290 Modern Food Microbiology
diameter. The enterotoxin preparation used by Stark and Duncan contained 1,000 erythemal units/ml.
To enhance readings, 1 ml of 0.5% Evans blue can be injected intracardially (IC) 10 minutes following
the skin injections and the diameters read 80 minutes later.
18 The specificity of the skin reactions can
be determined by neutralizing the enterotoxin with specific antisera prior to injections. The erythema
test was found to be 1,000 times more sensitive than the rabbit ileal loop technique for assaying the
enterotoxin of C. perfringens.
23
Sereny and Anton Tests
The Sereny method is used to test for virulence of viable bacterial cultures. It was proposed by
Sereny in 1955, and the guinea pig is the animal most often used. The test consists of administering,
with the aid of a loop, a drop of cell suspension, containing 1.5 × 10
10 to 2.3 × 10
10 /ml in phosphatebuffered saline, into the conjunctivae of guinea pigs weighing about 400 g each. The animal’s eyes are
examined daily for 5 days for evidence of keratoconjunctivitis. When strains of unknown virulence
are evaluated, it is important that known positive and negative strains are tested also.
A mouse Sereny test has been developed using Swiss mice and administering half of the dose noted
above. A Sereny test for shigellae and enteroinvasive E. coli (EIEC) strains has been developed and
found useful.
33
The Anton test is similar to the Sereny; it is used to assess the virulence of Listeria spp. Conjunctivitis
is produced when about 10
6 cells of L. monocytogenes are administered into the eye of a rabbit or
guinea pig.
2
ANIMAL MODELS REQUIRING SURGICAL PROCEDURES
Ligated Loop Techniques
These techniques are based on the fact that certain enterotoxins elicit fluid accumulation in the small
intestines of susceptible animals. Although they may be performed with a variety of animals, rabbits
are most often employed. Young rabbits 7–20 weeks old and weighing 1.2–2.0 kg are kept off food
and water for a period of 24 hours or off food for 48–72 hours with water ad libitum prior to surgery.
Under local anesthesia, a midline incision about 2 inches long is made just below the middle of the
abdomen through the muscles and peritoneum in order to expose the small intestines.
11 A section of
the intestine midway between its upper and lower ends or just above the appendix is tied with silk or
other suitable ligatures in 8 to 12-cm segments with intervening sections of at least 1 cm. Up to six
sections may be prepared by single or double ties.
Meanwhile, the specimen or culture to be tested is prepared, suspended in sterile saline, and injected
intraluminally into the ligated segments. A common inoculum size is 1 ml, although smaller or larger
doses may be used. Different doses of test material may be injected into adjacent loops or into loops
separated by a blank loop or by a sham (inoculated with saline). Following injection, the abdomen
is closed with surgical thread, and the animal is allowed to recover from anesthesia. The recovered
animal may be kept off food and water for an additional 18–24 hours, or water or feed or both may be
allowed. With ligatures intact, the animals may not survive beyond 30–36 hours.
8
To assess the effect of the materials previously injected into ligated loops, the animal is killed, and the
loops are examined and measured for fluid accumulation. The fluid may be aspirated and measured. The
reaction can be quantitated by measuring loop fluid volume to loop length ratios,
8 or by determining
diameter. The enterotoxin preparation used by Stark and Duncan contained 1,000 erythemal units/ml.
To enhance readings, 1 ml of 0.5% Evans blue can be injected intracardially (IC) 10 minutes following
the skin injections and the diameters read 80 minutes later.
18 The specificity of the skin reactions can
be determined by neutralizing the enterotoxin with specific antisera prior to injections. The erythema
test was found to be 1,000 times more sensitive than the rabbit ileal loop technique for assaying the
enterotoxin of C. perfringens.
23
Sereny and Anton Tests
The Sereny method is used to test for virulence of viable bacterial cultures. It was proposed by
Sereny in 1955, and the guinea pig is the animal most often used. The test consists of administering,
with the aid of a loop, a drop of cell suspension, containing 1.5 × 10
10 to 2.3 × 10
10 /ml in phosphatebuffered saline, into the conjunctivae of guinea pigs weighing about 400 g each. The animal’s eyes are
examined daily for 5 days for evidence of keratoconjunctivitis. When strains of unknown virulence
are evaluated, it is important that known positive and negative strains are tested also.
A mouse Sereny test has been developed using Swiss mice and administering half of the dose noted
above. A Sereny test for shigellae and enteroinvasive E. coli (EIEC) strains has been developed and
found useful.
33
The Anton test is similar to the Sereny; it is used to assess the virulence of Listeria spp. Conjunctivitis
is produced when about 10
6 cells of L. monocytogenes are administered into the eye of a rabbit or
guinea pig.
2
ANIMAL MODELS REQUIRING SURGICAL PROCEDURES
Ligated Loop Techniques
These techniques are based on the fact that certain enterotoxins elicit fluid accumulation in the small
intestines of susceptible animals. Although they may be performed with a variety of animals, rabbits
are most often employed. Young rabbits 7–20 weeks old and weighing 1.2–2.0 kg are kept off food
and water for a period of 24 hours or off food for 48–72 hours with water ad libitum prior to surgery.
Under local anesthesia, a midline incision about 2 inches long is made just below the middle of the
abdomen through the muscles and peritoneum in order to expose the small intestines.
11 A section of
the intestine midway between its upper and lower ends or just above the appendix is tied with silk or
other suitable ligatures in 8 to 12-cm segments with intervening sections of at least 1 cm. Up to six
sections may be prepared by single or double ties.
Meanwhile, the specimen or culture to be tested is prepared, suspended in sterile saline, and injected
intraluminally into the ligated segments. A common inoculum size is 1 ml, although smaller or larger
doses may be used. Different doses of test material may be injected into adjacent loops or into loops
separated by a blank loop or by a sham (inoculated with saline). Following injection, the abdomen
is closed with surgical thread, and the animal is allowed to recover from anesthesia. The recovered
animal may be kept off food and water for an additional 18–24 hours, or water or feed or both may be
allowed. With ligatures intact, the animals may not survive beyond 30–36 hours.
8
To assess the effect of the materials previously injected into ligated loops, the animal is killed, and the
loops are examined and measured for fluid accumulation. The fluid may be aspirated and measured. The
reaction can be quantitated by measuring loop fluid volume to loop length ratios,
8 or by determining