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Better beefsteak begins with healthy cattle.

On the surface, it seems nothing could be simpler: Take a cow, add plenty of grass and water, and presto! Abundant protein-laden beef is the reward.

But that scenario doesn't reckon with the unseen hordes of bacteria and viruses that can thwart the best laid plans of an unwary cattle producer.

One of the most prevalent of these microscopic troublemakers is a bacterium called Brucella abortus, the culprit behind bovine brucellosis, a serious problem in American cattle herds since at least the 1840's.

Sometimes called Bang's disease in honor of the Danish veterinarian who first isolated the disease organism, brucellosis causes cows to abort, interferes with fertility, weakens calves, and lowers milk yields.

Each year, the disease costs U.S. cattle producers an estimated $30 million, including animal losses and expenses for testing and vaccination.

Infected herds must be placed under quarantine. Additionally, regulations against interstate shipment within the United States and trade barriers imposed by foreign countries require U.S. cattle producers to certify that export cattle and embryos are brucellosis-free.

When a cow gives birth or aborts a calf, billions of B. abortus bacteria are shed in her body fluids or in the afterbirth. Other cows can become infected when they lick the afterbirth; newborn calves can be infected as fetuses or by drinking milk from an infected mother.

The disease typically gains entry to a healthy herd through the introduction of an infected heifer or calf. Animals can also become infected while mingling with other stock at fairs and livestock shows.

Precautions can be taken to impede invasion by this stealthy disease. Producers adding breeding stock to their herd must ascertain that the animals they buy are certified brucellosis-free. Calves are typically vaccinated between the ages of 5 and 9 months, a primary means of control.

But while commercial vaccines do protect cattle against brucellosis, there are significant problems with the currently used vaccine known as strain 19.

This vaccine uses a live strain of the bacterium, so the vaccine itself can cause abortion if given to a pregnant cow. The live bacterium also poses the threat of infection for humans handling the vaccine.

In addition, some cattle producers are reluctant to use the vaccine because of inability to distinguish in testing between animals that have been vaccinated and those that have been naturally infected.

"All of these factors together have dramatically slowed down eradication," says Norman F. Cheville, leader for brucellosis research at the National Animal Disease Center at Ames, Iowa.

"Vaccinated animals that react positively to blood tests for the disease may be classified as possibly diseased and consequently destroyed."

Fortunately, ARS researchers are gaining ground on a practical means of sorting out infected animals from vaccinated ones.

The scientists face two challenges. First, they must develop an effective vaccine with some sort of harmless mutation to help distinguish it from natural infection. Second, they must develop a diagnostic test capable of discerning that difference.

Addressing the first task, ARS molecular biologists Shirley M. Halling and Fred M. Tatum have genetically modified B. abortus vaccine strain 19 in hopes of deleting a specific protein immunogen. The protein immunogen causes the cow's immune system to produce the antibodies that typically indicate in testing that the cow is infected.

Currently, the protein immunogen is present in both the vaccine and in the disease-causing organism. If a vaccine lacking that protein is developed and used, Halling says, tests for the protein in an animal's blood could reveal whether the animal has received the altered vaccine or has been infected with a disease-producing strain of B. abortus.

The researchers are determining the effectiveness of three mutant vaccines on 24 cows and 24 calves. Two of the mutant vaccines used in the trials were genetically engineered by Halling and Tatum; the third is a natural mutant strain developed by scientist at Virginia Polytechnic Institute and State University.

"We're looking for the one that provides the best immunity and that can be easily identified by a blood test," says Cheville.

"Then we'll know positively if an animal has been vaccinated with the mutant vaccine or has been infected naturally by a virulent field strain. Such an accomplishment would be a major breakthrough toward the eradication of brucellosis."

In a related project directed by Halling, microbiologist Betsy J. Bricker has tackled the job of improving the diagnosis. She's discovered a small piece of genetic material - DNA - that's found in all Brucella, but not in other bacteria.

Bricker uses a technique called polymerase chain reaction (PCR) to track down the brucellosis organism. This technique makes many copies of targeted genetic material. When these copies are exposed to ultraviolet light, DNA of a particular size glows, unmasking any B. abortus lurking there.

Current bacteriological tests may take as long as 2 weeks to identify the organism. "But with PCR, the organism can be identified in 1 day," she says.

