Canine Bloat Study: Update 10/30/18



531241083185000Fred Hutchinson Cancer Research CenterMichael A. Harkey10/30/18Bloat, known to veterinarians as gastric dilatation-volvulus (or GDV), is an acute, life-threatening condition that occurs at high frequency in many large and giant breeds of dogs. Any dog owner that has witnessed GDV knows it is an unbearably stressful and painful experience. Great Danes are unusually susceptible to this condition. About 37% of Great Danes will experience bloat at some time in their life, and the majority of them will die without immediate medical intervention. German Shepherds, and many other large breeds have a lower incidence, around 3-5%, but that is still very high, and accounts for a large percent of emergency visits to the vet. Yet the causes of this condition have remained a mystery for decades. The goal of this study -- and all of our research -- is to identify the causes of GDV. That information will be essential to the development of therapies and breeding strategies to eliminate this condition. Over the last 4 years, we have completed two studies in Great Danes that have led to a commercially available genetic test for at-risk animals, and set the foundation for probiotic and dietary therapeutic strategies to minimize the occurrence of this deadly condition. Presently, we are conducting a similar study in German Shepherds. This is an update on our work. Our Working Hypothesis for the Cause of GDVWhile the causes of bloat are poorly understood, several risk factors have been described in the scientific literature, including age, dietary, behavioral, pre-existing health and genetic factors. The most significant risk factors appear to be genetic, since strong correlations with bloat exist for breeds, families and gender. Based on the existing scientific literature, we have developed a model of bloat that involves genetics, the gut bacterial population (microbiome), and some outside trigger. In our model, certain genes of the immune system regulate the composition of the bacterial population in the gut (the gut microbiome). This altered microbiome creates an unhealthy gut that is susceptible to GDV. Then some outside trigger, such as stress, or a heavy meal, precipitates the bloat event in a predisposed dog.Previous Work: The Great Dane Study: To test this hypothesis, we enrolled two groups of Great Danes, a “GDV” group in which all members survived bloat through surgical intervention, and a “control” group that had never experienced GDV. In a genetic study, five immune genes were sequenced from each dog, looking for genetic variations that associate with bloat. In a microbiome study, the gut bacterial population of each dog was analyzed from stool samples, looking for particular bacterial species or genera that are unusually low or high in the GDV group.The genetic study is now published in the scientific journal, AJVR. We have established three genes that contribute to GDV in Great Danes. For each of these genes, several alleles (molecular variants) are found in the Great Dane population, and in other breeds. One allele from each gene was found to significantly increase the risk of GDV in Great Danes. Those Danes that carried at least one of these risk alleles had a 3-fold higher risk of GDV. In fact, 62% of the dogs carrying a risk allele had to undergo emergency surgery to survive a GDV episode. This information will be crucial for owners and breeders that are trying to decide if preventative gastropexy surgery is appropriate for their dog, or if their dog should be bred. For this reason, we have designed genetic tests for these risk alleles. These tests are now available for Great Danes at VetGen, a company that specializes in the genetics of dogs.The second study, now published in the scientific journal, PLOS ONE, showed profound changes in the composition of the microbiome of Great Danes that had survived bloat, and remained healthy for several months. For example, the ratio of the two most abundant groups (phyla) of bacteria in the gut microbiome, bacteroidetes and firmicutes, is about 2-fold lower in dogs in the GDV group. Since these two bacterial groups account for over 70% of the bacteria in the gut, this is a massive change that could have significant effects on health. We cannot yet say that these changes in the gut microbiome cause GDV. However, the association suggests they are connected. If we do discover that bloat is caused by specific imbalances of the microbiome, then a whole array of therapeutic strategies will be available to combat the disease. For example, probiotics or specific dietary changes may be used to re-balance the microbiome, and thus, prevent GDV. This approach could theoretically block the genetic predisposition to GDV, since we believe that predisposition is mediated through the microbiome. AGSDCF-Funded Work: The German Shepherd Study Now, with generous support from the AGSDCF, we are asking if these same factors, DLA genes and gut microbiome, play a role in GDV in German Shepherds. We do not yet know if the data from Great Danes is breed-specific, or if it might apply to all dogs. If we find similar associations in German Shepherds then our hypothesis may hold true for dogs in general. If so, we may be able to establish a universal genetic test for predisposition to bloat. The microbiome data may lead to probiotic or dietary therapies to prevent bloat, even in predisposed dogs. The genetic study is well underway. We have collected DNA samples from about 500 German Shepherds, and have characterized the same genes that associated with bloat in Great Danes. This testing is three fourths complete, and the statistical analysis is just beginning. But we are starting to see trends already that look exiting.The story of the TLR5 gene in German Shepherds is turning out to be quite different than that of the Great Danes. The Danes have 2 variants (alleles) of the