Bioassay and Related Methods
291
the ratio of fluid volume secreted per milligram of dry weight intestine.
30 The minimum amount of C.
perfringens enterotoxin necessary to produce a loop reaction has been reported variously to be 28–40
µg and as high as 125 µg of toxin by the standard loop technique. The 90-minute loop technique has
been found to respond to as little as 6.25 µg and the standard technique to 29 µg of toxin.
18
This technique was developed to study the mode of action of the cholera organism in producing
the disease.
11 It has been employed widely in studies on the virulence and pathogenesis of foodborne
pathogens, including Bacillus cereus, C. perfringens, E. coli, and Vibrio parahaemolyticus.
Although the rabbit loop is the most widely used of ligated loop methods, other animal models are
used. The mouse intestinal loop may be used for E. coli enterotoxins. As used by Punyashthiti and
Finkelstein.
40 Swiss mice (18–22 g) are deprived of food 8 hours before use. The abdomen is opened
under light anesthesia, and two 6-cm loops separated by 1-cm interloops are prepared. The loops are
inoculated with 0.2 ml of test material, followed by closing of the abdomen. Animals are deprived of
food and water and killed 8 hours later. Fluid is measured and the length of the loops determined. Results
are considered positive when the ratio of fluid to length is 50 or more mg/cm. In this study, positive loops
generally had ratios between 50 and 100, but occasionally approached 200 or more. Alternatively, the
net increase in weight of loops in milligrams can be used to measure the intensity of a toxic reaction.
53
With the mouse loop, 1 µg of enterotoxin can be detected.
53 A rat jejunal loop assay has been presented
for detecting the ST b enterotoxin of E. coli. A linear dose response was found using 250- to 350-g
rats but only after the endogenous protease activity was blocked with soybean trypsin inhibitor.
49
The RITARD Model
The removable intestinal tie–adult rabbit diarrhea (RITARD) method was developed by Spira et al.
44
Rabbits weighing 1.6–2.7 kg are kept off food for 24 hours but allowed water. Under local anesthesia,
the cecum is brought out and ligated close to the ileocecal junction. The small intestine is now
brought out and a slip knot tied to close it in the area of the mesoappendix. Test material in 10 ml of
phosphate-buffered saline is injected into the lumen of the anterior jejunum. After injection, intestine
and cecum are returned to the peritoneal cavity and the incision closed. With the animal kept in a
box, the temporary tie is removed 2–4 hours after administration of the test dose, and the slip knot
in the intestine is released. Sutures are applied as needed. The animal is now returned to its cage and
provided with food and water. Animals are observed for diarrhea or death at 2-hour intervals up to 124
hours. At autopsy, small intestine and adjacent sections are tied and removed for fluid measurement.
Enterotoxigenic strains of E. coli produce severe and watery diarrhea, and the susceptibility of animals
to V. cholerae infections is similar in this system to that in the infant rabbit model.
The gist of the RITARD model is that the animals are not altered except that the cecum is ligated
to prevent it from taking up fluid from the small intestine, and a temporary reversible obstruction
is placed on the ileum long enough to allow the inoculated organism to initiate colonization of the
small intestine. The method has been successfully used as an animal model for Campylobacter jejuni
infection
9 and to test virulence of Aeromonas strains.
39
CELL CULTURE SYSTEMS
A variety of cell culture systems are employed to assess certain pathogenic properties of viable cells.
The properties often assessed are invasiveness, permeability, cytotoxicity, adherence/adhesion/binding,
and other more general biological activities. Some cell cultures are used to assess various properties
291
the ratio of fluid volume secreted per milligram of dry weight intestine.
30 The minimum amount of C.
perfringens enterotoxin necessary to produce a loop reaction has been reported variously to be 28–40
µg and as high as 125 µg of toxin by the standard loop technique. The 90-minute loop technique has
been found to respond to as little as 6.25 µg and the standard technique to 29 µg of toxin.
18
This technique was developed to study the mode of action of the cholera organism in producing
the disease.
11 It has been employed widely in studies on the virulence and pathogenesis of foodborne
pathogens, including Bacillus cereus, C. perfringens, E. coli, and Vibrio parahaemolyticus.
Although the rabbit loop is the most widely used of ligated loop methods, other animal models are
used. The mouse intestinal loop may be used for E. coli enterotoxins. As used by Punyashthiti and
Finkelstein.
40 Swiss mice (18–22 g) are deprived of food 8 hours before use. The abdomen is opened
under light anesthesia, and two 6-cm loops separated by 1-cm interloops are prepared. The loops are
inoculated with 0.2 ml of test material, followed by closing of the abdomen. Animals are deprived of
food and water and killed 8 hours later. Fluid is measured and the length of the loops determined. Results
are considered positive when the ratio of fluid to length is 50 or more mg/cm. In this study, positive loops
generally had ratios between 50 and 100, but occasionally approached 200 or more. Alternatively, the
net increase in weight of loops in milligrams can be used to measure the intensity of a toxic reaction.
53
With the mouse loop, 1 µg of enterotoxin can be detected.
53 A rat jejunal loop assay has been presented
for detecting the ST b enterotoxin of E. coli. A linear dose response was found using 250- to 350-g
rats but only after the endogenous protease activity was blocked with soybean trypsin inhibitor.
49
The RITARD Model
The removable intestinal tie–adult rabbit diarrhea (RITARD) method was developed by Spira et al.
44
Rabbits weighing 1.6–2.7 kg are kept off food for 24 hours but allowed water. Under local anesthesia,
the cecum is brought out and ligated close to the ileocecal junction. The small intestine is now
brought out and a slip knot tied to close it in the area of the mesoappendix. Test material in 10 ml of
phosphate-buffered saline is injected into the lumen of the anterior jejunum. After injection, intestine
and cecum are returned to the peritoneal cavity and the incision closed. With the animal kept in a
box, the temporary tie is removed 2–4 hours after administration of the test dose, and the slip knot
in the intestine is released. Sutures are applied as needed. The animal is now returned to its cage and
provided with food and water. Animals are observed for diarrhea or death at 2-hour intervals up to 124
hours. At autopsy, small intestine and adjacent sections are tied and removed for fluid measurement.
Enterotoxigenic strains of E. coli produce severe and watery diarrhea, and the susceptibility of animals
to V. cholerae infections is similar in this system to that in the infant rabbit model.
The gist of the RITARD model is that the animals are not altered except that the cecum is ligated
to prevent it from taking up fluid from the small intestine, and a temporary reversible obstruction
is placed on the ileum long enough to allow the inoculated organism to initiate colonization of the
small intestine. The method has been successfully used as an animal model for Campylobacter jejuni
infection
9 and to test virulence of Aeromonas strains.
39
CELL CULTURE SYSTEMS
A variety of cell culture systems are employed to assess certain pathogenic properties of viable cells.
The properties often assessed are invasiveness, permeability, cytotoxicity, adherence/adhesion/binding,
and other more general biological activities. Some cell cultures are used to assess various properties