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Technology tattles on mastitis.

Cells taken from the lining of dairy cow mammary glands are shedding new light on how milk is produced and how bacteria infect the cells and ultimately cause mastitis.

"Mastitis is a bacterial infection of the mammary glands of dairy cattle," says physiologist Albert J. Guidry, who is with the ARS Milk Secretion and Mastitis Laboratory in Beltsville, Maryland.

Mastitis costs U.S. farmers over $2 billion annually for treatment and lost milk production.

Preliminary studies have shown that cultures of mammary cells grown in single cell layers, or monolayers, can be used to show the effect of bacterial toxins on mammary cells. These toxins are substances manufactured by bacteria that can injure or kill healthy cells.

Scientists now have a method to compare and measure how bacterial toxins affect mammary cells.

They grow monolayers of cells in 96-well culture plates treated with a compound that makes healthy cells fluoresce. Fluorescence decreases when the cells are exposed to membrane-damaging bacterial toxin. The scientists then use a fluorometer to measure the decrease, for comparison with the fluorescence of mono-layers receiving no toxin.

The next step is to run similar tests using live, toxin-producing bacteria and compare the results," Guidry says. "Electron micrograph studies of the infected cells should reveal how the bacteria infect the cells and could lead to methods to control them.

"Previously, we could only speculate on the events taking place inside the infected mammary gland. It was a 'black box' that only grudgingly yielded its secrets," Guidry says.

"We wanted a system in which we could introduce infectious bacteria and directly observe cell and bacterial interaction. So now we have the inside layer of mammary secretory cells growing on a flat surface that we can observe under the microscope and measure various cell functions."

The scientists obtain the mammary epithelial cells at slaughter by enzyme digestion. They grow the cell suspension in a culture medium and freeze it for later use.

"Experimenting with these cells will reduce the number of live animals required for laboratory tests," says veterinarian Eduardo Cifrian, who is on leave from the National Institute for Research in Agriculture of Spain.

"Our tests have shown that these are normal mammary secretory/ excretory cells. They produce the milk proteins alpha and beta casein, alpha-lactalbumin, and lactoferrin. The cells also produce cytokeratin, which is unique to epithelial cells," Cifrian says.

This cell culture technique will enable scientists to determine how mammary cells produce milk. Studies are also under way to determine how growth hormone affects mammary cells.

Other scientists at Beltsville are exploring the possibility of using the cultures to test genetically engineered gene constructs before introducing them into embryos.

Recent studies using pigs and sheep have shown that it is possible to insert into farm animals genes that cause them to produce lifesaving biologicals--such as blood clotting factors for hemophiliacs--in their milk.

"Testing genetically engineered constructs in cultured cells derived from mammary glands would save both animals and time," says ARS geneticist Robert J. Wall.

An unusual feature of these cultured cells is the formation of mini-mammary glands. If the cells are maintained in culture for 10 to 12 days on certain types of culture media, small ducts or tubules grow and secrete protein as they would in the live animal.

Growing milk in culture bottles is not on the horizon. However, "understanding how the milk-producing cells respond to various nutrients and hormones and to bacterial infection may guide us to more efficient milk production and a practical, environmentally sound means to prevent mastitis," says Guidry.

Albert J. Guidry is at the USDA-ARS Milk Secretion and Mastitis Laboratory, Bldg. 173, BARC-East, 10300 Baltimore Ave., Beltsville, MD 20705-2350; phone (301) 5048285, fax (301) 504-9498.
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Author:Mazzola, Vince
Publication:Agricultural Research
Date:Sep 1, 1994
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