Clean Cows

Some cows we call pigs," Jeff Gandy says, walking down the middle of the milking barn between two rows of swishing tails.

cow in grass

"They're good cows," he adds. "They're just messy. They present a bit more of a problem than the other ones."

Gandy, manager of Penn State's mastitis research herd, stops behind a remarkably unsullied specimen with a yellow ear tag, and pats her haunch. "This one, she doesn't like to get dirty," he says. "She never comes in with mud on her."

He moves farther down the row, to a cow that is clearly much less concerned with appearances. "This particular cow," Gandy says, "likes to lie in the mud. That is why she has mastitis."

The accused—call her Bossy—turns her head and stares back mournfully. Her eyes look glassy. Her weight seems to droop on her frame, the bones prominent. Her piebald shanks are streaked with filth.

Gandy pats her softly, then squats down to feel her pink udder. Its right side, uninfected, is spongy and soft as jello. The left side, where the infection centers, is rigid to the touch and noticeably warmer. Gandy produces a shallow plastic cup and draws some milk into it. The liquid is not white but colorless, and filled with stringy, yellowish clumps.

"That's dead bacteria, with some fat, and blood serum," Gandy says. "That's about a high three on the infection scale, from the looks of it. She won't be any good to me for two or three more days. We'll probably lose 400 pounds of milk."

Bovine mastitis—inflammation of the mammary gland due to bacterial infection—is one of the biggest problems dairy farmers face. In terms of annual losses, according to a 1994 estimate, the disease costs $185 per cow in the United States, or $1.8 billion across the nation. "It is," says Lorraine Sordillo, "the number one deterrent to profitable dairy farming."

Sordillo is associate professor of veterinary science at Penn State and director of the Center for Mastitis Research. In addition to being partners against mastitis, she and Gandy happen to be married to each other. Mastitis, they stress, doesn't just come to cows who are sloppy. Bacterial invaders abound in a cow's environment: Streptoccocal organisms in soil and water and straw, Staph and other impurities in milking machines or on milkers' hands. In Bossy's case the culprit is coliform, encountered while lolling in manure-laden muck. (Coliform bacteria are routinely present in bovine, and human, intestines.) In all, however, more than 200 different species and strains of bacteria can cause the disease, says Sordillo.

Bacteria have easiest access to the sterile reaches of the mammary gland during and for about two hours after milking. Then the teat, otherwise closed tight, is relaxed and open, providing a clear pathway for invasion. As a precaution, the teats are routinely dipped in iodine before and after milking, but this doesn't always help.

Increased productivity, Gandy says, has heightened the problem. Today a good milk cow will produce 100 pounds of milk a day, 30,000 pounds over a 10-month lactation period. "These kinds of numbers were unheard of 20 years ago," says the third-generation dairyman. "Then, 60 pounds a day was good. And for my grandfather, 60 pounds was unheard of."

These big increases are the results of careful breeding, using artificial insemination techniques first developed at Penn State. Gandy points to a silver tank in the corner of his office. "That's bull semen," he says. He produces a trade-journal chart that assigns values to some two dozen characteristics, from pounds of milk produced to strength and condition of feet and legs. But "the increased stress of productivity compromises the immune system," Sordillo says. "The more milk produced, the more susceptible a cow is to disease."

Prevention is the best defense. "You try to keep the environment as clean and dry as possible," Gandy says. He beds his cows in sand, not straw. Sand is cooler and more sanitary, "and the cows love it." Even so, he acknowledges, "If you have a dairy farm, you have mastitis."

At any time one in three cows in a herd is likely to have the disease, Sordillo says. That's in a well-managed herd. "The course it takes depends on the bacteria involved. Sometimes a cow can eliminate it quickly. Sometimes it's in a form you can't see—it doesn't show up until you begin to get high white-blood-cell counts. Sometimes the cow gets sick, won't eat, gets dehydrated. There's a whole range in how the disease manifests itself."

Even in its milder forms, mastitis can cut into a cow's milk production. When the mammary tissue is damaged, Sordillo explains, it can't make as much milk. Once those cell counts get high enough, the milk that is produced can't be used anyway. "It's a very clear threshold," Sordillo says. "Every day, every time milk is picked up it is carefully screened for bacteria counts, inflammation cells, antibiotics—at least these three things. If it doesn't meet standards it is dumped."