The use of PCR in B. abortus diagnosis also eliminates the need for using live bacterial cells. Diagnosticians must use particular caution now in performing bacteriological tests with live cells because humans can contract brucellosis. Symptoms include extremely high fever and chills, fatigue, and an aching similar to that of arthritis.

In trials on 30 randomly selected field isolates of B. abortus, the PCR method correctly identified all 30 samples.

Bricker plans to design a test to identify other species of brucella such as B. melitensis, which infects goats, and B. suis, which infects pigs. She also hopes to expand the technique to identify B. abortus vaccine strain 19 or one of the new mutant vaccines.

Just as pregnancy poses obstacles to brucellosis vaccination, it can also interfere with herd vaccinations against infectious bovine rhinotracheitis (IBR), a severe respiratory disease of cattle.

Caused by bovine herpesvirus 1 (BHV 1), IBR can be blocked by live-virus vaccines. But most of these vaccines cannot be given to pregnant cattle because of the danger of abortion. And in some herd management systems, cattle producers may not know when their cows are pregnant or have just been bred.

At the National Animal Disease Center, veterinarian Janice M. Miller and microbiologist Cecelia A. Whetstone are testing a genetically altered version of BHV 1 that might be used to develop a safer vaccine against IBR.

Cooperating researchers at the University of Pennsylvania altered the virus by removing a piece of its genetic material that causes production of the enzyme thymidine kinase (TK). The ARS researchers then gave the altered virus to six pregnant cows and none of the cows aborted.

In contrast, give our of six pregnant cows given the normal TK-positive virus aborted. The researchers will now study at the TK-negative virus for its effectiveness as protection against IBR.

In the meantime, Miller recommends vaccinating young heifers against IBR at 4 to 10 months of age, before they reach breeding age.

At the Animal Diseases Research Unit at Pullman, Washington, microbiologist David T. Shen and colleagues are also addressing the question of bovine herpesviruses.

In addition to studying BHV 1, the Pullman researchers are tackling bovine herpesvirus 4, which has been implicated as a cause of infertility and abortion in cattle.

Shen and co-workers have produced specially designed probes that could be used to diagnose these two viruses. One type of probe, called a monoclonal antibody, can detect the presence of antibodies made by the cow's body in response to the virus.

The other, a nucleic acid probe made from a fragment of the virus' genetic material, will detect the virus itself.

"Because these probes are very sensitive, we might be able to track down the virus in a variety of samples from cattle, such as blood, tears, or semen," says Shen. Improved diagnosis could help researches prevent further spread of the potentially costly diseases, he adds.

Back at the National Animal Disease Center at Ames, close attention is also being paid to mastitis, an important and costly disease in dairy cattle and sheep as well as beef cattle. To complicate matters, the incidence of this bacterial disease is increased by stresses such as calving.

Veterinary medical officer Marcus E. Kehrli, Jr., and physiologist Judith R. Stabel have found in studies at Ames that cows are most susceptible to mastitis just before and after calving.

They hypothesized that a natural protein called G-CSF, made by various cells, could lend a helping hand to the cow's immune system in times of stress. G-CSF stimulates bone marrow to produce white blood cells that help fight off infections.

In tests, Kehrli gave daily injections of G-CSF to 10 dairy cows. The researchers noted that after these injections, the cow's number of white blood cells, called neutrophils, soared to 15 times more than normal.

"If we can increase the number and ability of white blood cells to protect against infections, we may reduce the need to use antibiotics," says Kehrli. "This means that costs to producers would be reduced."

Also figuring into the link between disease and stress is the breed of cattle involved, according to physiologist Michael T. Zavy.

Stress sets off a chain reaction in an animal. The hypothalamus in the animal's brain secretes a chemical called CRF that causes the animal's pituitary to release a hormone known as ACTH. This hormone in turn acts on the animal's adrenal gland, causing the production of substances called glucocorticoids.

Pinpointing the level of glucocorticoids is important because it's believed a chronic overabundance of glucocorticoids can interfere with the animal's natural immune response, leaving it wide open to a variety of infections.

In tests at the ARS Forage and Livestock Research Laboratory at El Reno, Oklahoma, Zavy studied differences in stress reaction between Bos taurus cattle, typically the British breeds, and the Bos indicus breeds, typified by Brahman cattle.

