The overall goal of intratracheal inoculation of Fischer 344 rats with Francisella tularensis is to determine vaccine efficacy against pulmonary tularemia. This method allows us to answer key questions in the development of a tularemia vaccine because we're able to administer the organism through the pulmonary route and therefore test vaccine efficacy against a biothreat agent. And so the main advantage of this technique is the ability to consistently deliver Francisella tularensis into the rat lung without the use of an aerosol-generating equipment.

The implications of this technique extend toward therapy and prevention of pulmonary tularemia because the Fischer 344 rat is considered a relevant model for vaccine development against this disease. Though this method can provide insights into vaccines and therapies against tularemia, it can also be applied to other pulmonary infectious disease models utilizing rats such as plague. In general, individuals new to this method will struggle with correct placement of the catheter into the trachea rather than the esophagus.

To prepare the catheter, begin by cutting a 20 gauge two inch needle and use a rotary tool to grind the tip of the needle to a blunt and smooth finish. Use 70%ethanol to clean the blunted needles and catheters and sterilize them under UV light for 15 minutes. Trim a 1, 000 microliter pipette tip to allow it to sit into a catheter opening and form an airtight seal.

Remove a filter from a 200 microliter aerosol barrier pipette tip and place it inside the trimmed pipette tip. Point the 1, 000 microliter pipette tip downward and ensure the filter can move freely inside the tip downward. After preparing F.tularensis inoculum and anesthetizing a rat according to the text protocol, place the rat dorsally on the rodent intubation stand.

Then attach the front teeth of the rat to the holder to keep the rat in place. With the dominant hand, use broad point dressing thumb forceps to move the tongue to the same side as the support hand. Using the support hand, secure the tongue with a laryngoscope against the rat's lower jaw to visualize the trachea and esophagus of the animal.

Next, insert the catheter containing the previously prepared 20 gauge blunted needle into the trachea. Remove the blunted needle from the catheter while ensuring the catheter remains in the trachea. Allow a few seconds to pass for the rat to be able to breathe around the catheter.

Firmly seat the trachea indicator onto the catheter opening. Movement of the aerosol barrier will indicate the catheter is inserted correctly into the trachea. Ensure that the rat is laying on the intubation stand such that the chest of the animal is facing perpendicular to the plane of the intubation stand.

Remove the trachea indicator and with a 200 microliter pipette tip firmly seated against the catheter opening, deliver 100 microliters of the inoculum containing F.tularensis. Attach a one milliliter slip tip tuberculin syringe filled with air and deliver 300 microliters of air to ensure inoculum reaches the lungs of the rat. Remove the catheter from the trachea and take the rat off of the surgical platform.

In this experiment, the humoral response to intratracheal inoculation of an attenuated strain of F.tularensis in the rat was determined by ELISA against UV inactivated bacteria. Total IgG response of the Fischer 344 rats to inactivated whole cell bacteria was assessed at days 14 and 28. Mock vaccinated rats received PBS.

The red area denotes reactivity of serum from these rats. An increase in serum antibody titers against F.tularensis post inoculation compared to naive mock vaccinated animals indicates the intratracheal vaccination efficacy. Once mastered, the intratracheal inoculation can be done in approximately one minute if it's performed properly.

While attempting this procedure, it is important to ensure that the catheter is placed within the trachea and not the esophagus. So, following this method, other procedures such as blood collection can be done to answer additional questions such as antibody response against a vaccine and/or the challenge. After its development, this technique paved the way for researchers in the field of tularemia vaccines to explore the efficacies of different vaccines against pulmonary tularemia.

After watching this video, you should have a good understanding of intratracheal delivery of Francisella tularensis in the Fischer 344 rat. So, keep in mind working with live animals and pathogenic agents can be extremely hazardous so it's very important to ensure that animals are anesthetized, properly restrained, and that all properly biocontainment procedures are done.