MICRO-202DVM 4thRabbit Ileal Loop TestASSIGNMENT#3Submitted to: Dr. Saif Ur RehmanSubmitted by: Waqas Nawaz11-arid-975
In the early 1950s, investigators discovered that injection of enterotoxin preparationsinto ligated segments of intestine (ileal loops) of rabbits (and later other animals includingpigs, dogs and calves) caused accumulation of fluid.This model has been used extensively to study the mechanisms of action of E.coli and otherenterotoxins.Procedure:1) Anesthetize the specific pathogen-free rabbit2) Open the peritoneal cavity by a sterile surgical technique3) Wash the bowel carefully with pre-warmed PBS and clamp it4) Measure the required number of loops (e.g. loop length=5cm, interloop length=5cm)and clamp the bowel proximal to the ileo-cecal junction5) Cut the length of the bowel making the loops and clamp all cut surfaces6) Reanastomose the remaining intestine and place it back into the peritoneal cavity7) Close the resected ends of the isolated intestine with sutures and to avoidcompromising the integrity of blood supply to the tissue, construct the requirednumber of loops by tying with ligatures8) While closing the loop tie, inject the bacterial suspension containing approximately 108viable bacteria in a 0.5ml volume of PBS into the proximal end of the loop9) Inoculate the positive control loops with 1µg of cholera toxin in 0.5ml of PBS; Whilenegative control loops receive only 0.5ml of PBS alone10)Replace the resected intestine into the peritoneal cavity; close the cavity and allow theanimal to recover11)After 18 hours, anesthetize the animal and remove the loops. Weigh the loops and placethe loop fluids in sterile containersAnalysis of Fluids from Loops: Measure the fluid from infected loops by volume, consistency, color and viscosity Determine the bacterial and cellular contents (erythrocytes and leukocytes) of the fluidby wet preparation, Gram and Giemsa staining Determine the predominant cell types in the fluid exudates by differential counts afterGiemsa staining Centrifuge the loop fluids to remove cellular components. Assay them for bicarbonate,pH and hemoglobin levels with a laboratory biochemical analyzer (Radiometer) and fortotal protein by the method of Bradford
Histopathology of Intestinal Tissue:After postmortem removal, weigh the loop tissue and cut longitudinally to release any ﬂuidcontained within. Note any gross changes and inspect the mucosal side for macroscopictissue damage. Wash the loop tissue in PBS and place small samples of loop tissue into 10%formaldehyde in PBS at pH 7.2. Embed the tissue in wax and stain thin-cut sections withhematoxylin and eosin. Examine under bright-ﬁeld illumination with a Nikon Axiophotmicroscope.Quantitation of Bacteria in Loops and Fluids:Assess the loop ﬂuid for number of viable bacteria by dilution and surface-viable countingby using a modiﬁcation of the technique of Miles et al. (25). Culture the loop tissue samplestaken in postmortem to demonstrate the presence of bacteria on or within loop tissue.Wash the tissue samples (0.5 g [wet weight]) with PBS before homogenization.Homogenize the tissue in 5 ml of broth (e.g. Luria Broth for Salmonella typhimurium) in astomacher and serially dilute the resulting suspension for surface viable counting. Plate allsamples on agar.Disadvantages: Excessively stressful for the animals Time consuming Cumbersome (difficult to handle) Difficult to standardizeRelatively expensive in terms of numbers of animals required, since only about 8 to 14supernatants may be tested per animal, not including positive and negative controls; also,each set of supernatants must be done in duplicate animals and the orientation ofsupernatants must be reversed from one animal to the next.