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Infectious Emergencies—
Creating the Tools To Study Immune Response
in Farm Animals
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Research associate Gloria Solano-Aguilar
(right) discusses data with immunologist
Joan Lunney after stained cells were
run and analyzed with a laser-based
flow cytometer.
(K9477-1)
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A multivehicle accident just happened
on U.S. Highway 100. Several witnesses grab their cell phones and dial 911. The
dispatcher on the other end radios the nearest highway patrol cars and alerts
fire and rescue squads to rush to the site. The first troopers on the scene
assess the damage and radio back for more patrol cars and emergency personnel,
while rescue workers use walkie-talkies to coordinate treatment of the injured.
Good communication turns chaos into coordination.
If, like Alice in Wonderland, you could shrink up and scramble down the
digestive or respiratory tract of a farm animal, you could witness emergency
scenes like this all along the way as viruses, bacteria, parasites—even
worms—try to penetrate the lining of the animal's gut. |
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Molecular biologist Dante Zarlenga
examines autoradiographic data
to confirm the proper construction
of reagents for studying changes
in bovine and swine cytokine
transcription.
(K9474-1)
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But instead of using radios and
walkie-talkies, cells of the immune system use proteins called cytokines to
orchestrate a response. You may have heard of some of them—the interferons
and interleukins, for example. Others, like chemokines, are less well-known.
Over the last decade, cytokines have become a hot area of interest in the
search for alternatives to antibiotics and other drug therapies for our food
animals, says ARS immunologist Joan K.
Lunney. She heads the Immunology and Disease Resistance Laboratory, at
Beltsville, Maryland.
The laboratory is at the forefront of using cytokines to answer basic questions
about the immune response of pigs and cattle. In pigs, for example, Lunney
wants to know at what age the immune system starts functioning and how well it
functions. The trick is to find which cytokines generate a protective immune
response and which either distract the immune system or cause an overresponse.
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Microbiologist Joseph Urban
initiates a subclinical oral
infection of young pigs with
a gastrointestinal parasite
to stimulate a strong cytokine
response at mucosal surfaces
in the gut. Support scientist
Ethiopia Beshah prepares to
rapidly process tissues from
infected animals for cytokine
gene expression.
(K9478-1)
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"We know many factors affect
the quality and quantity of cytokine response to an antigen or infection,"
says ARS microbiologist Joseph F. Urban, Jr. Some responses are appropriate and
protect the animal; others are inappropriate and further stress the animal.
"It's a balancing act—the yin-yang hypothesis of regulation."
Knowing which cytokines are key to maintaining this balance will enable
scientists to design therapies that stimulate the desired response or suppress
the undesired. Moreover, scientists will be able to select animals that have
the genetic background for appropriate responses.
Reagents to the Rescue
To look for changes in cytokine levels, one needs reagents. So Lunney's
laboratory has become a kind of reagent factory, preparing tiny molecules that
can be used to measure either the cytokines or changes in their production.
To measure a cytokine directly, the researchers make monoclonal
antibodies—molecules that attach to specific sites on a particular
cytokine. Using these monoclonals, they can measure the amounts of cytokines
produced at each stage of an infection or a vaccine trial. With a sensitive
fluorescence assay, they can even identify the exact cell making the cytokine.
To measure cytokine production indirectly, the researchers make "DNA
competitors," which enable them to detect changes in expression of the
gene that codes for the cytokine. When a cytokine isn't needed, levels of the
gene product—messenger RNA—are low. When the gene gets turned on,
messenger RNA levels rise.
Using the well-known PCR—polymerase chain reaction—the researchers
make millions of DNA copies of messenger RNA for a given cytokine. Then they
use PCR again to snip a chunk of bases—about 100 or so—out of the DNA
copy. This yields a deleted version, called a DNA competitor. To measure gene
expression for a specific cytokine, they use a known amount of its DNA
competitor to serve as a kind of internal standard in the PCR assay.
So far, laboratory personnel have produced DNA competitors for 11 cytokines in
pigs and 16 in cattle. The researchers draw from the large database of human
and mouse genes to prepare their reagents.
"We have sent DNA competitors for swine and cattle to hundreds of
investigators around the world," says ARS microbiologist Dante S.
Zarlenga. He works with cattle and swine cytokines and developed a simple
technique for making DNA competitors.
His colleague, microbiologist Louis C. Gasbarre, has used the reagents to
develop a framework of cattle immunity that researchers worldwide can build on.
It's Beyond Complex
Researchers at Lunney's laboratory are working to get the big picture of immune
response in cattle and pigs. And this picture is more than complex. It will
differ depending on whether the infectious agent is a virus, bacterium,
protozoan parasite, or worm, whether it's a combination of these agents, what
tissue is infected, and how long the infection has been around.
Zarlenga is working on what he calls third-generation assays from farm animals,
which will show changes in expression of many cytokines at once. He says the
technology already exists to "print" thousands of minuscule dots of
functional DNA on a single microscope slide.
Like magnets, these dots will attract their complementary DNA—the copies
researchers make from the cells they are studying. By adding fluorescent labels
to the complementary DNA, researchers can estimate the degree of cytokine
expression by the brightness of each dot.
The technology is up and running for human and mouse DNA, says Zarlenga. He and
Gasbarre are producing cytokine DNA from cattle for each of the spots on the
slide.
Meanwhile, Lunney collaborated with researchers at the University of
Illinois-Urbana and Pharmacia Animal Health in Kalamazoo, Michigan, to produce
monoclonal antibody panels for two pig cytokines. She says each antibody in a
panel attaches to a different site on the cytokine, allowing accurate
measurement. Trouble is, panels exist for less than one-third of the pig
cytokines and are only slowly becoming available.
"Most people want to measure the protein," Lunney notes,
"because you can actually determine which immune cell is making it."
Eventually, she hopes there will be antibody arrays for pigs and cattle like
the DNA arrays Zarlenga is working on.—By
Judy McBride,
Agricultural Research Service Information Staff.
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.
Joan K. Lunney,
Dante S. Zarlenga,
Louis C. Gasbarre, and
Joseph F. Urban, Jr., are with
the USDA-ARS Immunology and
Disease Resistance Laboratory, 10300 Baltimore Ave., Bldg. 1040,
Beltsville, MD 20705-2350; phone (301) 504-8201, fax (301) 504-5306. |
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"Infectious Emergencies" was published in
the June 2001
issue of Agricultural Research magazine.
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