Taking a Team Approach
When a mysterious condition began occurring in Pugs in the early 1970s, breeders and owners were taken aback. Apparently healthy, young adult dogs typically died not long after the onset of neurological signs, such as unsteadiness or seizures.
The condition resembled other canine diseases that cause seizures. Pug lovers consulted their veterinarians, trying to learn whether their dogs suffered from distemper, rabies, toxoplasmosis or even Rocky Mountain spotted fever. Dogs were euthanized on average 21 days after the onset due to uncontrollable seizures or a coma. It was heartbreaking for owners, particularly since little could be done to help affected Pugs.
Eventually the mysterious condition became known as Pug Dog encephalitis (PDE), which describes an aggressive and fatal inflammatory disease of the central nervous system. Technically known as necrotizing meningoencephalitis (NME), the condition also occurs in other toy breed dogs and is depicted by necrotizing, or dying, brain cells. Severe and progressive neurological signs — circling, head pressing, blindness and neck pain — are common. Affected dogs become lethargic and depressed. Though not all signs are seen in every dog, when several occur together in a young adult Pug, particularly seizures, loss of coordination and lethargy, PDE is suspected. The median age when signs appear is 18 months, but some dogs are diagnosed as young as 6 weeks of age and others as old as 9 years.
Ten years after it first appeared, PDE had been diagnosed in Pugs throughout the U.S., Australia and Europe. Despite the increasing commonality of the condition, breeders and owners had difficulty acknowledging that the disease was a problem in their beloved dogs.
"At first many Pug people did not accept the fact that we had such a horrible disease in our breed," says Charlotte Patterson, president of the Pug Dog Club of America (PDCA). "Because dogs are usually around 2 years of age before the disease strikes and it can only be confirmed by a brain necropsy, many owners already shocked by the rapid death of their dogs did not want to go that route."
By the early 1990s, members of the parent club wanted to learn more about PDE. Treatment options were limited, and virtually all affected Pugs eventually died from the disease. In 1995, PDCA began fundraising for PDE research and soliciting for research proposals through the AKC Canine Health Foundation.
"We started from square one," says Christine Dresser, D.V.M., PDCA health committee chairwoman. "I talked to lots of veterinary neurologists and internal medicine specialists and told them about our interest in this research."
Meanwhile, the Canine Health Foundation began requesting and reviewing research proposals from its network of researchers at veterinary schools across the country. The Canine Health Foundation also promised to support the research.
The parent club began holding raffles, auctions and bingo games, with fundraising earmarked for PDE research. "At our National Specialty in 2005 in San Antonio, we had a jail in which a sheriff arrested exhibitors for donations for warrants," Dresser says. "Those who were arrested had to donate to make bail. It was fun and successful." Pug chapters across the country generously contributed. Donors were recognized in the parent club's bimonthly Pug Talk magazine.
Turning to Genetic Research
The parent club turned to genetic research and found a devoted Pug owner and research ally in Kimberly Greer, Ph.D., research assistant professor at Texas A&M College of Veterinary Medicine & Biomedical Sciences. Greer, the proud owner of a Pug named "CiCi," conducted research in the school's Laboratory of Canine Genomics.
"I had researched the breed and knew that Pugs could get PDE," Greer says. "Being a geneticist, I was horrified that this loving breed had such a terrible disease with no treatment available and no genetic test for predicting which dogs carried the disease gene or which ones were at risk of getting the disease.
"I worried whether CiCi would develop PDE and anxiously awaited the 'magic' second year of her life so I would know fairly certainly that she was safe. At the time, we thought that if a Pug made it to age 2 without signs of PDE, the dog was safe. Of course, now we know that age is no guarantee and that Pugs even as old as 7 or 8 years of age can still develop PDE."
A study of PDE at Cornell University College of Veterinary Medicine aimed to learn whether PDE is caused by a virus. Since the brain lesions that occur in PDE resemble a viral form of encephalitis in people, herpes virus meingoencephalitis, the hypothesis seemed reasonable. The lead investigator was veterinary neurologist Scott Schatzberg, D.V.M., Ph.D., DACVIM.
No association was found, so Schatzberg concluded that PDE was not caused by the initial three major virus families for which they tested. Since then, Schatzberg and his colleagues have evaluated the brains of over 50 Pugs that died from the disease for numerous viruses and other infectious diseases. Still, no "triggering" viral infection has been identified.
Meanwhile, the disease continued to occur in Pugs, even in those with no known affected dogs in their pedigrees. Pug breeder Brenda Belmonte of Lake Bluff, Ill., recalls receiving a phone call late one evening from a puppy buyer whose 20-month-old fawn bitch named "Cookie" had suddenly become lethargic and hesitant to go down stairs. Two days later, the dog had a seizure.
"I thought it couldn't be PDE," Belmonte says. "There was not one incident of this disease in Cookie's pedigree. Both the sire and dam were from bloodlines I know quite well. I always have health tests done on my dogs prior to breeding and carefully research pedigrees. It did not make sense."
Indeed, Cookie had PDE. Only three days after the first seizure, the Pug was euthanized due to uncontrollable seizures. One week earlier, Cookie had been a healthy, energetic dog.
