Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Summary

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Abstract

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

Introduction

Influenza A viruses (IAV) cause respiratory disease in many species, including domestic birds, swine, and humans. Due to reassortment of the segmented IAV genome rapid viral evolution can occur and new IAV strains frequently emerge. Swine are a species that can serve as a mixing vessel for reassortment of IAVs from multiple host species.¹ There are currently three major subtypes of IAV commonly circulating among North American swine (H1N1, H1N2, H3N2), but multiple IAV introductions from humans have led to extensive IAV diversity within those subtypes.² Rapid evolution of IAVs infecting swine has been evident since 1998 when a triple reassortant IAV containing gene segments from human, avian and swine viruses³ became widespread among pigs in the United States.⁴ The internal gene segments from that triple reassortant IAV remain highly prevalent among IAVs currently infecting swine.⁵

Worldwide, IAV is a significant cause of respiratory disease in swine in which typical clinical signs include fever, anorexia, lethargy, coughing, labored breathing, sneezing, nasal discharge and poor weight gain. IAV can be particularly costly to sow farms where reproductive failure due to IAV induced fever and weak-born piglets have been documented.^6,7 Within the United States, IAV is commonly detected in commercial swine herds and the extensive antigenic and genomic diversity and continuing evolution among IAV infecting swine has hampered control of this virus.^8-11

Public health concerns about the emergence of a pandemic IAV strain resulting from reassortment in swine were realized in 2009 when a swine-lineage IAV containing gene segments from the triple-reassortant North American swine lineage and the Eurasian avian-like swine lineage caused a worldwide pandemic in humans.¹² The pandemic virus (A (H1N1)pdm09) has since reassorted with endemic swine IAV strains^13,14 and some of these newly reassorted strains have been transmitted back to humans.¹⁵ The frequency of reassortment events and emergence of new IAV strains with pandemic potential makes active surveillance of circulating IAV viruses in swine imperative, especially at the swine-human interface.

The swine-human interface is important for bi-directional interspecies transmission of IAV. Human-to-swine transmission occurring in commercial swine production is responsible for a large amount of IAV diversity currently present in the swine population. Agricultural fairs are the largest settings for the comingling of people and swine in the United States and are known sites for the zoonotic transmission of IAV.^15-21 In 2012, during the outbreak of a variant H3N2 IAV, 93% of cases reported attendance at an agricultural fair in the days prior to onset of disease.¹⁵ Genomic analysis of viral isolates from exhibition pigs compared with human isolates confirmed zoonotic transmission.²¹ Exhibition swine infected with IAV often do not show clinical signs of illness,^21-23 indicating the need for direct diagnostic tests.

Sampling of visibly ill pigs alone will not successfully identify IAV prevalence in swine and cannot be relied on to identify new strains of IAV emerging among swine. Active surveillance is absolutely necessary for detection of emerging strains of IAV in swine and to assess their threat for both swine and public health. Most IAV surveillance activities are voluntary and therefore minimally disruptive methods are needed. The three major ante-mortem sample collection procedures for IAV infecting swine are: nasal swabs, oral fluids, and nasal wipes. Current recommendations for sampling individual swine to detect IAV list the insertion of synthetic-fiber tipped swabs into the nostrils as the preferred method to gather nasal secretions and epithelial cells.^24,25 Because pigs may try to avoid this procedure, a team of trained personnel must restrain pigs either manually or with a snare depending on the size of the animal.²⁶ The restraint process is laborious for the personnel, and stressful for the pigs. Additionally, exhibition swine are often involved in multiple competitions at a fair so the perception of added stress on a competition animal can make owners resistant to the surveillance efforts.

With probabilities of IAV detection ranging from 80-100% in IAV infected herds, oral fluids have become a popular alternative to nasal swabs for the molecular detection of IAV in populations of swine.^27,28 Additionally, oral fluids may provide a wider window of IAV detection than nasal swabs following initial infection. However, IAV isolation from oral fluids has been problematic with only 50% of virus isolation attempts resulting in IAV recovery.²⁹

Using nasal wipes in lieu of nasal swabs during IAV surveillance in swine overcomes the limitations described above. Nasal wipes do not require the use of a restraining snare and can be performed without stressing the animals or witnesses of the procedure. Minimal technical training is needed to collect nasal wipes, which allows non-veterinary professionals, including swine owners, to collect the surveillance samples. Nasal wipes were previously compared to nasal swabs for the detection and isolation of influenza A virus³⁰ and the detailed protocol for this non-invasive method of sampling is described below.

Protocol

All swine used in the collection of the following data were protected under The Ohio State University Institutional Animal Care and Use Committee (animal use protocol number 2009A0134-R1).