Treatment for mastitis, once it is discovered, varies with the organism involved. "With coliform," Gandy says, "the best treatment is to just keep the cow stripped of milk, milk her four or five times a day. The bacteria is in the milk, so you get it out of there. Reducing the toxin load allows her body to stabilize and effectively clear out the infection." More commonly, though, the farmer has to give the immune system a fighting chance by administering a course of antibiotics. A cabinet in Gandy's office is cluttered with small tubes and boxes.

Unfortunately, Sordillo says, these drugs are often only marginally effective. "It depends on the bug, and on the time of treatment." Treatment during the "dry" period before a cow gives birth can be pretty effective. Once lactation starts, however, it's another story.

There are other problems, too. Over time, bacteria will develop resistance to a given antibiotic. And, she adds, "Nobody wants milk with antibiotics in it." A treated cow, Gandy says, is "out of the tank" for at least a few days, and no bones about it: If antibiotics are detected in the milk collected from a farmer's herd, that whole day's load is rejected. If it has already been pumped from the barn to the dairy company's truck, the farmer has to buy up whatever milk may be in the truck already—"and those tankers hold six or seven thousand gallons," Gandy says. "Most farmers have insurance in case this happens."

Sordillo, an immunologist whose specialty is the mammary gland, is trying to expand the farmer's repertoire for dealing with mastitis. She and her students in the pathobiology program are looking for new, non-antibiotic approaches to helping cows stay clear of infection.

Acow's mammary gland models the basic immune response: When foreign particles like bacteria invade, patrolling white blood cells called macrophages advance and surround them. "They 'uptake' the bacteria," says Gina Pighetti. "They reach out with these little pods and encircle it, and pull it in." Pighetti, a Ph.D. student in Sordillo's lab, grew up on a dairy farm near Milesburg, PA, where she still lives with her parents. ("I can be an inspiration to everybody," she smiles. "You don't have to be a science nerd to get into science.")

Once it has the enemy surrounded, the macrophage proceeds to digest it, breaking the bacterial particle down and presenting fragments of it to a T-cell. The T-cell, a higher ranking type of white blood cell, takes over the second stage of defense.

First, however, it has to be activated. The macrophage "turns on" the T-cell by secreting a protein called interleukin-1, one of a class of "biological response modifiers" known as cytokines.

mostly white cow in grass

Once activated, the T-cell undertakes the body's specific immune response. From the fragments presented by the macrophage, it identifies the invader. Then the T-cell starts making another cytokine, interleukin-2, which in turn signals its nucleus to start producing copies of itself, i.e., T-cell clones that can recognize, and kill, this particular foreigner. These specially equipped T-cells also release still more cytokines, which stimulate the macrophages to redouble their bacteria-gobbling efforts. Together, these defenders deploy to snuff out the infection.

The system works remarkably well, considering the degree to which it is challenged over the course of a 10-month lactation. "If you think about it," Sordillo says, "the mammary gland is milked two or three times a day. The host defenses are compromised every time. That's quite an infection load." Sometimes, however, the invaders are just too strong, and an infection takes hold.

"Lorraine likes to say that the immune system is like a scale," Gandy says. "You want to keep it so things are balanced. Sometimes the bacteria get the advantage." That's when antibiotics are typically sent in to restore order—holding the infection at bay by suppressing its growth until the body can gather enough strength to battle back.

To right the scales without this outside help, Sordillo suggests, a number of approaches are possible. "Some cows are more resistant to infection than others are," she notes. "So some geneticists are working on breeding programs aimed at enhancing resistance factors in succeeding generations." She and her students, however, are employing the tools of molecular biology, unraveling the hidden mechanisms that determine immune response. "We're looking at why some cows are more resistant, and how we can intervene with the ones that aren't."

They are interested particularly in cytokines, those powerful behind-the-scenes regulators. "Cytokines," Sordillo says, "are really the focus of the lab." Without their galvanizing presence, the immune response is pallid and ineffectual. Neutrophils don't seek and destroy invaders. T-cells don't proliferate. Infections easily grow large enough to tip the scales. When cytokines come on too strong, on the other hand, the body's defenses go too far. A cow with coliform mastitis like Bossy's can fall into deadly septic shock, the result of an uncontrolled inflammatory reaction. "At that point," Sordillo says, "the immune cells are causing more damage to tissue than the bacterial cells are.

