Jun 01, 2026

Protocol for collection of various environmental sample types for screening of Influenza A viruses

DOI
https://dx.doi.org/10.17504/protocols.io.n2bvjk7ybgk5/v1
  • Bhumika Prajapati1,
  • Rameshchandra Pandit1,
  • Amrutlal Patel1,
  • Abhinandan Patnaik1,
  • Shewane Bishnoi1,
  • Tejas Shah1,
  • Kalpesh Katudia1,
  • Roselin Neihsial1,
  • Jinal Thakor1,
  • Jill Gada1,
  • Rajvi Der1,
  • Parth Pandit1,
  • Kunjan Panchal1,
  • Bhagirath Dave1,
  • Debashrita Mittra1,
  • Harshal Purohit1,
  • Snehal Bagatharia1,
  • Amit Kanani2,
  • Dhwani Pandya1,
  • Madhvi Joshi1,
  • Chaitanya Joshi1
  • 1Gujarat Biotechnology Research Centre, Gandhinagar , Gujarat;
  • 2Deputy Director , Directorate of animal Husbandry , Government of Gujarat
Amrutlal Patel
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DOI: https://dx.doi.org/10.17504/protocols.io.n2bvjk7ybgk5/v1
Protocol Citation: Bhumika Prajapati, Rameshchandra Pandit, Amrutlal Patel, Abhinandan Patnaik, Shewane Bishnoi, Tejas Shah, Kalpesh Katudia, Roselin Neihsial, Jinal Thakor, Jill Gada, Rajvi Der, Parth Pandit, Kunjan Panchal, Bhagirath Dave, Debashrita Mittra, Harshal Purohit, Snehal Bagatharia, Amit Kanani, Dhwani Pandya, Madhvi Joshi, Chaitanya Joshi 2026. Protocol for collection of various environmental sample types for screening of Influenza A viruses. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjk7ybgk5/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 04, 2026
Last Modified: June 01, 2026
Protocol  Integer ID: 316287
Keywords: influenza, various environmental sample type, collection of various environmental sample type, applicability of different environmental sample type, environmental sample type, different environmental sample type, traditional clinical surveillance, environmental surveillance, complement to traditional clinical surveillance, poultry farm setting, standard biosafety measure, screening, useful in the surveillance setting, wetland, surveillance setting, virus
Funders Acknowledgements:
Gates Foundation
Grant ID: INV-064425
Abstract
Environmental surveillance has been identified as a cost‑efficient complement to traditional clinical surveillance for influenza A viruses (IAV). Literature suggests the applicability of different environmental sample types to be particularly useful in the surveillance settings where clinical  sampling is logistically challenging. This protocol provides a standard operating procedure (SOP) for collection of various  environmental sample types (surface water, water sediment, molted bird feathers, bird droppings, air etc.,) from wetland and poultry farm settings. The SOP also includes standard biosafety measures and quality assurance steps to ensure reproducibility across the studies and settings.
Image Attribution
Figure 1: Stock images of a) Wetland surface water collection, b) Poultry drinking water Collection
Figure 2: Stock images of a) Wetland faecal sample (bird droppings) collection, b) Poultry faecal sample collection

Figure 3: Sample image highlighting the feather region (quill/calamus) to be dipped in VTM during collection

Figure 4: Stock image of air sampling from wetlands

Figure 5: Stock images of electrostatic dust cloth (EDC) swabs collected from different poultry surfaces a) & b)

Figure 6: Stock images of poultry litter sample collection
Materials
1 Sterile 50 mL centrifuge tubes
2 Viral transport media (VTM)
5 Sterile wooden spoons
4 Liter jerry cans
5 Sterile gloves
6 Sterile N95 masks
7 Biohazard discard bags
8 Portable 4 ºC refrigerator
9 Portable air sampler (MD8 Airport portable air sampler, Sartorius) with sterile gelatin membrane filters 12602--80----ALK Gelatin membrane filter; Pore size: 3 microns, Diameter: 80 mm 10 Sterile Electrostatic dust cloth (5 cm x 5 cm blocks)
A Sample Collection of Wetland surface water/Poultry farm drinking water
Equip hands with sterile rubber gloves and conceal face with a fresh N95 mask to avoid accidental exposure or cross contamination during the sampling process. (Note: Sterilize gloves with 70-80% ethanol before initiating the sample collection)

Select the sampling locations within wetlands lake based on high bird activity to maximize the likelihood of detecting AIV shed into surface water.
Select the sampling locations within wetlands lake based on high bird activity to maximize the likelihood of detecting AIV shed into surface water.
Collect a minimum of 500 mL of water sample from the wetland waterbody surface/poultry water in a sterile 1 L jerry can. (Note: Sterilise gloves post sample collection with 70-80% ethanol).
Label collected samples clearly using waterproof permanent markers directly on the sample container (tube/bottle) including essential details:
○ Sample type
○ Location/site name
○ Date of collection
Post-collection, store the collected water samples in a potable refrigerator (4ºC) to maintain the cold chain, essential for sample preservation.
Discard used gloves and mask in a biohazard bag immediately after sample collection.
Use a fresh pair of sterile gloves and a mask for subsequent sample collection sites.
B Sample Collection of Bird droppings/ faecal matter from wetlands and poultry farms
Follow personal safety precautions as mentioned earlier in section A.
Try to collect the fresh fecal material if possible
Collect approximately 2-3 g of faecal matter in 3 mL of VTM using sterile autoclaved wooden spoons. (Note: Quantity of faecal matter may be augmented relative to the volume of sampling vial being used)
Label collected samples clearly using waterproof permanent markers directly on the sample container (tube/bottle), including essential details:
○ Sample type
○ Location/site name
○ Date of collection
Immediately store the collected sample VTMs in an ice box maintained at 4 ºC until further sample processing.
Discard the used gloves, mask and wooden spoons in biohazard bags post sample collection.
C Sample type: Molted feathers
Follow personal safety precautions as mentioned earlier in section A.

