Bioassay and Related Methods
295
for E. coli enterotoxins. For the LT of E. coli, CHO has been found to be 5–100 times more sensitive
than skin permeability and rabbit ileal loop assays.
21
Vero Cells
This monolayer consists of a continuous cell line derived from African green monkey kidneys; it
was employed by Speirs et al.
43 to assay for E. coli LT. Vero cell results compare favorably with
Y-1 adrenal cells (see below), and the test was found by these investigators to be the simple and
more economical of the two to maintain in the laboratory. Toxigenic strains produce a morphological
response to Vero cells similar to Y-1 cells. The use of Vero cells to study E. coli toxins is discussed in
Chapter 27.
A highly sensitive and reproducible biological assay for C. perfringens enterotoxin employing
Vero cells was developed by McDonel and McClane.
29 The assay is based on the observation that
the enterotoxin inhibits plating efficiency of Vero cells grown in culture. The inhibition of plating
efficiency detected as little as 0.1 ng of enterotoxin, and a linear dose-response curve was obtained
with 0.5–5 ng (5–50 ng/ml). The investigators proposed a new unit of biological activity—the plating
efficiency unit (PEU)—as that amount of enterotoxin that causes a 25% inhibition of the plating of
200 cells inoculated into 100 µl of medium.
Y-1 Adrenal Cell Assay
In this widely used assay, mouse adrenal cells (Y-1) are grown in a monolayer using standard cell
culture techniques. With monolayer cells in microtiter plate wells, test extracts or filtrates are added to
the microtiter wells followed by incubation at 37
◦ C. In testing E. coli LT, heated and unheated culture
filtrates of known positive and negative LT-producing strains are added to monolayers in microtiter
plates and results are determined by microscopic examinations. The presence of 50% or more rounded
cells in monolayers of unheated filtrates and 10% or less for heated filtrates denotes a positive response.
The specificity of the response can be determined by the use of specific antibodies in toxin-containing
filtrates. Details of this method for foodborne pathogens are presented in BAM.
17
Other Assays
An immunofluorescence method was employed by Boutin et al.
5 using 6-week-old rabbit ileal
loops inoculated with V. parahaemolyticus. The loops were removed 12–18 hours after infection and
placed in trays, cut into tissue sections, and cleaned by agitation. Tissue sections were fixed and
stained with fluorescein isothiocyanate-stained agglutinins to V. parahaemolyticus. The reaction of
the tagged antibody with V. parahaemolyticus cells in the tissue was assessed microscopically. By
use of immunofluorescence, it was possible to demonstrate the penetration by this organism into the
lamina propria of the ileum and thus the tissue invasiveness of the pathogen. Both Kanagawa-positive
and -negative cells penetrated the lamina according by this method.
The chorioallantoic membrane of 10-day-old chick embryos was used to assess the pathogenicity of
Listeria spp. L. monocytogenes causes death within 2–5 days with as few as 100 cells, and L. ivanovii
cells are lethal at levels of 100–30,000 per egg with death within 72 hours.
46 Culture filtrates of L.
monocytogenes and L. ivanovii release lactate dehydrogenase (LDH) in rat hepatocyte monolayers
following 3-hour exposures, but other listerial species had no effect.
24
295
for E. coli enterotoxins. For the LT of E. coli, CHO has been found to be 5–100 times more sensitive
than skin permeability and rabbit ileal loop assays.
21
Vero Cells
This monolayer consists of a continuous cell line derived from African green monkey kidneys; it
was employed by Speirs et al.
43 to assay for E. coli LT. Vero cell results compare favorably with
Y-1 adrenal cells (see below), and the test was found by these investigators to be the simple and
more economical of the two to maintain in the laboratory. Toxigenic strains produce a morphological
response to Vero cells similar to Y-1 cells. The use of Vero cells to study E. coli toxins is discussed in
Chapter 27.
A highly sensitive and reproducible biological assay for C. perfringens enterotoxin employing
Vero cells was developed by McDonel and McClane.
29 The assay is based on the observation that
the enterotoxin inhibits plating efficiency of Vero cells grown in culture. The inhibition of plating
efficiency detected as little as 0.1 ng of enterotoxin, and a linear dose-response curve was obtained
with 0.5–5 ng (5–50 ng/ml). The investigators proposed a new unit of biological activity—the plating
efficiency unit (PEU)—as that amount of enterotoxin that causes a 25% inhibition of the plating of
200 cells inoculated into 100 µl of medium.
Y-1 Adrenal Cell Assay
In this widely used assay, mouse adrenal cells (Y-1) are grown in a monolayer using standard cell
culture techniques. With monolayer cells in microtiter plate wells, test extracts or filtrates are added to
the microtiter wells followed by incubation at 37
◦ C. In testing E. coli LT, heated and unheated culture
filtrates of known positive and negative LT-producing strains are added to monolayers in microtiter
plates and results are determined by microscopic examinations. The presence of 50% or more rounded
cells in monolayers of unheated filtrates and 10% or less for heated filtrates denotes a positive response.
The specificity of the response can be determined by the use of specific antibodies in toxin-containing
filtrates. Details of this method for foodborne pathogens are presented in BAM.
17
Other Assays
An immunofluorescence method was employed by Boutin et al.
5 using 6-week-old rabbit ileal
loops inoculated with V. parahaemolyticus. The loops were removed 12–18 hours after infection and
placed in trays, cut into tissue sections, and cleaned by agitation. Tissue sections were fixed and
stained with fluorescein isothiocyanate-stained agglutinins to V. parahaemolyticus. The reaction of
the tagged antibody with V. parahaemolyticus cells in the tissue was assessed microscopically. By
use of immunofluorescence, it was possible to demonstrate the penetration by this organism into the
lamina propria of the ileum and thus the tissue invasiveness of the pathogen. Both Kanagawa-positive
and -negative cells penetrated the lamina according by this method.
The chorioallantoic membrane of 10-day-old chick embryos was used to assess the pathogenicity of
Listeria spp. L. monocytogenes causes death within 2–5 days with as few as 100 cells, and L. ivanovii
cells are lethal at levels of 100–30,000 per egg with death within 72 hours.
46 Culture filtrates of L.
monocytogenes and L. ivanovii release lactate dehydrogenase (LDH) in rat hepatocyte monolayers
following 3-hour exposures, but other listerial species had no effect.
24