Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

The goal of this procedure is to provide a robust in vivo model for the investigation of the molecular pathogenesis of E coli, K one neonatal sepsis and meningitis. This is accomplished by first orally administering the E coli K one to the neonatal rat. In the second step, the colonization is confirmed by perianal swabbing.

Next, the tissues and organs of interest are collected at the desired time points, and then in the final step, the samples are processed and preserved under the appropriate conditions. Ultimately, the extent of the colonization can be visualized and quantified using various techniques such as viability counts and QPCR. Main advantage of this technique over existing methods such as intra sternal and subcutaneous injection, is that it replicates the pathway of natural disease progression in infected humans.

Demonstrating the procedure will be fat margain a finally, a PhD student from my laboratory Retain all rat litters of 12 neonates per colony in individual cages with their lactating mothers under optimal conditions until the end point of the experiment to administer the bacteria for each animal, gently pick up the neonate vertically with one hand by the scruff of the neck, allowing the mouth to open. Then slowly insert a sterile pipette tip into the mouth and dispense 20 microliters of the inoculum. Over 30 seconds, return the animals to their mother immediately after feeding and assess the disease severity of each animal four to five times daily using the seven point scoring system outlined in the table.

If an animal scores less than or equal to three out of seven, immediately call the animal to minimize suffering. Record the animal is dead to confirm the colonization of the animals every 24 hours, beginning the first 24 hours after feeding. Pick up each animal by the scruff in turn as just demonstrated, and then gently swap the perianal area of the neonate with a PBS moistened sterile cotton tipped swab.

Return the animal to its mother and place the swab in 300 microliters of sterile PBS on ice. Then use the PBS to confirm colonization by eco like K one by serial dilution, followed by plating onto McConkey agar. Then to determine the number of viable eco, like K one, use a sterile microbiological loop to pick up an individual colony from one of the McConkey plates, and then transfer the colony into 200 microliters of sterile PBS.

Create a single cell suspension by vortexing, and then use a new sterile microbiological loop to subculture the cells onto a moula Hinton agar plate in a straight line. After allowing the plate to dry for 30 seconds, pipette 10 microliters of bacteria phage K one E onto the center of the line, and incubate the plate overnight at 37 degrees Celsius. The next day, examine the plate for bacteria phage mediated lysis to collect the GI tract.

Clean the abdomen of an inoculated animal after the animal is euthanized, then cut along the lower abdomen to the sternum, extending the incision across the sternum and down the right side from the sternum to the lower abdomen. Next, used iris curved dissection forceps to gently pull down the skin flap to expose, and then gently raise the peritoneum cutting vertically to expose the internal organs taking care not to damage any of the underlying organs. Now, gently transect the stomach at either side and place it into a pre wade biju, transect the colon at the rectum, and then use dissection forceps to carefully pull out the entire intestinal mass, placing the tissue in a sterile Petri dish.

Cover the tissue with 30 milliliters of PBS, and then identify the central mass of the mesenteric lymphatic system. Pinch the mass with fine dissection forceps, and then identify the most proximal part of the small intestine and pinch it with fine dissection forceps as well. Slowly pull the central mass of the mesenteric lymphatic system and the distal small intestine in opposite directions until the two tissues are completely separated.

Taking care not to stretch the small intestines. Transfer the mesenteric lymphatic system into 300 to 500 microliters of sterile PBS in a pre weighed tube on ice. Then transect the GI tract at the cecum to separate the small intestine from the colon.

Then transfer the colon into 300 to 500 microliters of sterile PBS in another freeway tube on ice, and then align the small intestine from the midpoint. Finally, collect the last two centimeters of tissue prior to the cecum for the representative distal small intestine sample tissue from five to seven centimeters above the midpoint for the proximal small intestine. Sample and tissue from three to five centimeters below the midpoint for the middle small intestine.

Sample into each of the tubes. After the tissue collection, weigh all the tubes gained to calculate the tissue weight by comparing the weight of the tubes before and after addition of the samples. Next, homogenize the tissues, washing the homogenizer between each sample, and then immediately store the samples on ice to calculate bacterial load plate.

Serial dilution of the homogenized samples onto Macon plates are overnight Incubation at 37 degrees Celsius the next day determine the Eli K one load by testing the susceptibility of individual colonies to the K one specific bacteria phage as previously demonstrated, the Coli K one systemic infection model described here replicates many of the features of a natural infection in humans. Importantly, the model displays a strong age dependency as illustrated in the graph. Two day old rat pups are highly susceptible to invasive disease, but over a seven day period, the animals become progressively more refractory to infection, but not GI tracted colonization as observed in humans.

Bacteria can also be visualized in blood samples by fluorescence microscopy before entering the central nervous system and after transit from the site of GI colonization to the blood compartment as demonstrated by these images of e Coli, A 1 92 PP cells in a blood smear from a postnatal day two pup following oral administration of the bacteria e coli, A 1 92 PP cells persist in the GI tract of postnatal day two, five and nine pups in large numbers as such, temporal aspects of the colonization in these three groups are very similar and reflect the capacity of the bacteria to replicate and maintain a population density within the gut. Thus, this model can be employed to investigate the virulence of different K one strains with respect to their capacity to invade the central nervous system and other organ systems from the site of colonization. Following this procedure, other methods, including the imaging of bioluminescent bacteria, using micro CT IVs can be performed to answer additional questions such as, how does the disease progress in real time?