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Avian Sample Collection for Influenza A and Newcastle 

Disease

January 3, 2023

This document contains sample collection recommendations from the National Veterinary Services 

Laboratories (NVSL) in Ames, IA, specifically for the detection of avian influenza A viruses (IAV) and 

avian paramyxoviruses (APMV-1), such as Newcastle disease (ND). This document supersedes all 

previous versions (WI-AV-0020) of Avian Sample Collection for Influenza A and Newcastle Disease

(now NVSL-WI-0023). 

• For guidance on the appropriate use of antigen capture immunoassay for influenza A, refer to 

https://www.aphis.usda.gov/animal_health/emergency_management/downloads/hpai/acia_testpolic

y.pdf 

• For guidance on the collection of environmental samples, please refer to the Post C&D 

Environmental Sampling Guidance. 

• For foreign animal disease (FAD)1 investigations, the fastest route for confirmation is by the 

collection of duplicate samples for submission to a National Animal Health Laboratory Network 

(NAHLN) lab and NVSL in parallel – refer to the Foreign Animal Disease (FAD) Investigation 

Manual (FAD PReP Manual 4-0) for further guidance. 

• For FAD investigations a minimum of two pools of 11-bird swab samples for gallinaceous poultry 

or four pools of 5-bird swab samples for any avian species are recommended.

1.1. Viral Transport Media (VTM)

1.1.1. Brain heart infusion broth (BHI; e.g., BD Bacto #237400) is the recommended VTM for 

these specimens as it contains a protein component which protects the virus from 

degradation during storage and shipping and is available from the NVSL for national avian 

influenza testing; the order form can be accessed here – refer to Table 1 – in summary: 

• 3 ml BHI Broth (40 tubes/box; no antibiotics): blue cap plastic tube for up to 5-bird 

swab pools from avian species.

• 5.5 ml BHI with antibiotics (40 tubes/box): black cap glass tube for up to 5-bird swab

pools for any avian species, and up to 11-bird swab pools from gallinaceous poultry 

only (refer to 3.1.2.); may also be useful for environmental samples. 2

• NOTE: BHI when stored appropriately and visually inspected is fit-for-purpose for 

several years. 

1.1.2. Other acceptable VTM include any salt-balanced, buffered media with a protein component 

such as tris-buffered tryptose broth (TBTB)3, nutrient broth (NB), and peptone broth (PB); or 

commercially available media, e.g., BDTM Universal Viral Transport 3 mL Collection Kit, and 

Primestore MTM (currently for Wildlife Services wild bird surveillance; and where approved 

by APHIS – contact NVSL for appropriate sample volume).

1 www.aphis.usda.gov/fadprep

2 Refer to HPAI Response Post C&D Environmental Sampling Guidance - Poultry May 5, 2022

https://www.aphis.usda.gov/animal_health/emergency_management/downloads/hpai/env_sampling_proced.pdf 

3 TBTB Formulation (NVSL media #10088): 1.21 g Trizma Base 77-86-1, 26 g Tryptose Broth, 1000 ml QH2O
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1.1.3. If not an FADI, In the absence of appropriate VTM, phosphate buffered saline (PBS) or 

saline solution (contact lens solution – not disinfectant) may be used as a last resort to keep 

the swab moist during transport – dry swabs are not acceptable (refer to 1.1.4.); NOTE:

PBS or saline should only be used when none of the preferable media are available as

negative results are not acceptable for confirmation of disease status; appropriate 

samples should be obtained. 

1.1.4. Dry swab specimens should be avoided – heat and desiccation can inactivate IAV and ND 

in ≤24 hours; therefore, negative results are not acceptable for confirmation of disease 

status; appropriate samples should be obtained.

2.1. Swab collection (Figure 1) 

• Target sample collection from birds with the following priority 4: 

1) Recent mortalities

2) Sick birds 

3) When birds specified in items #1 or #2 above are not available and sampling of 

apparently healthy birds is needed, target birds next to building inlets or in cages 

adjacent to sick/dead birds

• Use synthetic or semi-synthetic swabs (e.g., polyester, rayon, nylon) with a plastic handle 

(flocked or spun head).

• Avoid cotton or calcium alginate swabs or swabs with wooden handles which have been 

shown to inactivate virus and inhibit PCR invalidating the laboratory test results.

2.1.1. Oropharyngeal (OP) swabs are preferred for gallinaceous poultry (Figure 1b-1d) 

• Swab the oral cavity and opening of the trachea, avoiding the esophagus, and bring 

the swab up through the choanal cleft where the sinuses drain to capture material from 

the upper respiratory tract (refer to Figure 1a).

• Tracheal swabs (TR), if needed, are best obtained from fresh carcasses.

2.1.2. Cloacal (CL) swabs are preferred for domestic waterfowl and other wild birds unless H5 

goose/Guangdong lineage viruses are suspected in which case both OP and CL swabs are 

recommended (Figure 1e). 