Select the sampling locations within wetlands based on high bird activity to maximize the likelihood of detecting AIV from molted feathers in wetlands.
Collect 3 to 5 feathers and store in 3 mL VTM from a single sampling site, preferably molted from different bird species in the wetland setups. (Note: Make sure that the quill or calamus is dipped in the VTM)
The same sampling strategy is to be followed for feather collection from poultry fowls
Label collected samples clearly using waterproof permanent markers directly on the sample container (tube/bottle) including essential details:
○ Sample type
○ Location/site name
Date of collection Immediately store the collected VTMs containing feather samples in an ice box maintained at 4 ºC until further sample processing
Discard the used gloves and mask in biohazard bags.
D. Sample type: Wetland Pond sedimentation
Follow personal safety precautions as mentioned earlier in section A.
Collect sediment soil samples from banks of wetland water bodies in sterile 50 mL centrifuge tubes. (Note: Try collecting soil samples from different locations at the same sampling site to cover the maximum area)
Label collected samples clearly using waterproof permanent markers directly on the sample container (tube/bottle) including essential details:
○ Sample type
○ Location/site name
○ Date of collection
Store the collected sediment samples at 4 ºC until further pre-processing.
Discard the used gloves and mask in biohazard bags.
E. Sample type: Air sample from wetlands and poultry farms
Place the air sampler at a minimum height of 1 meter (use a tripod stand if available or hold the air sampler manually at a constant height and location) above ground level and within 1–2-meter distance ranges from the collection site.
Place a sterile gelatin filter (Pore size: 3 microns, Diameter: 80 mm) using the provided fitting apparatus for the air sampler using sterile forcep making sure that thefilter does not break or crack during the installation. (Note: Make sure that the gelatin filter is handled and installed while wearing sterile gloves)
Switch on the air sampler for 20 minutes with preset collection parameters (50 L/minutes), to collect a total air volume of 1000 L.
Remove the gelatin filter from the air sampler using clean forcep and collect it in 6 mL of VTM. Gently shake the VTM vial to dissolve the collected gelatin filter.
Label collected samples clearly using waterproof permanent markers directly on the sample container (tube/bottle) including essential details:
○ Sample type
○ Location/site name
○ Date of collection
Store the VTM vial at 4 ºC until further processing.
Repeat the same method at different locations, making sure that the air sampler is sterilised with 70-80% ethanol for each new sampling location.
F. Sample type: Electrostatic dust cloth (EDC) swab (from Poultry farm surface)
Follow personal safety precautions as mentioned earlier in section A.
Use the prepared sterile electrostatic dust cloth pieces to collect swabs from surfaces of poultry farms. (Note: Make sure to cover maximum area around the poultry environment)
Use different EDC swabs for different locations or depending on the number of cages present in a particular poultry farm.
Submerge the collected swab in sterile 50 mL centrifuge tubes.
Label collected samples clearly using waterproof permanent markers directly on the sample container (tube/bottle) including essential details:
○ Sample type
○ Location/site name
Date of collection
Store the collected swab at 4 ºC until further pre-processing.

Discard the used gloves and mask in biohazard bags.
G. Sample type: Poultry litter
Follow personal safety precautions as mentioned earlier in section A.
Collect approximately 2-3 g of poultry litter in 3 mL of VTM using
Label collected samples clearly using waterproof permanent markers directly on the sample container (tube/bottle) including essential details:
○ Sample type
○ Location/site name
Immediately store the sample VTMs in an ice box maintained at 4 ºC
Discard the used gloves, mask and wooden spoons in biohazard bags post sample collection.
Protocol references
1) Nazir J, Haumacher R, Ike AC, Marschang RE. Persistence of avian influenza viruses in lake sediment, duck feces, and duck meat. Applied and environmental microbiology. 2011 Jul 15;77(14):4981-5. https://doi.org/10.1128/AEM.00415-11

2) Spekreijse D, Bouma A, Koch G, Stegeman A. Quantification of dust‐borne transmission of highly pathogenic avian influenza virus between chickens. Influenza and other respiratory viruses. 2013 Mar;7(2):132-8. https://doi.org/10.1111/j.1750-2659.2012.00362.x

3) Nikitin N, Petrova E, Trifonova E, Karpova O. Influenza virus aerosols in the air and their infectiousness. Advances in virology. 2014;2014(1):859090. https://doi.org/10.1155/2014/859090

4 ) Marty-Quinternet S, Puget L, Debernardi A, Aubry R, Magy-Bertrand N, Prétet JL, Chirouze C, Bouiller K, Lepiller Q. Electrostatic wipes as simple and reliable methods for influenza virus airborne detection. Journal of Hospital Infection. 2021 Feb 1;108:15-8. https://doi.org/10.1016/j.jhin.2020.10.030

5) Azeem S, Guo B, Sato Y, Gauger PC, Wolc A, Yoon KJ. Utility of feathers for avian influenza virus detection in commercial poultry. Pathogens. 2023 Dec 7;12(12):1425. https://doi.org/10.3390/pathogens12121425
Acknowledgements
This study was funded by the Gates Foundation . We also would like to thank the GEER(Gujarat Ecological Education and Research) Foundation, Department of Forest and Environment ,Gandhinagar, for their support in wetland sampling.