"What we're looking for," she continues, is a way to exert some control over cytokines' effects—to enhance them, where it would be useful, and in other cases to suppress them." To do so, they need to better understand exactly how cytokines work.

Doctoral student Kim Shafer-Weaver earned her B.S. in animal biosciences at Penn State, with a minor in environmental resource management. ("My dad is a forester," she explains.) Now she is trying to determine just how cytokines make T-cells into killers. T-cells stimulated with IL-2, she explains, have been shown to be effective in destroying Staphylococcus aureus, an important cause of mammary gland infection. But how exactly does it accomplish the dirty work?

The question is an important one. "Because these cells are already in the mammary gland," Shafer-Weaver says, "they are strategically placed—if we can enhance their effectiveness maybe we can prevent infection instead of treating it." "What we're looking for," adds Sordillo, "is a naturally produced antibiotic factor."

To trace such a factor, Shafer-Weaver first had to isolate the different types of T-cells. (There are several types present in the mammary gland, each with a specific role in immune function.) Once she had a pure population of these cells, she stimulated them with IL-2 and watched how the cytokine affected the way they killed bacteria.

She found that IL-2 enhanced killing ability, but only with certain types of T-cells. Others were unaffected. "What this suggests," Shafer-Weaver says, is that some of these T-cells are so-called natural killers, killing by the release of some soluble antibacterial factor. "We're working on identifying that factor right now."

Pighetti, meanwhile, is scrutinizing another important factor in the immune response: diet. In particular, she is studying the role of the micro-nutrients selenium and vitamin E, both known for their beneficial properties as antioxidants.

Cells, like the organisms they constitute, require a certain level of oxygen in order to survive. Too much, however, is toxic. Higher than necessary levels of oxygen radicals (unstable, highly reactive groupings of oxygen atoms) can cause a breakdown of the cell membrane, interfere with protein functioning, and damage a cell's DNA, leading to increased risk of heart disease, cancer, and opportunistic infection.

Vitamin E and selenium, recent studies show, work together to keep oxygen levels healthy. "Animals with diets deficient in either of these micronutrients have lowered immune response," Pighetti notes. "An animal deficient in both," she adds, "will eventually die. It has an ineffective immune response."

The absence of these vital ingredients, it is known, interferes with the proliferation of T-cells. "But," she says, "we don't know why."

Does the absence of micronutrients somehow impede the expression of cytokines like IL-2? To find out, Pighetti conducted a series of trials with rats fed antioxidant-deficient diets. The ability of T-cells to replicate themselves was half of normal in the spleens of these rats, she recalls. On close inspection, though, the expression of IL-2 in these cells appeared unaffected. Nor was there any problem with the IL-2 receptors on the cell surface, which actually signal the cell to copy itself.

Pighetti remembers beginning to be discouraged about the course of her experiment. No explanation was presenting itself. Then, using radioactive iodine tracers, she noticed that the antioxidant-starved T-cells had trouble internalizing iron, a mineral needed to synthesize DNA and protein. This could well be the reason for low T-cell proliferation, she concludes. "Without iron, cells can't breathe—they can't produce the energy needed to function."

Chunlei Su, another of Sordillo's Ph.D. students, began his study of animal disease in his native China, and continued for his master's degree at Brigham Young University in Utah. There his adviser was Beverly Roeder, a graduate of Penn State. She suggested that he take his Ph.D. with Sordillo. Su is taking an approach to mastitis that nicely complements the work of his lab mates: He's getting a better bead on the enemy by studying Staph aureus, the bacterial culprit in 20 to 40 percent of all mastitis cases.

"Staph aureus is a special problem because it is contagious," he explains. "It is transferred during milking—on the milker's hand or through the milking machine. It is difficult to eliminate." S. aureus, Su adds, typically does not develop into full-blown mastitis. Instead, it causes lingering sub-clinical infection, increasing somatic cell counts in a cow's milk and decreasing the amount of milk produced by up to 30 percent.

close-up of white and brown cow

Antibiotics have proved ineffective against chronic S. aureus mastitis. Frequently the only way to stop its spread through a herd is to get rid of the infected cows. As a result, over the last 30 years researchers have focused on trying to develop a vaccine against S. aureus. "A lot of research has been done," Su says, "but so far the results have not been very promising." Up to now, he contends, attempts to develop an effective vaccine have been hampered by the limited number of S. aureus strains that have been looked at.

