Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

The overall goal of this procedure is to collect nasal wipes from pigs for influenza, A virus surveillance, providing a low stress alternative to nasal swabs. This method can enhance surveillance and provide insights into the strains of influenza. A virus circulate among swine because it is simple to perform and does not require specialized training.

The main advantage of this technique is that the samples for diagnostic testing can be collected from individual animals with minimal restraint. The implications of this technique extend toward improving participation in influenza A virus surveillance programs, because a slow stress method is more appealing to swine owners. Though this method was originally developed to use with exhibition swine, it can be applied for virus surveillance and commercial swine farms, agricultural affairs, and live animal markets.

To begin first dissolve 37 grams of brain heart infusion, or BHI powder in 900 milliliters of purified water. Stir the solution at 70 degrees Celsius to dissolve the powder, completely autoclave the broth for 15 minutes in a liquid cycle, and then allow it to cool. Next dissolve 6.02 grams of penicillin, G, sodium salt, and 10 grams of streptomycin sulfate in 100 milliliters of purified water at room temperature inside the hood.

Filter the antibiotic solution through a 0.22 micron polyether cell phone membrane into a sterile bottle. Add all 100 milliliters of this solution to the 900 milliliters of BHI. Broth and mix well test the pH of the medium and adjust it to between 7.2 and 7.6 with concentrated sterile HCL or sodium hydroxide if required, perform a PCR assay with primer specific for influenza, A virus as described in the text protocol to make sure that the medium is free of any viral genetic material.

Dispense five milliliters of the viral transport medium into eight milliliter sterile polyethylene vials. Freeze the vials at minus 20 degrees Celsius in cryo boxes until sample collection. To prepare for sample collection, thaw an appropriate number of the transportation vials on ice.

One tube per pig. Transport the tubes and chilled ice packed containers prior to exposure to the animals. Wear biosafety level two personal protective equipment, including coverall, respirator, earplugs, and boot covers.

Enter the pen and confine the pig to a small area without restraining it. Using a gloved hand, remove a sterile gauze pad from its wrapper, holding the pad with only the fingertips. Wipe it across the pig's snout and external nares with as much pad surface as possible.

Collect approximately one milliliter of the nasal secretions. When wiping the snout, attempt to enter the external nares with the gauze pad and collect all visible nasal secretions. Then fold the gauze pad with the same hand.

And after opening the cap of the vial with the other uncontaminated hand carefully insert the folded pad inside of the vial. Recap the vial without contaminating the surface. Next, shake the vial so that the gauze is completely submerged in the liquid medium.

Label each vial with the animal identifier and to make sure that all necessary information for identification and description of the animal subject is correctly filled in. Remove the contaminated glove and then place the vial in its receptacle inside of the tilt container. Since the protocol requires the gauze pad to be held in hand, it is imperative to change gloves in between each sample to reduce contamination, the sample collector must also take care to avoid contaminating the gauze pad by touching animals or objects, Aliquot the samples if necessary, and then place them on dry ice as soon as possible After collection.

Store the vials at minus 80 degrees Celsius for the identification and isolation of influenza. In the collected samples. Follow the steps as indicated in the text protocol comparison of the number of pigs that were positive and negative for the identification of influenza virus using quantitative real-time.

PCR showed that 235 samples were positive for influenza when samples were collected by snout, wipes, or nasal swabs. 282 samples were negative for the virus using both collection methods. Only 36 samples showed different results demonstrating a high degree of concordance between both collection methods.

The following table shows the number of pigs found positive and negative for influenza virus. When influenza was cultured from snout, wipes, or nasal swabs. A high degree of correlation was also found using influenza culture.

After watching this video, you should have a good understanding of how to collect nasal wipes from swine for influenza A virus testing.