Greer was vested in wanting to better understand PDE. Her Pug family now included two rescue Pugs in addition to CiCi, and she was active in agility with her dogs. When she accepted a job as assistant professor of biotechnology at the School of Natural Sciences and Mathematics at Indiana University East in Richmond, she continued focusing on the genetics of PDE. She, along with members of the parent club, began collecting samples of DNA from affected and unaffected dogs.
The genetic analysis entailed searching for associations among the affected dogs. Did the dogs live in similar environments? Did signs of PDE have a seasonal onset? Were there similarities among the dogs' vaccination histories or medical records? Were there gender or coat-color correlations? Did the necropsy results show similarities? What treatments were used, and what were the outcomes?
Cookie's owner submitted the Pug's information, including the necropsy results that confirmed the diagnosis of PDE. Belmonte contributed DNA samples from Cookie's sire and dam, littermates and other relatives.
Answers began to stream in. Greer discovered that fawn female Pugs younger than 7 years of age are more likely to develop PDE than older, male and non-fawn-colored Pugs. No correlation could be found suggesting allergens or environmental factors. Affected dogs treated with anticonvulsant therapy to help block the seizures lived significantly longer than those that did not receive treatment. Statistically, about 1.2 percent of Pugs die from the disease.
MRI (magnetic resonance imaging) scans of the dogs' brains taken before they died and necropsies taken after they died did not indicate consistencies in physical signs or brain lesions, other than they became progressively worse. Greer concluded that MRI scans were good indicators of disease severity and how long a dog might live.
Genetic analysis was complicated. PDE did not have a simple dominant or recessive mode of inheritance nor was it sex-linked.
Insights came when Greer and her colleagues discovered an association with the dog leukocyte antigen (DLA) of dog chromosome 12. Markers for the mutation are at or near the region containing the DLA class II genes, which are important in immune function, specifically in recognizing self from non-self tissues. Diseases associated with DLA class II genes include autoimmune hemolytic anemia, immune arthritis and hypothyroidism.
The discovery indicated that PDE is a form of autoimmune disease. "We already suspected this due to certain substances found in the brain tissue and cerebrospinal fluid of affected dogs," Greer says. "Humans also have immune-mediated diseases associated with this region of the brain. The most well-known is multiple sclerosis, which shares some, but not all, similarities with PDE. It is possible that PDE could result from genetically susceptible dogs being exposed to environmental triggers that cause an autoimmune response. We still are looking at these factors."
Dogs with two identical copies of the PDE associated high-risk marker (S/S) in this region have a lifetime risk of 12.75 for developing the disease over Pugs that have only one or no copies (N/S or N/N) of the marker. Though the PDE gene mutation is yet to be discovered, a marker test that determines susceptibility to PDE was developed in 2010 by Greer and collaborators at the University of California-Davis Veterinary Genetics Laboratory. Though the test is not 100 percent definitive, it provides valuable information.
Breeders should be careful not to remove all Pugs with PDE risk markers from their breeding programs, Greer advises. "Forty percent of Pugs have the S genotype in either a heterozygous (N/S – 29 percent) or homozygous (S/S – 11 percent) state," she says. "Eliminating such a large proportion of the Pug population would lead to a loss of genetic diversity that would potentially have even more devastating consequences to the breed than does PDE."
The goal should be to use excellent N/S or S/S dogs bred to N/N dogs to produce litters without PDE. This provides a choice of dogs to progressively decrease the frequency of the PDE gene in future matings to N/N dogs. It also helps avoid giving rise to another genetic disease.
Making Better Breeding Decisions
The PDE marker test benefits breeders by providing information that helps them make more informed selective breeding decisions. It benefits Pug lovers by helping to reduce the incidence of PDE in the breed.
Though the test came after Cookie died from PDE, Belmonte is grateful for the opportunity to test for the disease prior to future matings. "The marker test has opened dialogue among many Pug breeders," she says. "There are still unknowns, but at least now we can make educated decisions when considering dogs to breed. I have all my dogs tested, and I will not breed a bitch or allow a stud dog to be used on a Pug that has not been tested for PDE."
The parent club supports the marker test and is working to get it added to the breed's required health tests in the Canine Health Information Center (CHIC) database. "Getting health clearances is in the best interest of the health of the breed," Patterson says. "The information greatly increases the depth and breadth of pedigree analysis. Having the marker test for PDE is a huge step forward."
Research efforts to identify the PDE gene mutation as well as other aspects of the once mysterious condition continue. Greer hopes to learn why one in eight Pugs with high-risk susceptibility markers develops the disease while others do not. Schatzberg, who is now associate professor at the University of Georgia College of Veterinary Medicine and chief of neurology at the Veterinary Emergency and Specialty Center in Santa Fe, N.M., continues to study the genetics of PDE in collaboration with Matt Huentelman, Ph.D., of the Translational Genomics Research Institute in Phoenix. They recently found potential genetic association at two or three additional locations in Pugs and are studying the disease in other toy breeds.
"We recently were surprised to learn that there seems to be two different groups of NME-affected Pugs that carry the risk locus first described by Dr. Greer and her colleagues and that Pugs seem to succumb to the disease for different genetic reasons," Schatzberg says.
Though there is more to learn about PDE/NME, much has been accomplished over the past 40 years. While breeders and owners initially were reluctant to accept knowledge of the disease, they eventually began working together to fundraise and support research. Their collective efforts have contributed to what is known today about the disease and will undoubtedly steer future discovery.