Since many herds in the southern United States include a combination of Brahman and British blood, Zavy's study included 10 calves from Brahman fathers and Angus mothers, 12 from Brahman fathers and Hereford mothers, and 22 from Angus fathers and Hereford mothers.

Over a 45-day period, the calves were separated from their mothers, weaned, and hauled on cattle trucks for 21 hours. Throughout the test period, 11 blood samples were taken from each calf to check levels of a specific glucocorticoid, cortisol. In addition, each animal received an equal dose of ACTH during initial handling, weaning, transportation, and recovery to stimulate cortisol release.

"By doing all this, we could compare the amount of cortisol secreted at different times," says Zavy. "If the animals all reacted to the stress in the same way, they should theoretically have equal amounts of cortisol."

Overall, the tests indicated weaning and transport were most stressful for both Bos taurus and Bos indicus cattle. But they also uncovered some differences in how the two species handled stress.

"On a day-to-day basis, Bos indicus animals had up to 50 percent higher levels of cortisol, as well as higher levels of norepinephrine and epinephrine, than Bos taurus," Zavy says.

"But when we gave the ACTH, the Bos taurus animals mobilized more cortisol than the Bos indicus. It wouldn't be unreasonable to say this difference could affect disease-resistance capabilities among the different breeds. But this work is just a starting point; there's still a long way to go."

While transport is admittedly stressful, stopovers at the sale barn can also take a toll, says Andy Cole, an animal scientist and research leader at the ARS Bovine Respiratory Disease Research Unit at Bushland, Texas.

"The typical feeder calf in the United States is weaned at 6 to 7 months of age and goes to the auction barn, where it's purchased by a buyer who's putting together an order for a feeder or stocker operation," explains Cole.

"On average, it takes 4 days for an order to be assembled. That's 4 days when calves from as many as 60 different farms are mingled together and stressed from being separated from their mothers."

The result: A bacterium called Pasteurella haemolytica, commonly found in the calf's nasal cavity, may take advantage of the animal's stressed condition and invade its lungs, causing pneumonia. In addition, other bacteria and viruses, probably passed on from the calf's new penmates, attack and infect.

Fortunately for farmers, 14 years of research at Bushland have uncovered secrets for easing the calf's journey down the marketing path.

"The typical order buyer will feed the calves low-quality hay at the sale barn," Cole says. "But our data indicates that if you give them a nutritionally balanced concentrate plus good quality hay, the number of animals that get sick will be reduced up to 20 percent, and death rates are cut an average of 30 percent."

Cole says a typical feed concentrate might include corn as its grain, along with a protein supplement and cottonseed hulls or ground alfalfa, for an overall protein content of about 14 percent. Using a 50-percent-grain feed concentrate of this type costs about 30 cents per calf, he adds.

One common method of preconditioning the calf for the rigors to come has been weaning it about 30 days before sale and switching it to feed concentrate at the farm.

"But our data indicate that's not economical," says Cole. "However, if you give them limited amounts of feed concentrate in addition to the milk from their mothers the last 60 days on the farm, they get more nutrition in their rumen, they become familiar with the concentrate, and they're stressed less when they're taken from their mothers."

One of the chief culprits in calves' stress-related ills is bovine viral diarrhea, or BVD. This virus is receiving close scrutiny from microbiologist Hwei-Sing Kwang and veterinary medical officer E. Travis Littledike at the Roman L. Hruska U.S. Meat Animal Research Center (MARC) at Clay Center, Nebraska.

"The mortality rate on BVD is usually low, but it sets the cattle up for many other diseases," says Littledike. "Cattle producers vaccinate their herds against this disease, but there's a big controversy over which vaccines to use. We found evidence that the strain of BVD we have here at MARC wasn't controlled by the vaccine we'd been using for the last 8 years."

Complicating the picture are carrier animals who do not respond to the vaccines.

"When a calf is exposed to the virus while still in its mother's uterus between the 45th and 125th day of pregnancy, the calf's immune system isn't programmed yet," Littledike explains. "So when the immune system is finally programmed, it assumes that virus is a normal part of the animal's body and does not make antibodies against the virus."

The carrier animal can spread the virus to its herd mates through coughing, saliva, feces, or any other body secretion, notes Kwang.

"If we can pull the carriers out of the herd before breeding season begins, that should prevent further BVD problems in the herd, unless more BVD-infected animals are brought into the herd," Littledike adds.

