...From the pages of Agricultural Research magazine
An Udder Solution for Bossie's Woes
Dairy scientist Max Paape
and cell biologist Yan Wang
use fast protein liquid
chromatography to purify
bovine recombinant CD14.
It's almost impossible to protect dairy cows from E. coli and
other coliform bacteria. These gram-negative bacteria lurk in bedding
and other damp areaseven in the cleanest dairy barns--waiting
for a nice, warm udder to infiltrate. And they make life miserable for
3 million U.S. dairy cows that show visible signs of acute infection
. . . not to mention costing an estimated $1.4 billion in annual losses
for Bossie's owners from incapacitated cows and milk that can't be sold.
Coliform bacteria account for about 40 to 50 percent of mastitis cases
in the United States, and 80 percent of these cows will become visibly
sick, says Agricultural Research
Service dairy scientist Max Paape. Of the 3 million cows infected
annually, the bacteria put about 300,000or one-tenthout
of commission entirely. And many die from shock induced by the bacterial
toxin, or endotoxin.
"Standard therapies haven't been successful in relieving symptoms and reducing mortality from acute coliform mastitis," Paape says. "Vaccines have had limited success in reducing clinical symptoms, but they don't eliminate the coliform organisms."
That's about to change. Late last year, ARS filed a patent application
on a recombinant gene that promises both effective treatment for infected
cows and prevention in future cows bioengineered with the gene. It codes
for a proteinsoluble CD14that's naturally suspended in cows'
milk and blood plasma.
The protein binds to the endotoxin and neutralizes it. That prevents
the cow's immune system from overreacting to the toxin, a reaction that
bungles the infection-fighting process and could put the animal into
shock, explains Paape. He is at ARS' Immunology and Disease Resistance
Laboratory in Beltsville, Maryland.
CD14 also sensitizes the lining of a cow's mammary glands to very low
levels of endotoxinproduced by just a few bacteria. Once sensitized,
these mammary cells start an attack against the infiltrating bacteria
before they can get a hoofhold and pour out enough endotoxin to make
the cow sick, explains cell biologist Yan Wang, who conducted this research
with Paape for her doctoral dissertation. She is now at the National
Institute of Allergy and Infectious Diseases in Rockville, Maryland.
Paape and Wang are co-inventors of the recombinant gene and its applications, along with colleague Dante Zarlenga, a molecular biologist who specializes in cloning and designing genes.
A Bet That Paid Off
Paape had already found the CD14 protein embedded in the membranes
of white blood cells in cows. And the soluble kind was known to increase
during coliform infections in humans and laboratory animals, says Wang.
So she and Paape predicted that they'd find the soluble protein in cows'
milk. They also bet it could temper the animals' acute reaction to coliform
endotoxin while initiating an appropriate response to the infiltrating
bacteria. They bet right.
They enlisted Zarlenga's expertise to clone the gene for soluble CD14.
He used as a template the gene for the type of CD14 that stays bound
to membranes. Because the gene for the membrane-bound form is longer
than the one for the soluble version, he and Wang clipped off the extra
bases on one end before inserting the recombinant CD14 gene into bacterial
Then they transferred the gene to insect cells in order to produce
enough of the protein to test.
Tissue culture studies showed that the recombinant CD14 protein binds
to endotoxin, effectively neutralizing it. The researchers expect it
will do the same when injected into a sick cow's blood, but they don't
have enough of the protein yet for such a study.
"For the first time, veterinarians will have a product to prevent
acute endotoxin shock in dairy cows," says Paape. Wang emphasizes
that CD14 "is a protein found naturally in cows, so any side effects
should be minimal."
Prevention Preferred Over Cure
CD14 works to prevent infection, too. Paape and Wang incubated the
protein with endotoxin in a culture dish to form a complex. When they
injected the protein-endotoxin complex into cows' teats, it stimulated
the mammary cells to launch an appropriate responseone that brings
in the white blood cells that gobble up coliform bacteriawithout
calling in the cavalry.
"It needs more testing in cows," says Wang, "but I think
it's very promising for both treatment and prevention."
While it's not feasible for dairy producers to inject CD14 into their
cows' teats regularly, the gene for CD14 can be designed and inserted
into tomorrow's dairy cows so that it produces the protein only in their
mammary cells. And that's not science fiction.
ARS colleagues in the Gene Evaluation and Mapping Laboratory at Beltsville
have already produced a cow with engineered immunity. "Annie"
is a clone of a Jersey cow whose mammary cells produce a protein that
promises to prevent infections from Staphylococcus.
Two of those colleagues, ARS physiologist Robert Wall and support scientist
Juli Foster-Fry, are constructing a designer CD14 gene and will insert
it into mice. If tests show that it works, Wall plans to insert it into
cows. Ultimately, his laboratory wants to bioengineer a cow that is
protected against all mastitis-causing organisms. And that's when you'll
see Bossie smile!By Judy
McBride, formerly with ARS.
This research is part of Animal Health, an ARS National Program
(#103) described on the World Wide Web at http://www.nps.ars.usda.gov.
Max J. Paape and
Dante Zarlenga are
with the USDA-ARS Immunology
and Disease Resistance Laboratory, 10300 Baltimore Ave., Bldg. 1040,
Room 105, Beltsville, MD 20705-2350; phone (301) 504-8302, fax (301)
"An Udder Solution for Bossie's Woes" was published in the June 2002 issue of Agricultural Research magazine.