Bioassay and Related Methods
289
to their mothers, they are observed for diarrhea. Diarrhea after 6–8 hours is a positive response. If
death of animals occurs, a large volume of yellow fluid is found in the small and large intestines.
The quantitation of enterotoxin is achieved by ascertaining the ratio of intestinal weight to total body
weight. Young pigs have been used in a similar way to assay porcine strains of E. coli for enterotoxin
activity. Infant rabbits have been employed to detect Shiga-like toxins of E. coli.
34
Monkey Feeding
The use of rhesus monkeys (Macaca mulatta) to assay staphylococcal enterotoxins was developed
in 1931 by Jordan and McBroom.
26 Next to humans, this is perhaps the animal most sensitive to
staphylococcal enterotoxins. When enterotoxins are to be assayed by this method, young rhesus
monkeys weighing 2–3 kg are selected. The food homogenate, usually in solution in 50-ml quantities,
is administered via stomach tube. The animals are then observed continuously for 5 hours. Vomiting in
at least two of six animals denotes a positive response. Rhesus monkeys have been shown to respond
to levels of enterotoxins A and B as low as approximately 5 µg per 2–3 kg of body weight.
31
Kitten (Cat) Test
This method was developed by Dolman et al.
15 as an assay for staphylococcal enterotoxins. The
original test employed the injection of filtrates into the abdominal cavity of very young kittens (250–
500 g). This procedure leads to false-positive results. The most commonly used method consists of
administering the filtrates IV and observing the animals continuously for emesis. When cats weighing
2–4 kg are used, positive responses occur in 2–6 hours.
10 Emesis has been reported to occur with
0.1 and 0.5 µg of staphylococcal enterotoxin A (SEA) and SEB per kilogram of body weight.
4 The
test tends to lack the specificity of the monkey-feeding test because staphylococcal culture filtrates
containing other byproducts may also induce emesis. Kittens are much easier to obtain and maintain
than rhesus monkeys, and in this regard the test has value.
Rabbit and Guinea Pig Skin Tests
The skin of these two animals is used to assay toxins for at least two properties. The vascular
permeability test is generally done by use of albino rabbits weighing 1.5–2.0 kg. Typically, 0.05–0.1
ml of culture filtrate is inoculated intradermally (ID) in a shaved area of the rabbit’s back and sides.
From 2 to 18 hours later, a solution of Evans blue dye is administered IV, and 1–2 hours are allowed
for permeation by the dye. The diameters of two blue zones are measured and the area approximated
by squaring the average of the two values. Areas of 25 cm
2 are considered positive. E. coli LT gives a
positive response in this assay.
16 Employing this assay, permeability has been shown to be a function
of the E. coli diarrheagenic enterotoxin.
Similar to the permeability factor test is a test of erythemal activity that employs guinea pigs. The
method has been employed by Stark and Duncan
45 to test for erythemal activity of C. perfringens
enterotoxin. Guinea pigs weighing 300–400 g are depilated (back and sides) and marked in 2.5-cm
squares, and duplicate 0.05-ml samples of toxic preparations are injected ID in the center of the
squares. Animals are observed after 18–24 hours for erythema at the injection site. In the case of
C. perfringens enterotoxin, a concentric area of erythema is produced without necrosis. A unit of
erythemal activity is defined as the amount of enterotoxin producing an area of erythema 0.8 cm in
289
to their mothers, they are observed for diarrhea. Diarrhea after 6–8 hours is a positive response. If
death of animals occurs, a large volume of yellow fluid is found in the small and large intestines.
The quantitation of enterotoxin is achieved by ascertaining the ratio of intestinal weight to total body
weight. Young pigs have been used in a similar way to assay porcine strains of E. coli for enterotoxin
activity. Infant rabbits have been employed to detect Shiga-like toxins of E. coli.
34
Monkey Feeding
The use of rhesus monkeys (Macaca mulatta) to assay staphylococcal enterotoxins was developed
in 1931 by Jordan and McBroom.
26 Next to humans, this is perhaps the animal most sensitive to
staphylococcal enterotoxins. When enterotoxins are to be assayed by this method, young rhesus
monkeys weighing 2–3 kg are selected. The food homogenate, usually in solution in 50-ml quantities,
is administered via stomach tube. The animals are then observed continuously for 5 hours. Vomiting in
at least two of six animals denotes a positive response. Rhesus monkeys have been shown to respond
to levels of enterotoxins A and B as low as approximately 5 µg per 2–3 kg of body weight.
31
Kitten (Cat) Test
This method was developed by Dolman et al.
15 as an assay for staphylococcal enterotoxins. The
original test employed the injection of filtrates into the abdominal cavity of very young kittens (250–
500 g). This procedure leads to false-positive results. The most commonly used method consists of
administering the filtrates IV and observing the animals continuously for emesis. When cats weighing
2–4 kg are used, positive responses occur in 2–6 hours.
10 Emesis has been reported to occur with
0.1 and 0.5 µg of staphylococcal enterotoxin A (SEA) and SEB per kilogram of body weight.
4 The
test tends to lack the specificity of the monkey-feeding test because staphylococcal culture filtrates
containing other byproducts may also induce emesis. Kittens are much easier to obtain and maintain
than rhesus monkeys, and in this regard the test has value.
Rabbit and Guinea Pig Skin Tests
The skin of these two animals is used to assay toxins for at least two properties. The vascular
permeability test is generally done by use of albino rabbits weighing 1.5–2.0 kg. Typically, 0.05–0.1
ml of culture filtrate is inoculated intradermally (ID) in a shaved area of the rabbit’s back and sides.
From 2 to 18 hours later, a solution of Evans blue dye is administered IV, and 1–2 hours are allowed
for permeation by the dye. The diameters of two blue zones are measured and the area approximated
by squaring the average of the two values. Areas of 25 cm
2 are considered positive. E. coli LT gives a
positive response in this assay.
16 Employing this assay, permeability has been shown to be a function
of the E. coli diarrheagenic enterotoxin.
Similar to the permeability factor test is a test of erythemal activity that employs guinea pigs. The
method has been employed by Stark and Duncan
45 to test for erythemal activity of C. perfringens
enterotoxin. Guinea pigs weighing 300–400 g are depilated (back and sides) and marked in 2.5-cm
squares, and duplicate 0.05-ml samples of toxic preparations are injected ID in the center of the
squares. Animals are observed after 18–24 hours for erythema at the injection site. In the case of
C. perfringens enterotoxin, a concentric area of erythema is produced without necrosis. A unit of
erythemal activity is defined as the amount of enterotoxin producing an area of erythema 0.8 cm in