"Getting a collection of field strains is a lot of work," Sordillo explains, "and most molecular biologists are not clinically oriented. Our approach is to combine field work and molecular biology techniques, so we've developed the relationships with diagnostics labs, which gives us easier access to many field isolates of the bacteria." Already, Su has gathered some 400 strains, from collaborators around Pennsylvania and the United States, as well as from South Korea, Israel, France, the Czech Republic, and Denmark. ("The individual farms are kept confidential," Sordillo stresses.) Using DNA fingerprinting techniques to identify their similarities, he has grouped these strains into genotypes, or families. Now, within these families, Su is screening individual strains by exposing them to neutrophils in culture and noting which bacteria are most resistant to being killed.

Su has found that the strains that cause mastitis are limited to a handful of S. aureus families. By further screening in the implicated genotypes, he now hopes to identify the specific genes or factors that make these strains virulent. Once these factors are identified, the theory goes, they can be used to develop a vaccine that works.

"There are many virulence factors involved in the disease process," Su acknowledges. "It is unlikely that any one factor can make an effective vaccine. But a combination of several factors is probably the best way to control the disease."

Last summer, Shafer-Weaver took a break from work in the lab and traveled with Sordillo to New Hampshire, where she displayed her findings on cytokine function at a Gordon conference on mammary gland biology. (The Gordon conferences are bi-annual international meetings on cutting-edge research in selected fields.) "Mine was the only immunology poster there," she remembers. She was one of only four graduate students chosen by the judges to present their research to the assembly.

The attention was gratifying. "A lot of people when they see a poster with a cow on it will pass right by," Shafer-Weaver says. "When they see you're in Ag they think you're not doing high-profile, top-notch research. But immunity is immunity, and a T-cell is a T-cell. If youÆre interested in the immunology of the mammary gland, the bovine model is an excellent model for studying the human system too."

Their results do indeed, Sordillo says, have implications beyond the barn. Mastitis afflicts humans too, she notes. "One of three women who breastfeed get it. It's a major reason why they stop breast-feeding prematurely. Few studies have been done in the area of human lactation and mastitis, but those that have been done suggest that this is a significant problem."

The knowledge gained by studying the mastitis model, she adds, can be translated to other diseases involving inflammation. Dave DeRosa, who recently finished a masterÆs degree with Sordillo, was recruited by several major drug companies to work in the area of arthritis. "They're looking at ways to modify inflammation and immunity," Sordillo says. Others of her students have gone on to medical school, and one, recently, to work in a molecular immunology lab.

Sordillo herself is now in the early stages of a collaboration with enzymologist Channa Reddy, Penn State distinguished professor of veterinary science. Backed by a USDA human nutrition grant, the two are investigating the causes of atherosclerosis—the hardening of the arteries associated with heart disease—by looking at bovine aortic cells. "We're examining the effects of micronutrients like selenium and vitamin E on endothelial cell metabolism and vascular integrity," Sordillo reports. "I'm interested in the immunology, and Dr. Reddy is interested in the biochemistry. What we are doing, really, is studying the fundamentals of inflammation.

"This is what our program is all about," she adds. "We go from the whole animal down to the molecular level, and we get a broad picture of the mechanisms involved in the progression of disease."

Gina M. Pighetti, Kimberly A. Shafer-Weaver, and Chunlei Su are Ph.D. students in veterinary science in the College of Agricultural Sciences, 108 Henning Building, University Park, PA 16802; 814-865-0211. Lorraine M. Sordillo, Ph.D., is associate professor of veterinary science and director of the Center for Mastitis Research, 101 Henning, 863-2165; lms10@psu.edu.

Sordillo's laboratory is funded by grants from NIH, USDA, the Pennsylvania Department of Agriculture, and several pharmaceutical companies.

For more information on the pathobiology program, visit the department of veterinary science at http://www.cas.psu.edu/docs/CASDEPT/VET/vet.html. For more about mastitis, go to the National Mastitis Council at http://www.nmconline.org/.

Last Updated January 01, 1998