disposition of Poultry in the Event of Positive Lab Results

1) The VS administrative unit in the field or the VS staff responsible for quarantine 

oversights will consult with the supervising director on options to dispose of the 

shipment.

2) The VS administrative unit in the field or VS staff responsible for quarantine 

oversights will notify the importer in writing of the final disposition decision and any 

associated user fees.

7. Inquiries

Questions should be directed to the avian import staff officer at 301-851-3300, opt.2, or by email 

at VS-Live.Animals_Import.Permits@aphis.usda.gov.