Immunology, Allergy, Aging and Nutrition
Ebenezer Satyara, PhDS

Today, aging and stress are major causes of immune challenges in otherwise healthy animals and humans. Unlike immune deficiency caused by malnutrition, immune deficiencies due to life-stage and stress need a comprehensive strategy and cannot be addressed simply by correcting nutritional problems. Lowered immune status and allergies seen in the aging and following chronic stress is characterized by reduced antigen presenting cell function, increased inflammation resulting in less efficient or altered immune response, leading to increased susceptibility to infections, increase in allergies, autoimmune disease, and cancers. Over 65% of the body’s immune cells are present in the gut, making the gut the ‘largest immune organ’. Microbiota present in the gut has a fundamental role in the development, education, and function of the overall immune system, and as a result, the immune system has evolved to maintain a symbiotic relationship with these diverse, ever evolving commensal microbiota. Nutrition affords us the opportunity to simultaneously impact both the gut-associated immune system and gut microflora. The receptors of the innate immune system present in the gut are the primary targets of strategies for immunomodulation via diet. Diet interacts with the immune system at multiple levels, starting by providing basic nutrients, moving on to providing higher level of key nutrients such as protein, vitamins and minerals, leading to a more focused modulation of the immune system. A framework outlining this interaction, along with relevant examples of dietary ingredients that impact various key pathway that influence both the immune system and gut microflora are presented with an overall view how nutrition impacts immunity/allergy in aging pets.

The gut immune function involves:

1. Barrier function to keep out pathogens
2. Surveillance: managing what gets across and differentiating self from pathogens
3. Defense: dealing with an intruder

Factors to impact macrophages and regulator T cells needed to decrease inflammation

1. Deficient nutrition= decreased immune function
2. Excessive nutrition e.g., obesity (fat cells release leptins and other cytokines=immunosenece)
3. Life stage: pups=50% decreased immune response, seniors=decreased immune response, also disease and stress= decrease immune response

Four Stages where nutrition impacts the immune system:

Passively support

1. Balanced, complete diet with high quality protein, vitamins and minerals
2. Optimize macro and micronutrients: increased protein, calorie restriction, antioxidants, vitamins and minerals. Example: colostrum study where Huskies were fed bovine colostrum which resulted in increased immune response and decreased inflammation to canine distemper vaccines (stronger and longer immunity both gut IgA and titer). Another colostrum study in old dogs given Lyme vaccine showed increase titer with increased T regulator cells and decreased inflammation. The IL10 in colostrum, increases cytokines reducing inflammation. Another study showed positive response to improve immunomodulation with probiotics and gut health. A 44-week study with 4 breeds and 14 puppies also showed improved response to a primary canine distemper vaccine, longer immunity and increased IgA. More studies are needed to determine how to use these products and in addition, they are varying in quality and are highly heat sensitive. Another example, short chain fatty acids produced by microflora with fiber provide essential food for the colonocytes. Broccoli and cabbage with AHR ligands provide a pathway to produce regulator T cells, and Turmeric and green tea with NRF -2 activators provide the immune system with information.

Actively help immune system:

3. Active immodulation (enables macrophages and manage inflammation)
4. Personalized immunonutrition: e.g.: in IBD (inflammatory bowel disease in dogs) the particular ingredient that the dog inappropriate reacts to can be avoided. The future will personalize nutrition for specific disease and optimize health with diet and specific ingredients.

Canine Atopic Dermatitis (CAD) and Food Allergies (FA)
Craig Griffin, DVM DACVD

 CAD is a genetic predisposed inflammatory, redness and/or pruritus (itching) of the skin. There are numerous genetics involved, and clinical presentation can vary in different breeds. The disease often includes paws, ears, face, forelegs, axilla, inguinal and perineal area. CAD is the top canine insurance claim and involves 10-15% of all dogs. The classic age of onset is 6 months to three years old. CAD is lifelong and often progressive due to abnormalities in skin; therefore, treatment should never stop.

Diagnosis is made by compatible history, exam, ruling out other causes, intradermal skin testing and serum testing. None of which is perfect, although intradermal testing is considered the gold standard. This can provide information on what allergens can be avoided or immunized against (receiving personalized allergy shots).

