Plant Produced Protein for Dairy Cow Infection    

Researchers have developed a new approach to thwart
bacteria that cause coliform mastitis, an inflammatory
udder disease of dairy cows.

Coliform mastitis is the most prevalent form of clinical
mastitis in the dairy industry and is mainly caused by the
Escherichia coli bacterium. Use of antibiotics to control
mastitis infections can be expensive and carries with it
concerns about the emergence of antibiotic-resistant
bacteria. Yet mastitis is expensive too, costing dairy
farmers an estimated $2 billion annually from incapacitated
cows and milk that can’t be sold.

Now, scientists have endowed a potato virus with a gene
that - when introduced into a host plant - prompts the plant
to produce a therapeutic protein called “CD14.” First
isolated by Dante Zarlenga of the ARS Bovine Functional
Genomics Laboratory, this beneficial protein can be
extracted from the plant and used to treat mastitis. The
engineered, or recombinant, virus is called “PVX/CD14,”
for “potato virus X carrying the gene of therapeutic protein

The researchers extracted enough CD14 protein from the
inoculated plants for field tests. Their colleagues, dairy
scientist Max Paape and microbiologist Douglas
Bannerman with the ARS Bovine Functional Genomics
Laboratory, Beltsville, Maryland, tested purified protein’s
ability to alleviate mastitis in dairy cows.

CD14 is known to help the immune system fight infection,
but it is present in the cow’s mammary gland at low levels.
CD14 binds to a molecule known as “lipopolysaccharide,”
located on E. coli’s outer membrane. The scientists
hypothesized that increasing the level of CD14 in the milk
would enhance protection.

When inserted into the mammary gland through the
opening of a cow’s teats, CD14 binds to the E. coli and
triggers the cow’s immune response, which fights mastitis
inflammation. That process helps to neutralize and clear
toxins produced by the bacteria, lessening the chances of
an excessive immune response.

The researchers infused the CD14 protein into one of a
test cow’s four teats, or “quarters.” All four quarters were
subsequently exposed to E. coli. Fewer viable bacteria
were recovered from the quarter that received the CD14
treatment than from those that did not receive the plant-
derived protein.

The researchers chose tobacco plants, Nicotiana
benthamiana, to be their CD14-producing factories. They
inoculated the young plants with laboratory-produced RNA
of their recombinant virus by rubbing a small drop of the
RNA onto the plants’ leaves. Once the viral RNA enters the
test plants through small tears in the leaves, it begins to
spread and the CD14 gene begins to make the protein.
The protein can then be extracted from mashed-up leaves.

Researchers say this is the first report of a functionally
active animal receptor protein being produced in a plant.
When a plant is infected with a virus, thousands of copies
of viral RNA are made in each plant cell. When the
recombinant virus reproduces itself in a plant cell, it also
makes the target protein, CD14. This is how the virus turns
a plant into a bio-factory that rapidly generates proteins of

Separating and extracting the therapeutic protein from the
host plant is possible because Nemchinov first tagged the
CD14 protein with the amino acid histidine. The histamine-
tag tracker enables to harvest high levels of the desired
plant-produced protein.

The researchers are able to purify about 1,000 micro
grams of CD14 from 10 grams of leaf tissue taken from
one plant. That means each of these plants provides
enough protein to potentially treat about 10 cows with a
dosage of 100 micro grams each. Fifty plants would yield
purified protein to treat a herd of 500 cows.

One large greenhouse can accommodate enough host
plants for large-scale purification of therapeutic protein.
Another advantage of the greenhouse approach is that the
plants inoculated with laboratory-derived infectious RNA
could be used to inoculate more plants to scale-up protein
production. Researchers have applied for patent
protection on the plant-derived CD14, and they are now
seeking partners to help further develop and test the
protein for safety, effectiveness, and proper dosage.

The CD14-based product may eventually be commercially
developed for use by dairy farmers as a treatment to
prevent cows from becoming infected during their dry
period. Dairy cows are milked for 305 days and then enter
a 60-day dry period, during which they are most
susceptible to coliform infections. The plant-made CD14
could be incorporated into a polymer and infused into the
udder during dry-off. The polymer would allow slow release
throughout the dry period to help fight infections.

The researchers wish to work with a commercial partner to
produce large volumes of plant-made CD14 and then
conduct further tests to determine the most effective
dosages in cows to achieve maximal protection from
infection. The dose that needed may not be as much as
the 100 micro grams per dose that they used in their first
tests. Further studies may show that same result can
achieve the same results with less protein.

By expressing an easily purified therapeutic protein in
plants, the ARS team has developed a novel preventive
approach to treating mastitis that may provide a cost-
effective alternative to antibiotic use.

                                                                                          November 2007
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