Researchers Aim To Find Genetic Marker For Anasarca

When her bitch, CH Rocket City's Angel Among Us, gained 10 pounds in the last couple of weeks of pregnancy, Pat Rzonca of New Caney, Texas, didn't think too much about it. Rzonca, who has bred Bulldogs under the Rocket City prefix since 1998 and is an AKC judge, knows that a bitch's weight gain in the later stages of gestation relates partly to the number of puppies she carries.

Born by cesarean section, one puppy in the litter of four was a "water puppy." Swollen almost two times larger than the size of its litter mates, this puppy's body was filled with fluid that accumulated between the cells throughout its body during gestation. The condition, known as anasarca, is not new to Bulldog breeders.

Experts believe genetic and environmental conditions contribute to the development of anasarca. Though the disorder ranges in severity, the majority of puppies are stillborn or die shortly after birth. Epidemiological data on the prevalence of anasarca among dog breeds is not available, but brachycephalic breeds, such as Bulldogs, are considered susceptible.

Two years after the anasarca puppy was born, Rzonca bred another bitch from a different pedigree. An ultrasound taken two weeks before the puppies were due indicated the bitch was carrying a healthy litter. When the puppies were born by cesarean section, Rzonca was alarmed that four of the five puppies had anasarca. Despite their receiving immediate veterinary care, none of the anasarca puppies survived.

"Two were huge anasarca puppies and two had extra fluid," Rzonca says. "It was devastating to know that I had done everything to prevent anasarca, yet I still produced it. These puppies' organs were engorged with fluid. After they were born, they went into organ failure."

One of the concerning aspects of anasarca is that bitches must have cesarean sections due to the size of the puppies, says Dan Bandy, the Bulldog Club of American health liaison. "A bitch cannot naturally whelp a 2-pound water puppy," he says. "We hope that the genetic basis for this disorder is found and that one day we can get back to more natural whelpings of litters."

Efforts to discover a genetic marker for anasarca in Bulldogs are under way at the University of Illinois. The research, which is funded by the Bulldog Club of America Charitable Fund, is led by Jonathan Beever, Ph.D., professor of molecular genetics in the Department of Animal Sciences. Beever discovered the gene that causes anasarca in some cattle breeds in 2006. He initially thought that the cattle gene would provide insights about anasarca in Bulldogs, but this did not prove true.

"Even though the puppies bear significant similarities to cattle with anasarca, there are multiple genetic or environmental mechanisms that may lead to the same appearance," Beever explains.

Anasarca also occurs in humans. The condition, called fetal hydrops, is similar to canine anasarca. Most human fetal hydrops cases are immune-mediated due to a genetic incompatibility between the Rh factor in the baby's and mother's blood. Rh disease has declined since the 1970s, when an anti-D IgG (Rho(D) immune globulin) injection was introduced to give RhD-negative mothers during pregnancy or within 72 hours of delivery. The antibodies in the injection remove any maternal antibodies that attach red blood cells. Secondary causes of non-immune fetal hydrops include blood diseases, metabolic disorders, cardiovascular abnormalities and parvovirus.

Once Known as 'Walrus Puppies' 
A second-generation Bulldog breeder, Elizabeth Hugo-Milam of Newcastle, Okla., saw her first anasarca puppy nearly 40 years ago. "My parents bred Bulldogs in the 1960s, and I remember one being born in a litter when I was about 5 years old," she says. "Back then, they were called 'walrus puppies,' and everyone knew about them."

Today, Hugo-Milam, who breeds under the Hug-O-Bull prefix, sees an occasional anasarca puppy in her own breedings. "If you breed long enough, you're going to see it," she says. "I think the disease prevalence in Bulldogs is the same as it was 40 years ago."

Unfortunately, pregnant bitches exhibit few, if any, signs of carrying anasarca puppies, which causes breeders and veterinarians to be caught off guard. Bandy, a veterinary technician at Town and Country Veterinary Clinic in Shawnee, Okla., manages the clinic's reproduction service, Show Dogs on Ice. "Some bitches carrying anasarca puppies have an onion skin-type sheen around their teats in late pregnancy," he says. "Not all bitches exhibit this sheen, and there is no scientific data that supports the significance of this observation."

Bandy's own experiences breeding Bulldogs have resulted in anasarca puppies. "One of my first litters produced three normal puppies and one severely affected anasarca puppy that was stillborn," he says. "A few years later, I bred a female from the first litter that produced six normal puppies and one affected puppy, which died within 48 hours."

