Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

Summary

This protocol describes intratracheal inoculations of Fischer 344 rats with Francisella tularensis. This procedure mimics pulmonary exposure of humans to this potential biothreat agent and can be used to test vaccine and therapeutic efficacy against pulmonary tularemia.

Abstract

Pulmonary infection with the bacterium Francisella tularensis can lead to the serious and potentially fatal disease, tularemia, in humans. Due to the current lack of an approved tularemia vaccine for humans, research is focused on vaccine development utilizing appropriate animal models. The Fischer 344 rat has emerged as a model that reflects human susceptibility to F. tularensis infection, and thus is an attractive model for tularemia vaccine development. Intratracheal inoculation of the Fischer 344 rat with F. tularensis mimics pulmonary exposure in humans. The successful delivery into the rat trachea is critical for pulmonary delivery. A laryngoscope with illumination is used to properly intubate the tracheae of anesthetized rats; the correct placement within the trachea is determined by a simple device to detect breathing. Following intubation, the F. tularensis culture is delivered in a measured dose via syringe. This technique standardizes pulmonary delivery of F. tularensis within the rat trachea to evaluate vaccine efficacy.

Introduction

F. tularensis (Ft) causes the human disease, tularemia. When the bacteria are acquired through the pulmonary route, this leads to pneumonic tularemia, which has high morbidity and mortality¹. F. tularensis is considered a biothreat agent due to the danger associated with aerosolized forms, and there is currently no vaccine approved for human use in the U.S. An intensive effort is currently underway to develop vaccines and therapeutic measures against pneumonic tularemia, to protect the human population against the illicit use of this bacterial biothreat.

Much of the tularemia research has focused on....

Protocol

This work was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Animal protocols involving rodents were approved by the University of Texas at San Antonio Institutional Animal Care and Use Committee (IACUC) under protocol MU009(RA).

1. Prepare Catheter, Trachea Indicator, and F. tularensis Inoculum

1. Prepare Catheter (20 G x 2 in)
   1. Cut the 20 G x 2 in needle and grind the tip of the needle to a blunt and smooth finish with a rotary tool.
      NOTE: The length of the blunted needle should allow....

Results

The humoral response to intratracheal inoculation of F. tularensis in the rat can be determined by enzyme-linked immunosorbent assay (ELISA) against UV-inactivated bacteria, as described previously¹¹. Total Immunoglobulin G (IgG) response of Fischer 344 rats to inactivated whole cell bacteria was assessed post-intratracheal inoculation with an attenuated strain of Fn (10⁷ CFU inoculum) at day 14 and day 28 (Figure 1.......

Discussion

The Fischer 344 rat is becoming an important model for tularemia vaccine development³. Exposure to F. tularensis through the pulmonary route is critical for demonstrating efficacy against weaponized forms of F. tularensis, because these are delivered as aerosols. Intratracheal inoculation of the rat facilitates exposure of the rat lungs to F. tularensis without the need for large, expensive, and complicated aerosol generating equipment. All experiments utilizing select a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
GreenLine fiber optic blade size 0	Carefusion	5-5231-00	Macintosh American profile
GreenLight system laryngoscope handle	Carefusion	4559GSP
Exel International Safelet I.V. Catheter	EXEL INTERNATIONAL	26743
Slip Tip Sterile Syringes 1mL	BD	309659
Broad Point Dressing Thumb Forceps	Thermo Scientific	76-302
200 μL barrier tip	GeneseeScientific	24-142
1,000 μL pipette tip	Olympus Plastics	24-173
Dremel 3000-2/28 Rotary tool kit	Dremel	3000228
Rodent Intubation Stand	Braintree Scientific	RIS 200
Isoflurane	Butler Schein	NDC 11695-6776-2
Rodent anesthesia machine	Surgivet	VTC302 Classic T3
Rodent Anesthesia chamber	Braintree Scientific	AB 1