60 Part I: Principles
system enhanced the sensitivity of the PCR assay by
up to 100-fold (Ge et al. 2002). In the future, the use
of robotic equipment will result in automation of the
PCR-ELISA procedure, allowing for fast, sensitive,
accurate, and large-scale screening of microorgan-
isms that produce the Shiga toxins. This method also
can be applied for detection of any other food path-
ogens, using specific biotin-labeled PCR primers.
BOVINESPONGIFORMENCEPHALOPATHY
DETECTION
The transmissible spongiform encephalopathies
(TSEs), are a group of neurodegenerative diseases
affecting many animals, including humans, and are
characterized by the formation of microscopic
“holes” in the brain tissue. Members of the TSE
family include (1) the diseases that afflict humans:
kuru, Gertsmann-Sträussler-Scheinker (GSS), fatal
familial insomnia (FFI), and Creutzfeldt-Jakob dis-
ease (CJD); and (2) the diseases that afflict animals:
scrapie (sheep and goats), wasting disease (elk and
deer), mink encephalopathy (mink), feline spongi-
form encephalopathy (cats), and bovine spongiform
encephalopathy (BSE) (Prusiner 1998). Bovine
spongiform encephalopathy affects cattle and is
commonly known as mad cow disease. The symp-
toms associated with BSE are weight loss, drooling,
head waving, aggressive behavior, and eventually
death. All TSEs, including BSE, are caused by a new
infectious agent called a “prion protein” (PrP)
(Prusiner 1982). Dr. Stanley Prusiner discovered pri-
ons in 1984 and was awarded the 1997 Medicine
Nobel Prize for his research. Prions are endogenous
glycoproteins found abundantly in the brain tissue
of all mammals and may function as neuron helpers.
They can manifest in two different protein confor-
mations: (1) PrPc, the normal form, which is non-
pathogenic, protease sensitive, and high in -helical
content, and (2) PrPSc, the misfolded form, which is
disease-inducing, protease resistant, and high in -
sheet content. PrPSc has infection properties, and
when it comes in contact with PrPc, it starts a chain
reaction, transforming PrPc into PrPSc (Horiuchi
and Caughey 1999). Prions are not completely de-
stroyed by sterilization, autoclaving, disinfectants,
radiation, or cooking. They are totally degraded only
with incineration at temperatures greater than
1000°C or treatment with strong sodium hydroxide
solutions (Dormont 1999).
Scientists believe that BSE can spread among cat-
tle through contaminated feed containing the dis-
ease-inducing form of prion. It is a common practice
in many countries to feed cattle with the remains of
other farm animals as a source of protein. The
hypothesis for the spread of BSE in cattle is that
body parts of sheep infected with scrapie were
included in cattle feed, and PrPSc jumped species to
infect bovines (Bruce et al. 1997). The disease
spread throughout the world when England sold
BSE-contaminated cattle feed to other countries.
Since it first appeared in 1986, the risk of BSE infec-
tion has resulted in the destruction of 3.7 million
animals in the United Kingdom. In humans, CJD is
an inherited disease caused by a mutation in the pri-
on protein gene, PRNP, and affects one in a million
people. It is believed that the human victims may
have contracted a new variant of CJD (nv-CJD)
from eating meat products contaminated with BSE.
The pathogenic prion protein that causes BSE is
nearly identical to the prion that causes nv-CJD
(Johnson and Gibbs 1998). Between 1996 and 2003,
156 cases of nv-CJD have been suspected or con-
firmed in many countries, mainly in Great Britain.
One of the main problems in dealing with and
containing the BSE epidemic is that there are no
tests for detection of the disease in a live animal.
Prions do not trigger any detectable specific immune
response, and levels of abnormal prions in other
parts of the body, such as the blood, are too low to
detect. The only means of diagnosis in live animals
is observation of BSE symptoms in the animal.
Because of the lack of reliable live-detection meth-
ods, all animals in a herd that may have been in con-
tact with BSE-contaminated feed must be destroyed,
causing great losses for the cattle industry.
The primary laboratory method used to confirm a
diagnosis of BSE is the microscopic examination of
brain tissue after death of the animal. In addition to
microscopic examination, there are several tech-
niques used to detect the PrPSc, among which, the
Western blot test and immunocytochemistry (devel-
oped by Prionics AG, Switzerland) are the most
commonly used (Kübler et al. 2003). Other current-
ly approved BSE tests include (1) a test developed
by CEA, a research group in France, in which a
sandwich immunoassay technique for PrPSc is done
following denaturation and concentration steps and
(2) a test developed by Enfer Scientific, in Ireland, in
which polyclonal antibodies and an enzyme-coupled