2.1.3. The NVSL prefers submission of the entire swab suspension for diagnostic testing. After 

collecting the sample, swirl the swab vigorously in the VTM, squeeze the excess liquid from 

the swab inside the specimen tube and collect the swab in an appropriate container for 

proper disposal at the laboratory. Avoid leaving swabs or other collection devices in the 

tube; swabs left in the media may reduce the volume available for testing.

2.1.4. Clearly label containers with appropriate ID using a waterproof marker or other label 

(barcode ID labels are available from NVSL on the same request form used for BHI).

3.1. Pooling procedures5

3.1.1. Swab samples may be pooled in accordance with Table 1 and by:

• the same species, 

• the same premises, and

4 Refer to HPAI Red Book,

https://www.aphis.usda.gov/animal_health/emergency_management/downloads/hpai_response_plan.pdf.

5 Pooling procedures are based upon work conducted by Spackman et al. in coordination with the NVSL 

(BMC Veterinary Research 2013, 9:35 http://www.biomedcentral.com/1746-6148/9/35). 
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• the same sampling route – do NOT pool TR/OP and CL together, 

• additionally, pool mortalities, sick birds, and apparently healthy birds separately

3.1.2. The 5-swab pool in at least 3 mls of VTM was validated for both TR/OP and CL swabs from 

gallinaceous poultry and domestic ducks tested for IAV and ND; collection of up to 6 swabs 

in 3 mls VTM following the guidance in 3.1.1. allows for collection of the recommended 11 

samples per Secure Supply plans and NPIP surveillance using two 3ml VTM tubes rather 

than three. 6 

• A single 11-bird swab LAB POOL may be generated from one 5-bird swab pool and 

one 6-bird swab pool at the testing laboratory (testing labs should refer to NVSL 

SOP-AV-0068 for further details).

3.1.3. The 11-bird swab pool is only valid for IAV/ND testing of TR/OP swabs from 

gallinaceous poultry and must be in 5 mls BHI; NOTE: This has not been evaluated for 

other diseases such as infectious bronchitis, infectious laryngotracheitis, or mycoplasma. 

NOTE: The NVSL supplies 5.5ml BHI which contains antibiotics and is not appropriate for 

testing of bacterial diseases. For FADIs, a minimum of 2 11-bird swab pools per barn is 

required; or 4 5-bird swab pools for non-gallinaceous species. 

3.1.4. Tissues: pool by system (respiratory, enteric, reproductive) typically from a single bird; it is 

not recommended to pool tissues from more than one bird especially for free-living 

waterfowl.

4.1 Specimen transfer and storage

• Maintain cold chain for all samples. 

• Specimens should be held on ice pack immediately following collection until transferred to 

the testing laboratory or other refrigerated storage.

• Tubes should be stored and transferred in an upright position to reduce chances of 

leakage.

• IAV and APMV-1 have been shown to be stable in BHI when stored at refrigeration (4oC) 

for up to 96 hours, with consideration given to the length of time needed at the laboratory 

for sample processing.

• If samples have been frozen (-70oC), they should remain frozen until delivered to the 

testing laboratory.

• Specimens should never be stored in the freezer portion (-20oC) of a standard 

refrigerator/freezer unit with an automatic defrost cycle (specimens will go through 

freeze/thaw, which is detrimental to the survival of virus and viral nucleic acid).

5.1 Forwarding samples to the NVSL in Ames, IA

5.1.1. Email ahead of shipment to: nvsl.ai.nd@usda.gov - Please include: 1) number of samples, 

2) 10-4 form7, 3) tracking number, 4) pertinent case and contact information on the day the 

package is shipped. Please use 8 AM delivery option if shipping by FedEx.

a. For FAD1 investigations or samples from poultry, include in the email subject line: 

6 While pools of 6 swabs have not specifically been evaluated, it is considered an acceptable practice 

where seeking to conduct recommended 11-bird swab surveillance using 2 pooled samples instead of 3 

(refer to 3.1.3); NOTE: Testing of a single 11-bird swab pool only applies to TR/OP swabs from 

gallinaceous poultry. 

7 http://www.aphis.usda.gov/library/forms/pdf/VS_Form10_4.pdf 
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State FAD reference number (or 2-digit state if no FAD number)/Priority X/Fed Ex 

tracking #. 

b. If wildlife, email subject line should contain: 

2-digit State/ wild bird/Fed Ex tracking #. 

5.1.2. If the investigation is Priority 1 or A, email first to nvsl.ai.nd@usda.gov. If there is no reply, 

it is critical to directly speak to someone in Diagnostic Virology at the NVSL, call 515-337-

7551 (after normal business hours the number will roll to security personnel who can get in 

contact with relevant personnel). Other contacts for DVL can be found at the following site: 

USDA APHIS | NVSL Diagnostic Virology Laboratory. 

5.1.3. Please contact the Diagnostic Virology Laboratory at the number above for further 

instructions when sending driver/courier after hours/holidays/weekends. 
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Table 1. Preferred specimens for Influenza A and Newcastle disease diagnostics.

