Dr. Waqas Nawaz PMAS Arid agriculture university rawalpindi, Pakistan

1. MICRO-202
DVM 4th
Rabbit Ileal Loop Test
ASSIGNMENT#3
Submitted to: Dr. Saif Ur Rehman
Submitted by: Waqas Nawaz
11-arid-975

2. In the early 1950s, investigators discovered that injection of enterotoxin preparations
into ligated segments of intestine (ileal loops) of rabbits (and later other animals including
pigs, dogs and calves) caused accumulation of fluid.
This model has been used extensively to study the mechanisms of action of E.coli and other
enterotoxins.
Procedure:
1) Anesthetize the specific pathogen-free rabbit
2) Open the peritoneal cavity by a sterile surgical technique
3) Wash the bowel carefully with pre-warmed PBS and clamp it
4) Measure the required number of loops (e.g. loop length=5cm, interloop length=5cm)
and clamp the bowel proximal to the ileo-cecal junction
5) Cut the length of the bowel making the loops and clamp all cut surfaces
6) Reanastomose the remaining intestine and place it back into the peritoneal cavity
7) Close the resected ends of the isolated intestine with sutures and to avoid
compromising the integrity of blood supply to the tissue, construct the required
number of loops by tying with ligatures
8) While closing the loop tie, inject the bacterial suspension containing approximately 108
viable bacteria in a 0.5ml volume of PBS into the proximal end of the loop
9) Inoculate the positive control loops with 1µg of cholera toxin in 0.5ml of PBS; While
negative control loops receive only 0.5ml of PBS alone
10)Replace the resected intestine into the peritoneal cavity; close the cavity and allow the
animal to recover
11)After 18 hours, anesthetize the animal and remove the loops. Weigh the loops and place
the loop fluids in sterile containers
Analysis 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 fluid
by wet preparation, Gram and Giemsa staining
 Determine the predominant cell types in the fluid exudates by differential counts after
Giemsa staining
 Centrifuge the loop fluids to remove cellular components. Assay them for bicarbonate,
pH and hemoglobin levels with a laboratory biochemical analyzer (Radiometer) and for
total protein by the method of Bradford

3. Histopathology of Intestinal Tissue:
After postmortem removal, weigh the loop tissue and cut longitudinally to release any fluid
contained within. Note any gross changes and inspect the mucosal side for macroscopic
tissue 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 with
hematoxylin and eosin. Examine under bright-field illumination with a Nikon Axiophot
microscope.
Quantitation of Bacteria in Loops and Fluids:
Assess the loop fluid for number of viable bacteria by dilution and surface-viable counting
by using a modification of the technique of Miles et al. (25). Culture the loop tissue samples
taken 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 a
stomacher and serially dilute the resulting suspension for surface viable counting. Plate all
samples on agar.
Disadvantages:
 Excessively stressful for the animals
 Time consuming
 Cumbersome (difficult to handle)
 Difficult to standardize
Relatively expensive in terms of numbers of animals required, since only about 8 to 14
supernatants 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 of
supernatants must be reversed from one animal to the next.