Targeting E. coli
At the National Animal Disease
Center in Ames, Iowa, visiting
scientist Joachim Pohlenz, a
pathologist from the School
of Veterinary Medicine in
Hannover, Germany, selects
and trims intestinal tissues
to be processed and examined
for signs of E. coli O157:H7
Getting at bacteria before they have a chance of getting into people
is the focus of ARS studies in
Ames, Iowa, aimed at stopping a particularly nasty E. coli-related
Microbiologist Evelyn Dean-Nystrom and veterinary medical officer William
Stoffregen of ARS's National Animal Disease Center (NADC) have worked
to pinpoint where E. coli O157:H7 bacteria lurk in calves. Nystrom
is also working with scientists at the Uniformed Services University
of the Health Sciences in Bethesda, Maryland, to develop and test an
oral vaccine that eliminates E. coli O157:H7 bacteria from cattle.
Nystrom works at NADC's Preharvest Food Safety and Enteric Diseases
Research Unit, while Stoffregen works at the center's Bacterial Diseases
of Livestock Research Unit.
Enterohemorrhagic E. coli O157:H7 is the most common infectious cause of bloody diarrhea in people in the United States. Hemolytic uremic syndrome, a potential consequence of E. coli O157:H7 infection, is the primary cause of acute kidney failure in U.S. children.
Microbiologist Evelyn Nystrom
and technician Bryan Wheeler
vaccinate a calf orally with
cells mixed with milk replacer.
Where Bacteria Hide
E. coli is normally found in the intestines of all animals,
including humans, where it suppresses growth of harmful bacteria. But
E. coli O157:H7 is a rare variety that produces large quantities
of potent Shiga toxins, which can cause severe damage to small blood
vessels and kidney tissue.
Undercooked or raw ground beef has been implicated in many E. coli
O157:H7 disease outbreaks among humans. In the United States, Shiga
toxin-producing E. coli causes diarrhea in more than 100,000
people each year, with E. coli O157:H7 responsible for more than
70,000 of these cases.
"In cattle, these bacteria almost always have no easily discernible effect," says Nystrom. "That's a major reason why E. coli O157:H7 is hard to detect in them."
Nystrom and Stoffregen found that, in addition to intestines, calves'
gall bladders may be good indicators of whether an E. coli O157:H7
infection has taken place. The gall bladder stores and secretes bile,
which includes salts used to break down food.
Veterinarian Bill Stoffregen shows
microbiologist Evelyn Nystrom
epithelial surface lesions and
brown-stained E. coli O157:H7
bacteria in the gall bladder
of an infected calf.
| The researchers found that signs
of bacterial infection were present in the gall bladders of 12 of 13 calves
used for their study. Four days after Nystrom and Stoffregen inoculated
weaned calves with E. coli O157:H7 bacteria, most had developed
cholecystitis (inflammation of the gall bladder), and many had lesions
and E. coli O157:H7 bacteria in their gall bladders.
"This discovery identifies the gall bladder as a possible niche for E. coli O157:H7 infection in cattle and as a potential source of Shiga toxin-producing E. coli contamination of beef products," says Stoffregen. "Including gall bladders in samples cultured for E. coli O157:H7 may help identify infected cattle at slaughter," adds Nystrom.
Intimin Is Vaccine's Key Ingredient
The key ingredient of the oral vaccinedeveloped at the Bethesda
university's Department of Microbiology and Immunologyis intimin,
a protein on the outer membrane of the O157:H7 strain. The bacteria
need intimin to attach themselves to intestinal tissue.
Nystrom assisted with this study early on by showing that calves injected
with purified bacterial intimin would develop antibodies against it.
"This confirmed previous studies in mice that showed that intimin-specific
responses reduced adherence of E. coli O157:H7 bacteria to both
cultured tissue cells and to intestinal cells in the intact animal,"
Nystrom's work also revealed that intimin-fighting antibodies interfere
with E. coli O157:H7 colonization and lessen intestinal damage
in newborn pigs. Earlier studies found that pregnant pigs vaccinated
against bacterial intimin developed antibodies against it in their sera
and colostrum. Also, newborn piglets experimentally challenged with
a Shiga toxin-negative E. coli O157:H7 strain, and who ingested
colostrum from intimin-vaccinated pigs, had fewer of the inoculated
bacteria in their intestines than did piglets nursed by nonvaccinated
Microbiologist Alison O'Brien, who chairs the university department,
is working closely with Nystrom in efforts to prove that the vaccine
is effective in cattle and to develop a plant containing the vaccine
that cattle will eat. "We want an inexpensive, effective, easily
administered vaccine to prevent cattle from becoming infected with E.
coli O157:H7, thus blocking transmission of these organisms to humans,"
O'Brien says. Corn is a potential candidate for development into an
intimin-producing edible plant for livestock, she adds.
Nystrom says a vaccine directed against intimin will not affect colonization
by beneficial, non-disease-causing E. coli bacteria because these
bacteria do not produce intimin.
The vaccine was developed by Nicole A. Judge, a graduate student in
O'Brien's laboratory who transferred the gene that encodes for intimin
into a non-nicotine tobacco cell line. Tobacco cells are the standard
ones used to determine whether plants can express a foreign antigen.
In the lab, the cell line was freeze-dried into powder that was then
rehydrated with buffer, mixed with milk, and given to calves.
Nystrom explains that the vaccine as first created could not be produced
in sufficient quantities to make enough intimin to be effective in cattle.
Wayne Curtis, a Pennsylvania State University professor, was contracted
to address this problem. He successfully scaled up production of intimin-expressing
tobacco cells so that a vaccine could be effective for calves.
"E. coli O157:H7 is a very dangerous infection, and cattle are an important source of it," says Nystrom. "One way to reduce the risk of infections in humans is to reduce the level of these bacteria at the source point. Through vaccines such as this one and by pinpointing where the bacteria hide in cattle, we can contribute greatly to making beef an even safer consumer product."By Luis Pons, Agricultural Research Service Information Staff.
This research is part of Food Safety (Animal and Plant Products),
an ARS National Program (#304) described on the World Wide Web at www.nps.ars.usda.gov.
Evelyn A. Dean-Nystrom
and William C. Stoffregen
are at the USDA-ARS National
Animal Disease Center, 2300 Dayton Ave., Ames, IA 50010-0070; phone
(515) 663-7376 [Nystrom], (515) 663-7844 [Stoffregen], fax (515) 663-7458.
"Targeting E. coli Infections at Their Source" was published in the August 2004 issue of Agricultural Research magazine.