"But because a carrier's immune system doesn't usually produce antibodies against the virus, it's hard to identify them with tests that check for antibodies. The virus has to be isolated and identified in the blood to detect carriers."

In addition to costing as much as $25 per test, current methods to detect infection in an animal can take up to 2 weeks for results. In contrast, Kwang has devised a system that offers an answer in 1 to 2 days.

Kwang's system, which involves producing recombinant proteins in bacteria from a piece of DNA of a BVD strain, might also help clarify whether the antibodies in a calf's blood are the result of an earlier vaccination or actual infection.

One telltale sign is the presence of specific proteins produced by the viral RNA. Animals naturally tend to produce antibodies against three such proteins - p80, gp53, and gp48 - in greater abundance than against other proteins from the virus.

Kwang and Littledike have found no evidence of p80 in blood samples from animals that have been vaccinated with a killed strain of BVD. However, p80 was detected in animals that had been naturally infected or had received a modified like BVD vaccine. All three methods of exposure resulted in antibodies to gp53 and gp48.

Down the road, the scientists hope to speed the diagnostic process to a few hours' time. Meanwhile, they say they may be able to use the viral proteins to pinpoint the elusive carriers in large herds.

"We could inject a rabbit with a recombinant protein like gp53 or gp48 and use the rabbit antibodies against that protein to test the blood sample from an infected carrier," says Kwang. "If the rabbit antibodies reacted to the gp53 or gp48 protein in the blood, it would mean the virus is present in the suspected carrier's blood."

Internal problems of a more tangible type - parasites - are engrossing microbiologist Louis C. Gasbarre and animal scientist Andrew C. Hammond. Hammond is research leader at the ARS Subtropical Agricultural Research Station at Brooksville, Florida, while Gasbarre works at ARS' Helminthic Diseases Laboratory at Beltsville, Maryland.

The two researchers are cooperating on studies of how possible genetic differences among cattle affect their natural ability to resist parasite infection.

One of their chief research tools is a unique herd of Angus cattle known as the Wye Herd and owned by the University of Maryland. Once held by a private owner on Maryland's Eastern Shore, this closed herd has been so carefully developed that its genetic makeup is very well defined.

From 1986 to 1988, the researchers monitored every calf born in the Wye herd for parasite infection. Although most calves' natural defenses against internal parasites began working by a certain age, some animals continued to have unusually high numbers of parasites.

"We estimate 15 percent of the calf population is responsible for 80 percent of parasite transmission," says Gasbarre. "About 50 percent of the calves we studied had under 100 parasite eggs per gram of feces, and about 80 percent had under 200 eggs per gram. But a few had well over 1,000 eggs per gram."

By studying the calves' "family tree," the researchers have found strong indications that the genetics of the sire heavily influence the calf's chances of increased parasite infection.

"This trait is about one-third genetically controlled," Gasbarre says. One immediate suspect in the transmission of this trait is a specific complex of genes responsible for an animal's natural internal defenses against threats ranging from tumors to allergies.

The researchers have classified the Wye Angus by their combinations of these crucial genes, known as BoLA for bovine lymphocyte antigens.

Selective breedings done with the Angus at Brooksville and at the Wye Research and Education Center at the University of Maryland have demonstrated little correlation between the sire's BoLA and parasite infection levels of its offspring. However, those experiments have verified that the sire strongly influences parasite levels.

"We're absolutely convinced that there's a strong genetic effect involved in parasite resistance, but it's not strictly from the BoLA genes." Gasbarre says. "It's other genes from the sire."

Ultimately, the scientists hope to discover differences at the cellular level that control whether a beef animal is more likely to have heavy parasite infection.

"Once we known that difference, we can look for some sort of marker," says Gasbarre. "The marker may not be actually responsible for the trait, but it would be something you could check for in a calf. Then you might treat those animals more intensively, or even remove them from the herd." - By Sandy Miller Hays, Linda Cooke, and Julie Corliss, ARS.

For Addresses and telephone numbers of ARS scientists mentioned in this article, please contact the Editor, Bldg. 005, BARC-West, 10300 Baltimore Ave., Beltsville, MD 20705. Phone (301) 344-3280.
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Title Annotation:research into cattle diseases
Author:Hays, Sandy Miller; Cooke, Linda; Corliss, Julie
Publication:Agricultural Research
Date:Dec 1, 1991
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