The overall goal of the following experiment is to study host pathogen interactions in a bovine model of respiratory infection. This is achieved by inducing a respiratory infection in calves by intra bronchial inoculation with the pathogen of interest during the course of the disease. The animal is sampled repeatedly by bronchoalveolar lavage.
Next bronchial brushings and transbronchial lung biopsies are obtained to recover epithelial cells and alveo or lung tissue samples respectively. Ultimately, pathogen specific respiratory disease can be confirmed, for example, by inflammatory changes in lavage, cytology, and by immunohistochemical detection of the pathogen. In transbronchial lung biopsies is The main advantage of this large animal model compared to other existing animal models and rodents, is that in our model, we are able to study the course of disease over a long time period in the same individual through repeated sampling.
While this method can provide insight into the host pathogen interactions in vivo, it can also be applied to other systems such as alternative pathogens or host species. Maintaining anesthesia and assisting with the sampling will be seen a fisher, she's a veterinarian from our laboratory. Before beginning the procedure, fill three syringes with one, two, and five milliliters of the inoculum.
Then after anesthesia has been confirmed, pull out the animal's tongue and stretch the neck. Next, using slight rotating movements, place a metal speculum in the cal's mouth. Push the speculum forward under site control, using a flashlight to determine when the larynx is visible.
Next, insert a Teflon tube into the working channel of an endoscope. The tube should not protrude from the tip of the endoscope. Insert the endoscope through the speculum.
Slight adjustments to the speculum may be necessary to enable the endoscope. To pass the larynx, use the bronchus trache, which branches off to the right side of the lung to help align the image on the monitor. Then attach the syringe containing five milliliters of inoculum to the end of the Teflon tube.
Navigate the tube into the branches where the inoculum will be deposited and administer the appropriate amount of inoculum. Next, attach the syringe containing one milliliter of inoculum to the tube. Navigate to the bronchus traches and deposit the inoculum in the coddle branch.
Remove the endoscope and the speculum. Then attach the last syringe to an actuator and spray one milliliter of the inoculum into each nostril. Place the animal back in its stall in the prone position for waking.
The recovery stable should be air conditioned. Since the animal's ability for thermoregulation is decreased under general anesthesia To collect valve samples at the desired time point for sampling warm five syringes each containing 20 milliliters of sterile isotonic saline in a 38 degree Celsius water bath. Then insert a lavage catheter into the working channel of the endoscope and insert the endoscope into the metal speculum.
Next, navigate forward into the main bronchus of the left lung until the wedge position is reached and the endoscope cannot be pushed any further ahead. Then one after another. Attach the syringes with the warm sodium chloride solution to the lavage catheter for instilling and directly aspirating the fluid.
Store the bronchoalveolar lavage fluid in siliconized glass bottles on ice immediately after recovery to prevent the alveolar macrophages from attaching to the glass surface. Note both the amount of instilled saline and the amount of recovered fluid. Then remove the lavage catheter from the working channel to collect epithelial cells.
Next, navigate the endoscope to the desired sampling location. For example, in this experiment to the bifurcation trache, when the endoscope is in place, cover a bronchial brush with the tube and insert it into the working channel of the endoscope until the brushes tip appears on the monitor. Push the brush and the plastic tube forward about five centimeters.
Then push the handle of the brush to uncover it from the plastic tube. When the brush is in position, navigate it to the location that is to be brushed. Then gently push and pull the brush back and forth while navigating the endoscope.
To ensure contact between the brush and the wall of the bronchus to rub off the epithelial cells. Stop rubbing when bleeding occurs. Then cover the brush with the tube and pull it out of the working channel.
Gently roll the epithelial cell coated bristles over up to five microscope slides and fix the smears in cold methanol for 10 minutes. Then after allowing the slides to air dry, store them at minus 20 degrees Celsius to perform a trans bronchial lung biopsy. Next, navigate the endoscope to the desired sampling location.
For example, the pars Alis of the LOAs crans as in this experiment, before inserting the biopsy forceps into the working channel, open and close it a few times to ensure its proper function, and then push the forceps into the coddle branch of the bronchus traches until a slight resistance occurs. Now, pull back two to three centimeters and open the forceps. Push the forceps forward again, about two centimeters, and then close them.
Pull back and remove the forceps from the working channel. Use a needle to carefully remove the tissue from the biopsy forceps and store it in a suitable fixation medium to prevent the formation of autolytic processes. Finally, after allowing the calf to recover in its stall, once again, monitor the animal for pneumothorax for the next 24 hours, providing feed and fresh water.
When the animal has regained full consciousness, frozen sections of transbronchial lung biopsies can be stained immunohistochemical. To visualize the inoculated pathogen, note the brown chlamydial inclusions in the lung of this animal that has been inoculated with chlamydia C four days prior to sampling.Immunohistochemical. Staining of the lavage cells allows the visualization of intracellular pathogens.
For example, in this image, chlamydial inclusions present in the alveolar macrophages sampled from a calf. Nine days after intra bronchial inoculation with chlamydia, CTAC can be observed. Bronchoalveolar lavage cells can be differentiated by cytological staining.
Alveolar macrophages are the predominant cell type in the lavage fluid. However, the number of neutrophilic granulocytes increases when inflammatory processes are present. Once mastered, the inoculation can be done in 10 to 15 minutes and the sampling within 20 to 30 minutes if performed properly.
After watching this video, you should have a good understanding on how to perform intra bronchial inoculation and calves, and on how to sample bronchoalveolar batch fluid, bronchial brushings, and transbronchial lung biopsies in the anesthetized animal.