Recognizing anasarca as a health concern in the breed, the Bulldog Club of America turned to a Bulldog enthusiast, Michael Hughes, Ph.D., a research pathologist in the Department of Pathology at the University of Southern California, to help coordinate the genetic research. "Mistakenly, in the past, anasarca has been described as fluid under the skin," Hughes says. "This condition is caused by a buildup of interstitial fluid, or fluid between cells, that causes the tissue to become waterlogged. While anasarca is not common, it's also not rare."

Experts believe that anasarca may occur due to genetics and environmental factors. A once popular theory attributed fluid retention in bitches to high-sodium diets that cause edema, or swelling, in puppies. This proved untrue. Yet another theory was that canine parvovirus triggers anasarca.

"Parvovirus is a cause of human anasarca, and while there are no studies that rule out parvovirus exposure as a cause of non-immune anasarca in dogs, I would be surprised if it is not one of the non-immune causes," Hughes says.

Once a genetic marker for anasarca is identified, researchers may then turn to an environmental study, Hughes says. "In humans with anasarca, researchers found that a small percentage is due to genetics, while most are caused by environmental factors," he says. "This may be the case in Bulldogs as well."

In Beever's laboratory, genetic profiling of DNA from 15 litters of healthy and anasarca-affected puppies involves SNP (single nucleotide polymorphism) chip analysis. Comparing the DNA of healthy and affected dogs, the researchers look for regions of difference in hopes of finding a genetic marker.

"SNP markers are like mile markers on a highway," Beever explains. "Of the 15 litters being studied, 13 puppies are affected, about one puppy per litter. The ratio of three normal puppies to one affected puppy supports a recessive mode of inheritance."

More DNA from affected puppies, as well as their sires and dams, would help advance the research, Beever says. "The condition is proving to be more complex in Bulldogs than we originally thought," he says. "In some litters, 90 to 100 percent of the puppies are affected by anasarca. We can't help but think that multiple genes may be involved, with environmental influences playing a role, too."

Locating the genetic marker for anasarca in Bulldogs may not be far off, Beever predicts. "If we receive the DNA samples we need, we may locate the marker relatively quickly. Once we locate the region, finding the gene should be much easier."

When the causative gene mutation is identified, a genetic test can be developed to identify carriers. This will allow breeders to make selective breeding choices that will reduce the likelihood of producing anasarca puppies.

Late-Stage Gestation Disease

In his genetic research of anasarca in cattle, Beever found that the condition develops around the fifth month of gestation, or about halfway through the pregnancy. The fluid retention in cattle starts in the head and moves through the body eventually reaching the tail. "I do not know if this is the case with Bulldogs, but the canine condition does appear to develop in the later stages of gestation," he says.

Severely affected puppies that are stillborn often have other abnormalities, such as cleft palates. Those that are born alive die shortly after birth because they are not able to excrete the excess fluid in their bodies. During gestation, the puppy survives because it is tethered to the dam by the umbilical cord. Once that cord is severed, anasarca affected puppies are unable to excrete the enormous amount of excess fluid and subsequently drown in their own fluid.

Puppies with mild edema can survive and should live normal lives if they receive immediate veterinary care, which may include administering a diuretic to reduce fluid retention and potassium. Efforts include elevating the puppy's temperature to 100 degrees Fahrenheit and gently massaging to stimulate circulation.

Severely affected puppies are completely swollen with fluid and may weigh up to five times more than normal. The average weight of a healthy Bulldog puppy is between 14 and 16.9 ounces compared to up to 33.8 ounces, or more than 2 pounds, for an anasarca puppy.

"The extensive edema and size of the puppies prevent a natural birth," Hughes says. "These litters must be born by cesarean section. As a precautionary measure, an ultrasound should be taken two weeks before a bitch is due. If there is a problem, it should be apparent."

Breeders who produce litters with anasarca-affected puppies are left wondering what they could have done differently. "I have bred 25 homebred champions, but I just wonder how many more I would have if so many puppies had not been taken by anasarca," Rzonca says.

Purina appreciates the support of the Bulldog Club of America and particularly Dan Bandy, chairman of the BCA Health Committee, in helping to identify topics for the Purina Pro Club Bulldog Update newsletter.

How to Participate in Anasarca Research

Bulldog breeders and owners may participate in genetic research to identify a marker for anasarca, a condition that causes swelling or massive
edema in puppies due to fluid accumulation between cells during gestation.
The researchers request DNA samples from affected puppies and from sires
and dams that have produced affected puppies.

The research is being conducted at the University of Illinois. For information and instructions on submitting DNA samples, please contact Dr. Michael
Hughes, coordinator of anasarca genetic research for the Bulldog Club of
America, at mwhughes@usc.edu.