Sampling 

source

Preferred 

Specimen Sample Collection Comment

Gallinaceous 

poultry (e.g., 

chickens, 

turkeys, 

pheasants, 

quail)

Tracheal or 

oropharyngeal 

(TR/OP) 

preferred

• FOR FADs – typically 5 swabs/ 

pool in at least 3 mls of VTM 

• Up to 11 swabs/pool in at least 

5mls of VTM pooled is valid only 

for TR/OP swabs from 

gallinaceous species a

Virus usually shed via respiratory 

route; may be strain dependent

Cloacal swab 

(CL) may be 

used

Up to 5 swabs/pool at least 3 mls of 

VTM pooled by sample route and 

species

Domestic 

waterfowl 

(production)

CL preferred, 

TR/OP swab 

may be used

Up to 5 swabs/pool from a single 

flock and species in at least 3 mls of 

VTM

Virus usually shed via the enteric 

route; may be strain dependent – e.g., 

for H5 goose/Guangdong lineage, 

both OP and CL are recommended

Wild/captive 

waterfowl 

speciesc 

TR/OP and CL

swabs may be 

used

• Collect USDA Wildlife Services

Surveillance samples by pooling 1 

CL and 1 OP swab from a single 

bird in one 3ml VTM tubeb

; this 

approach may also be used for 

captive waterfowl that are openly 

housed 

• Captive flocks in closed, common

housing may be pooled 5 

swabs/pool in at least 3mls VTM 

by sample route and species

Wild migratory waterfowl are the 

natural reservoir for influenza A 

viruses (typically enteric shed)

Other wild/free 

living/captive 

/pet species

Typically, CL 

swabs; fresh 

fecal samples 

may be used – 

call the NVSL 

for guidance

• Captive flocks in closed, common 

housing may be pooled 5 

swabs/pool in at least 3mls VTM 

by sample route and species

group (e.g., passerines)

Shedding of influenza from non-host 

species can be variable and 

dependent on other factors such as 

immune status and virus strain

Any avian 

species Tissue samples

Pool by system from a single bird 

(e.g., respiratory, enteric, 

reproductive) b - mince tissue and 

place in 3mls VTM

vND viruses may replicate to higher 

titres in tissues; brain tissue is 

preferred if neurological signs are 

noted a The 5-bird swab pool in 3 mls of VTM was validated for IAV and ND testing of both TR/OP and CL swabs from 

gallinaceous poultry and domestic ducks; pooling of up to 6 swabs from the same species, location, and sampling 

route in 3 mls allows for collection of 11 samples per Secure Supply plans and NPIP surveillance using two tubes 

rather than three. Either 3ml (any for domestic species) or 5.5 ml with abx (for TR/OP swabs from gallinaceous 

poultry only) may be used for zone surveillance. 

b Antibody from one bird may neutralize virus from another (e.g., mixed backyard poultry); avoid potential of mixing 

viruses from different birds in a single sample when sampling migratory waterfowl.

c The H5 goose/Guangdong HPAI viruses often shed higher via the OP route in waterfowl; sampling of both routes is 

recommended. 
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Figure 1. 

Swab collection: a) schematic of oral cavity; b-d) tracheal/oropharyngeal (TR/OP) swabs 

preferred for gallinaceous poultry; e) cloacal (CL) swabs preferred for domestic waterfowl and 

wild birds. From: Methods in molecular biology, vol. 436: Avian influenza virus. E. Spackman, 

ed. Humana Press, Totowa, NJ. pp. 95. 2008.
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Appendix 1: BHI Stability

Stability studies were conducted on 3 separate lots of BHI media containing antibiotics (NVSL media # 

50067) and 3 separate lots containing no antibiotics (media # 10009). Each lot of media was spiked with 

live virus then frozen and thawed two days later to mimic shipping conditions from field to lab. Virus 

isolation was conducted and harvested material was evaluated for bacterial contamination and 

confirmation of virus recovery. Testing was conducted three separate times for each of the 3 lots of media 

over the course of approximately one year. 

BHI broth was determined to be stable a minimum of 6 years from manufacture date. Expiration dates 

are provided on the outer box containing BHI tubes prepared at the National Veterinary Services 

Laboratories for quality management. The expiration of unopened BHI tubes may be extended in 6 month 

increments up to 6 years past expiration using the following criteria.

1) The expiration on the box applies to the tubes provided in the original shipment. Tubes can be 

individually labeled with the expiration date after receipt.

2) Individual laboratory policies for expired reagents should be used if applicable. If the policy is 

to NOT use expired reagents, then a deviation including a plan must be put into place to 

proceed. A laboratory may have a policy to requalify media or obtain documentation 

supporting an extended expiration. If there is no policy regarding expired media, follow the 

recommendations below.

3) BHI expiration can be extended as long as the unopened media has been stored at 4°C or 

colder (-20°C recommended), the tubes have not been compromised, no obvious color 

change is observed, and the media does not appear to be cloudy.

4) The following should be documented: storage conditions of the expired media since the time 

of receipt, who confirmed the criteria in bullet 3 and when the media was evaluated.