The overall goal of this procedure is to document passage of infectious PreOn material through the digestive system of American Crows. This is accomplished by first gaging crows with infectious PreOn material. The second step is to collect the crow feces for four hours post gavage and prepare it for injection into mice.
Next mice are inoculated intraperitoneal with one milliliter of crow fecal homogenate. The final step is to monitor mice daily until they express clinical signs of mouse scrapy. Ultimately, western blot analysis is used to confirm the presence of infectious PreOn in the mouse model and verify disease transmission.
The idea that crows could transport. CWD came to us one winter day when after driving to a captive elk facility where we were doing some research and seeing roadkill elk being fed on by crows, and later that day, crows coming outta the sky outta nowhere, landing in these feed troughs, feeding right next to the elk, defecating on the grain that the elk were consuming that on top of the observation from the literature that a lot of the crows that summer throughout Colorado, Wyoming winter in a concentrated area in New Mexico, it just so happens that that area in New Mexico is where some CABD has popped up and it's a hundred a mile hundreds of miles away from the epicenter of crack wasting disease. So these two observations got us thinking that potentially crows could play a a role in transporting CWD around the landscape.
A visual demonstration of these techniques is critical as incorrect intraperitoneal and oral gavage techniques can result in the death of study animals. To begin, add four drops of blue dye to a scrambled egg and mix it well. Then draw five milliliters of the dye and egg mixture into a two inch gavage needle with one person gently restraining the crow.
Open the beak of the crow wide enough to see down its throat and carefully insert the gavage needle into its esophagus. If any altered breathing is noticed, remove the syringe and try again as it may have been placed in the trachea. Once it is certain the gavage needle is in the esophagus, slowly express the contents of the syringe.
Check the crow every 30 minutes until blue-green stained feces are no longer being excreted. The test crows took approximately four hours to quit excreting blue dyed feces. Next, add one part uninfected C 57 black six mouse brain to four parts sterile PBS and some glass beads into a bullet blender.
Homogenizer homogenize the sample for one to five minutes until the smooth consistency is achieved. Repeat the process using terminally ill R ML Chandler strain infected C 57 black six mouse brains. Next centrifuge the homogenous at 3000 times G for one minute.
To remove the larger particulate matter. Then remove the supernatant and dilute it with an equal volume of sterile one XPBS to generate a 10%weight per volume of brain matter. In PBS store the samples at negative 80 degrees Celsius until needed 17 hours prior to gavage.
Remove the food but not the water from the crow pens. Randomly allocate the crows into treatment groups and orally gavage each crow with five milliliters of freshly thaw, normal or infected mouse brain homogenate using a two inch gavage needle. Then transfer the crows into individual cages.
Collect and pool all feces within each cage at four hours post gavage. Next homogenize the pooled feces for each crow in a blue bullet homogenizer with glass beads until a uniform texture is achieved. Then take 500 microliters of the mixture and dilute it one to 20 with 9.5 milliliters of sterile PBS centrifuge, the diluted fecal homogenate for 15 minutes.
Add 1, 300 times G and then transfer the supernatant to a fresh tube. To minimize the threat of a secondary microbial infection, add one unit of penicillin and one microgram of streptomycin per milliliter of homogenate. Then sonicate the samples in a 3000 MP ator at a power setting of 70 for 30 seconds to disrupt the membrane of any remaining microbes.
First, randomly assign the mice to one of the four groups shown here. Group one will receive infected crow feces. Group two will receive non-infected crow feces.
Group three will receive infected mouse brain and group four will receive normal mouse brain. Then for MN groups one and two, draw up one milliliter of the scrapy positive or scrapy negative crow feces. Homogenate using a 25 gauge needle according to the group number of the mouse.
Then scruff the mouse by its dorsal neck fur. Using the thumb and index finger and gently rotate it to expose the ventral side. Elevate the posterior end of the mouse so that its head is slightly lower.
Insert the needle one centimeter through skin, one centimeter lateral of midline and one to two centimeters anterior to the sacroiliac joint. Then pull back slightly and inject the homogenous slowly into the mouse body cavity. Next, prepare the frozen mouse brain homogenate with and without scrappy for injection into the animals in groups three and four.
By first diluting the haws stock mixtures one to 10 with sterile PBS. Then inject one milliliter of the diluted scrapy positive brain homogenate into mice in group three and one milliliter of the diluted scrapy negative brain homogenate into the mice. In group four.
Monitor mice daily until they express clinical signs of mouse scrapy. Clinical signs may include kyphosis, ataxia, stiff tail, lack of grooming, emaciation and lethargy. Score my daily for each of the six clinical signs.
When visible signs are evident. Zero equals no visible signs. One equals moderate and two equals a severe level of a sign.
Euthanize mice when total daily scores for each sign, reach greater than or equal to eight for one day greater than or equal to six continuously for three days or at 365 days. Post inoculation, harvest brains immediately following euthanasia and store them at negative 80 degrees Celsius. Then properly dispose of carcasses to confirm a scrappy diagnosis.
Thaw the frozen mouse brains and individually homogenize them in a blue bullet homogenizer with glass beads until uniform texture is achieved. Next, prepare 125 microliters of a proteinase K digestion solution by combining 109.39 microliters of PBS with 12.5 microliters of 500 millimolar EDTA at pH eight and 3.1 microliters of a 50 microgram per milliliter proteinase K solution. Then add three microliters of the digestion solution to 17 microliters of the brain.Homogenate.
Incubate the samples at 45 degrees Celsius on a shaking heat block for 30 minutes. Once the incubation is finished, inactivate the digestion solution by adding eight microliters of SDS page loading buffer, and incubate the samples at 95 degrees Celsius for five minutes. Then load the samples onto a 12%SDS page.
Gel electrophoresis the proteins and transfer the gel to an IMON PVDF membrane Once transferred, block the PVDF membrane with 5%non-fat milk in PBS with 0.2%between 20 and rock for one hour at room temperature. Then probe the membrane with borrow 224 and IPRP monoclonal antibody conjugated to horse radish peroxidase diluted one to 20, 000 in superblock and rock for one hour at room temperature following incubation. Rinse the membrane for one hour in PBS with 0.2%between 20.
Visualize the proteins by incubating the membrane with chemiluminescent substrate for five minutes. Then imaging the membrane on a G box gel documentation system. Diseased mice were identified by manifestation of clinical mouse scrapy signs and disease confirmation was completed by Western blood analysis.
Normal mouse brain homogenous with no scrapy are shown here in the left two lanes with and without the aid of protease K Without protease K three bends are seen. However, since the normal protein is susceptible to proteinases, the bends are not seen in the second lane. Lane three shows a protease digested scrapy positive control sample.
The proteases cause a shift in the three original bands due to the loss of the n and c termini of the proteins. But the infected proteins are unable to be completely digested as they were in the normal brain sample. The proteinase K digested brain from a mouse inoculated with feces from a scrappy inoculated crow is shown on the right and confirms the presence of prions in the sample as the bands are not digested.
After watching this video, you'll have a greater understanding of how to document the passage of infectious prion material through the digestive system of crows. Then verify infectivity with an interperitoneal mouse bioassay.