gene, which we call “A” and “B”. Carriers of the B allele are 2-3 times more likely to experience a serious episode of bloat, requiring emergency intervention. But we have never seen this allele in German Shepherds. Instead, some members of this breed carry a third allele, “C”. Carriers of the C allele, are protected from bloat compared to those carrying the A allele. This difference may explain why German Shepherds are about 10 times less likely to experience GDV than Great Danes. A similar story is unfolding for the DLA88 gene. So far, the allele that puts Great Danes at risk for GDV, has not been detected in German Shepherds. As with the TLR5 gene, this finding may help explain the lower incidence of GDV in German Shepherds.The genetics of the DLA88 and DRB1 genes is more complex than that of TLR5. These genes are highly variable; over 100 alleles of each gene exist in the total dog population. However, due to selective breeding, each breed, carries a limited set of these alleles, and these genetic sets are different for each breed. So, comparison of genetic risk in different breeds can be like comparing apples and oranges. Thus, as described above for DLA88, a “risk” allele in one breed may not be seen in another breed. As expected, many genetic associations with GDV are emerging in the German Shepherd study, which were not seen in the Great Dane study. This may complicate the search for a universal genetic marker for GDV. But so far, we have found one risk allele that is common to Great Danes and German Shepherds. The 1201 allele of the DRB1 gene has emerged as a risk allele for GDV in both breeds. This could turn out to be a universal genetic marker for GDV risk among dogs The initial testing of the gut bacterial populations (microbiomes) in these dogs is complete. However, a lot of complex statistical analysis is still required before we can begin to see associations with GDV. This work should be done in the next few months.Related Research in Our LabWe have two related studies in progress. Together, these related studies are designed to broaden our understanding of the genetic risks for bloat to all breeds of dogs, and to develop methods for rapid genetic testing for predisposition to GDV and other diseases.Cornell mixed breed study. We have acquired a unique set of DNA samples from the Veterinary Biobank at Cornell. These samples represent 100 dogs covering a wide variety of breeds. Fifty of the dogs were treated for bloat at the Cornell Veterinary Clinic. The other 50 are healthy controls that are matched (in pairs) for breed, age and gender to each of the GDV dogs. This is a statistically powerful sample set for determining if the genetic risk factors found in Great Danes and German Shepherds, are universal risk factors for dogs. It would be the basis of a cross-breed genetic test for GDV risk, that could be made widely available to dog owners. The plan is to isolate and sequence the three target genes from each sample, to determine which variants associate with GDV. Next-Generation DLA Typing. The immune genes that are the focus of our bloat project, are also known to play a role in many human and dog diseases, such as Inflammatory Bowel disease, Crohn’s disease, and Anchylosing Spondylitis. These genes, known cumulatively as MHC genes, are extremely complex and variable, each having hundreds to thousands of genetic variants within the human or dog populations. In humans they are called HLA (Human Leukocyte Antigen) genes and in dogs, they are known as DLA genes. Their job is to distinguishing foreign cells (potential infectious agents targeted for destruction) from one’s own cells (safe from the wrath of the immune system). It is this set of genes that must be matched between donor and recipient for a successful tissue or organ transplant. As you can see, these genes are vitally important in many aspects of human and dog health. But because of their complexity, these genes are difficult and expensive to sequence. During the tenure of these studies, a new sequencing technology has developed and matured to such a level that it is now more cost effective, more accurate, and orders of magnitude faster than the methods we have been using.? This is the so-called “Next Gen Sequencing”, which involves simultaneous sequencing of up to 1.5 million DNA samples and automated computer analysis of the results in an over-night run.? Dr. Dan Geraghty, my colleague at Fred Hutchinson Cancer Research Center, has developed an HLA typing service based on this technology (Nelson et al., 2015). Together, we are translating that technology to dogs.? Development of this technology for DLA (dog genetic typing) would reduce the cost per dog (by about 10 fold) and massively increase the number of dogs that we could include in a study such as the GDV study.? In turn, this would greatly increase the statistical power of the study.? In addition, this approach would generate long-term clinical and research benefits for all canine DLA-related issues, by drastically decreasing the time and cost of DLA typing.?? We have generated a working Next-Gen protocol for one dog MHC gene, known as DLA88. Our first test run on 300 dogs was spectacularly successful. We not only demonstrated that the method works, but also discovered 20 previously unknown alleles of this gene. Several other DLA genes, including three that are important for transplant success, remain to be worked out. We are preparing to tackle this job now. This is one of those cases where the initial goal may be a boring technological advance, but the long-term payoff for canine health could be huge.Michael Alan Harkey, PhDClonal Tracking and Canine Resource DevelopmentMail Stop D1-100Fred Hutchinson Cancer Research Center1100 Fairview Avenue North, P.O. Box 19024Seattle, WA?? 98109-1024Phone:? (206) 667-3369FAX:???? (206) 667-5978mharkey@ ................
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