1. Preparation of Viral Transport Medium and Sample Collection Vials

1. Add 37 g of Brain Heart Infusion to 900 ml of purified water and mix thoroughly with a stir bar and magnetic stirrer while heating to 70 °C to completely dissolve the powder.
2. Autoclave the broth at 121 °C for 15 min. Cool on the bench top until the broth reaches room temperature. Refrigerate at 4 °C overnight if needed.
3. Dissolve, by mixing with a magnetic stir bar, 6.02 g of Penicillin G sodium salt and 10 g of streptomycin sulfate into 50 ml of purified water at room temperature. Add additional purified water to bring the total volume to 100 ml. Filter the solution through a 0.22 µm polyethersulfone membrane into a sterile bottle.
4. Aseptically add the filtered penicillin and streptomycin solution into the cooled Brain Heart Infusion Broth and mix thoroughly (stir bar or swirl flask by hand) to make the viral transport medium.
5. Aseptically test the pH of the viral transport medium; if needed, adjust the pH to 7.4 ± 0.2 using HCl or NaOH.
6. Perform quality assurance testing on the viral transport medium by testing for influenza A virus matrix protein by rRT-PCR, as described by Zhang and Harmon.²²
7. Dispense 5 ml of the viral transport medium into sterile high-density polyethylene bottles with 8 ml capacity. Label the vials for use in the field.
8. Store the ready-to-use vials in standard cryo-boxes at -20 °C until sample collection.

2. Collection of Nasal Wipes from Swine

1. Thaw the appropriate number of vials immediately prior to sample collection; one vial will be needed per pig sampled. Thawing at room temperature takes approximately 30-45 min. Keep the thawed vials cool on ice packs until use.
2. Don appropriate personal protective equipment as dictated by the situation (e.g. coveralls, boot covers, respirator, ear plugs, etc.).
3. Enter the animal area. If needed, confine the pig to a small area but do no restrain. Do not rouse resting animals.
4. Put on a pair of disposable exam gloves. Take care not to contaminate the gloves by touching pigs, people, or inanimate objects.
5. Use a gloved hand to remove a sterile 5.08 cm × 5.08 cm (2 in. × 2 in.) cotton gauze pad from its wrapper. Sterile gauze pads should be used whenever possible. Individually wrapped gauze pads are more convenient to use than those wrapped with two gauze pads per package.
6. Hold the cotton gauze pad with the fingertips of one hand exposing as much of the pad as possible for sampling.
7. Wipe the gauze pad across the pig’s snout, taking extra care to enter the external nares if possible. Collect visible nasal secretions (approximately 1 ml) with the same gauze pad.
8. Using the same hand, fold the gauze pad upon itself to facilitate placement in the vial containing viral transport medium.
9. Using the other hand, remove the cap from the vial and place the folded gauze pad into the vial. Recap the vial and shake the vial to ensure the mixing of the gauze pad and viral transport medium.
10. Remove the gloves and place in an appropriate receptacle. Change gloves between each pig.
11. Verify the vial identifier. Record pig identification number, age, sex, clinical signs, and other notes deemed necessary by investigator.
12. Chill the samples immediately after collection. Consider making aliquots of the sample before freezing to prevent multiple freeze-thaw cycles. As soon as possible after collection, place the samples on dry ice for transportation to the laboratory. Store the vials at -80 °C until testing is initiated.

3. Detection of Influenza A Virus Nucleic Acid 

1. Thaw the samples in a 37 °C dry bead bath for 5 min and then finish thawing the samples on the bench top at room temperature for 20-30 min. The simultaneous addition of several vials to the beads will cause the bath to heat; caution should be exercised as to not overheat the vials.
2. Remove 100 µl of viral transport medium for RNA extraction.
   1. Use a high-throughput (96 well plate format) magnetic bead platform for extracting RNA from a large number of samples.³¹
3. Use real-time reverse transcriptase PCR assays for the rapid detection influenza A virus.³¹

4. Isolation of Influenza A Virus from Nasal Wipes

1. Thaw the samples as described above in Section 3.1
2. Treat thawed samples with gentamicin sulfate (1,000 μg/ml), amphotericin B (22.5 μg/ml), and kanamycin sulfate (325 μg/ml).
3. Inoculate into Madin-Darby Canine Kidney (MDCK) epithelial cells as described previously.³²

Results

Successful use of this method yields rRT-PCR results that, accompanied with the use of an internal control during RNA extraction and rRT-PCR, show samples did not contain PCR inhibitors from any environmental debris picked up during sampling. After sample inoculation, virus isolation wells should be free of visible environmental debris from the sample. There is reasonable agreement between rRT-PCR results and virus isolation results with the understanding that PCR often yields a higher IAV positive rate than virus isolat...

Discussion

Collecting samples from pigs using polyester-tipped nasal swabs has proven useful in conducting IAV surveillance; however, the use of the nasal swab procedure hinders surveillance efforts due to the required use of a snare for restraint. Nasal wipes represent a refinement of current swine sampling techniques to minimize stress on people and pigs during sample collection. While the method has been developed for and validated in exhibition swine settings, it can be easily applied to other situations where ante-mortem detec...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BBL Brain Heart Infusion	Becton, Dickinson and Company	211059
Penicillin G Sodium Salt	MP Biomedicals, LLC	021194537	1500 u/mg
Streptomycin Sulfate	AMRESCO LLC	0382
Gentamicin Solution	Mediatech, Inc.	30-005-CR	50 mg/ml
Amphotericin B Solution	Fisher	S 24 25	250 ug/ml
Kanamycin Sulfate	Teknova	K2105	5000 ug/ml
TPP Rapid Filtermax System	TPP Techno Plastic Products AG	99150
Nalgen Diagnostic Bottles	Thermo Scientific	342002-9025	HDPE with white PP closure
Dermacea Gauze Sponge, 8 ply	Covidien	441211	5.08 cm × 5.08 cm (2 in. × 2 in)
Nitrile Exam Gloves	Saftey Choice	19-170-010 (A-D)