288 Modern Food Microbiology
and vomited (after ca. 106 minutes, total of 2 times). The ferret was shown previously to respond to
SEB when injected intravenously.
Suckling (Infant) Mouse
This animal model was introduced by Dean et al.
12 primarily for Escherichia coli enterotoxins and
is now used for some other foodborne pathogens. Typically, mice are separated from their mothers
and given oral doses of the test material consisting of 0.05–0.1 ml with the aid of a blunt 23-gauge
hypodermic needle. A drop of 5% Evans blue dye per milliliter of test material may be used to
determine the presence of the test material in the small intestine. The animals are usually held at 25
◦ C
for 2 hours and then killed. The entire small intestine is removed, and the relative activity of test
material is determined by the ratio of gut weight to body weight (GW/BW). Giannella
19 found the
following GW/BW ratios for E. coli enterotoxins: <0.074 = negative test; 0.075–0.082 = intermediate
(should be retested); and >0.083 = positive test. The investigator found the day-to-day variability
among various E. coli strains to range from 10.5% to 15.7% and about 9% for replicate tests with the
same strain. A GW/BW of 0.060 was considered negative for E. coli ST a by Mullan et al.
32 In studies
with E. coli ST, Wood et al.
51 treated as positive GW/BW ratios that were >0.087, whereas Boyce
et al.
6 held mice at room temperature for 4 hours for Yersinia enterocolitica heat-stable enterotoxin
and considered a GW/BW of 0.083 or greater to be positive. In studies with Y. enterocolitica, Okamoto
et al.,
36 keeping mice for 3 hours at 25
◦ C, considered a GW/BW of 0.083 to be positive.
In using the suckling mouse model, test material may also be injected percutaneously directly in the
stomach through the mouse’s translucent skin or by administration orogastrically or intraperitoneally.
For the screening of large numbers of cultures, the intestines may be examined visually for dilation
and fluid accumulation.
38 Infant mice along with 1- to 3-day-old piglets are the animals of choice for
E. coli enterotoxin ST a ; ST b is inactive in the suckling mouse but active in piglets and weaned pigs.
7,27
The infant mouse assay does not respond to choleragen or to the heat-labile toxin (LT) of E. coli. It
correlates well with the 6-hour rabbit ileal loop assay for the ST a of E. coli.
Suckling mice have been used for lethality studies by employing IP injections. Aulisio et al.
3 used
1- to 3-day-old Swiss mice and injected 0.1 ml of diluted culture. The mice were observed for 7 days;
deaths that occurred within 24 hours were considered nonspecific, whereas deaths occurring between
days 2 and 7 were considered specific for Y. enterocolitica. By this method, an LD 50 can be calculated
relative to numbers of cells per inoculum. In the case of Y. enterocolitica, Aulisio et al. found the LD 50
to be 14 cells, and the average time for death of mice to be 3 days.
Rabbit and Mouse Diarrhea
Rabbits and mice have been employed to test for diarrheagenic activity of some foodborne pathogens.
Employing young rabbits weighing 500–800 g, Pai et al.
37 inoculated orogastrically with approximately 10
10 cells of Y. enterocolitica suspended in 10% sodium bicarbonate. Diarrhea developed
in 87% of 47 rabbits after a mean time of 5.4 days. Bacterial colonization occurred in all animals
regardless of dose of cells.
Mice deprived of water for 24 hours were used by Schiemann
41 to test for the diarrheogenic activity
of Y. enterocolitica. The animals were given inocula of 10
9 cells/ml in peptone water, and fresh drinking
water was allowed 24 hours later. After 2 days, feces of mice were examined for signs of diarrhea.
Infant rabbits have been used by Smith
42 to assay enterotoxins E. coli and Vibrio cholerae. Infant
rabbits 6–9 days old are administered 1–5 ml of culture filtrate via stomach tube. Following return
and vomited (after ca. 106 minutes, total of 2 times). The ferret was shown previously to respond to
SEB when injected intravenously.
Suckling (Infant) Mouse
This animal model was introduced by Dean et al.
12 primarily for Escherichia coli enterotoxins and
is now used for some other foodborne pathogens. Typically, mice are separated from their mothers
and given oral doses of the test material consisting of 0.05–0.1 ml with the aid of a blunt 23-gauge
hypodermic needle. A drop of 5% Evans blue dye per milliliter of test material may be used to
determine the presence of the test material in the small intestine. The animals are usually held at 25
◦ C
for 2 hours and then killed. The entire small intestine is removed, and the relative activity of test
material is determined by the ratio of gut weight to body weight (GW/BW). Giannella
19 found the
following GW/BW ratios for E. coli enterotoxins: <0.074 = negative test; 0.075–0.082 = intermediate
(should be retested); and >0.083 = positive test. The investigator found the day-to-day variability
among various E. coli strains to range from 10.5% to 15.7% and about 9% for replicate tests with the
same strain. A GW/BW of 0.060 was considered negative for E. coli ST a by Mullan et al.
32 In studies
with E. coli ST, Wood et al.
51 treated as positive GW/BW ratios that were >0.087, whereas Boyce
et al.
6 held mice at room temperature for 4 hours for Yersinia enterocolitica heat-stable enterotoxin
and considered a GW/BW of 0.083 or greater to be positive. In studies with Y. enterocolitica, Okamoto
et al.,
36 keeping mice for 3 hours at 25
◦ C, considered a GW/BW of 0.083 to be positive.
In using the suckling mouse model, test material may also be injected percutaneously directly in the
stomach through the mouse’s translucent skin or by administration orogastrically or intraperitoneally.
For the screening of large numbers of cultures, the intestines may be examined visually for dilation
and fluid accumulation.
38 Infant mice along with 1- to 3-day-old piglets are the animals of choice for
E. coli enterotoxin ST a ; ST b is inactive in the suckling mouse but active in piglets and weaned pigs.
7,27
The infant mouse assay does not respond to choleragen or to the heat-labile toxin (LT) of E. coli. It
correlates well with the 6-hour rabbit ileal loop assay for the ST a of E. coli.
Suckling mice have been used for lethality studies by employing IP injections. Aulisio et al.
3 used
1- to 3-day-old Swiss mice and injected 0.1 ml of diluted culture. The mice were observed for 7 days;
deaths that occurred within 24 hours were considered nonspecific, whereas deaths occurring between
days 2 and 7 were considered specific for Y. enterocolitica. By this method, an LD 50 can be calculated
relative to numbers of cells per inoculum. In the case of Y. enterocolitica, Aulisio et al. found the LD 50
to be 14 cells, and the average time for death of mice to be 3 days.
Rabbit and Mouse Diarrhea
Rabbits and mice have been employed to test for diarrheagenic activity of some foodborne pathogens.
Employing young rabbits weighing 500–800 g, Pai et al.
37 inoculated orogastrically with approximately 10
10 cells of Y. enterocolitica suspended in 10% sodium bicarbonate. Diarrhea developed
in 87% of 47 rabbits after a mean time of 5.4 days. Bacterial colonization occurred in all animals
regardless of dose of cells.
Mice deprived of water for 24 hours were used by Schiemann
41 to test for the diarrheogenic activity
of Y. enterocolitica. The animals were given inocula of 10
9 cells/ml in peptone water, and fresh drinking
water was allowed 24 hours later. After 2 days, feces of mice were examined for signs of diarrhea.
Infant rabbits have been used by Smith
42 to assay enterotoxins E. coli and Vibrio cholerae. Infant
rabbits 6–9 days old are administered 1–5 ml of culture filtrate via stomach tube. Following return