Treatment is a systematic approach:

1. Eliminate fleas, mange, parasites, environmental irritants, etc.
2. Look at patient history: pattern on body, seasonality, GI signs are not uncommon (30-50% have concurrent food sensitivity). The GI signs seen can be soft stools, increased number of stools, flatulence, borborygmi (increased gut sounds), mucous or blood in stool or vomiting. In additions, conjunctivitis and sneezing is commonly seen.
3. Address secondary infection
4. Diet trial: Only 40% of clients are found to do this perfectly, where absolutely no other foods or chews cross the pet’s lips, including switching to topical heartworm, flea and tick preventatives for three months. There are no perfect foods and there are a couple types of food trials:
   1. Hydrolyzed diets keep the protein size very small less then 10-12 Daltons which is believed to prevent reaction. Most recently, extensively hydrolyzed have become available providing an even less likely chance of body reaction.
   2. Limited ingredient contains only a couple uncommonly used proteins. Veterinary prescription diets are made in factories that completely clean machinery prior to producing to prevent contamination during production. In addition, testing the food by PCR or other analysis to assure no contamination.
   3. Elemental diets are most commonly used in human infants. Purina EL recently released is modeled after this with no animal proteins, highly digestible and low allergen small proteins, as in hydrolyzed.
5. Address allergic aspects (covered in later lectures)

If not treated for life, a progression of symptoms can be seen which require changes in treatment, another diet trial, or allergy testing. The chronic case can develop many secondary changes that also greatly contribute to pathology and clinical symptoms. Most notable is the role of secondary microbial disease has and the development of bacterial and yeast infections which can greatly complicate the case.

Pyoderma and the Role of the Microbiome in Atopic Dermatitis
Galia Sheinberg DMV DLACVD

CAD is a common inflammatory and pruritic skin disease that causes frequent visits to the veterinarian where more options are now available to treat the allergic component, but treating the secondary pyoderma (bacterial infection) has remained a challenge. In recent times the role of the skin microbiome and dysbiosis (changes in the skin microbiome) has been recognized. Dysbiosis is seen in the skin in CAD and the gut in IBD. It results in inflammation, decreased variety of bacteria and secondary infection. The proposed change in the skin in CAD are:

1. Skin barrier dysfunction including increased transdermal water loss, immune activation, decreased antimicrobial peptides, and altered microbiome (dysbiosis)
2. Dysbiosis does not mean infection or pyoderma. Pyoderma is increase in staphylococcus associated with exacerbation in signs. Dysbiosis is associated with increased staph bacteria. Canine Staph pseudointermedius is part of the normal canine flora but overgrowth can cause infection.
3. Pyoderma is always secondary to allergies and the associated itching can permit spreading.
4. Treating pyoderma with antibiotics (based on culture) temporarily restores the microbiome to a more normal state but does not eliminate the abnormalities, just controls. The goal is to eliminate overgrowth and support the barrier function. Methicillin and Multi drug resistant bacteria are now common due to the over and inappropriate use of antibiotics.
5. Types of pyodermas:
   Superficial with pustules, papules and collarettes (circular lesions which can be mistaken for ringworm)
   Deep with furunculosis, ulcers and draining tracts
6. Diagnosis is made primarily by cytology, with skin scrapings to rule out mites, and fungal screening tests (woods lamp, fungal culture and fungal PCR)
   1. Treatment goal is to restore the skin barrier and treat the primary cause. WAVD (World Association of Veterinary Dermatology) created guidelines, see their open access articles on their website.
   2. #1 is topical treatment for all, and in 2/3 of cases works as well or better then oral antibiotics. Topical treatment should be the only treatment for superficial pyoderma and always part of deep pyoderma. This includes shampoos, sprays, mousse, and wipes daily or as directed. Chlorhexiderm, benzoyl peroxide, and bleach are commonly used. Bleach, diluted appropriately, has been safe and effective for resistant bacteria. Topical does not contribute to resistance.
   3. Control pruritus
   4. Oral antibiotics only if deep, concurrent disease, or cannot do topical. Empirical antibiotics are no longer recommended, if using oral antibiotics, culture first. Bacterial resistance is worsening and is affecting humans and all organisms.

Local and Systemic Changes in Lipid Profile as Potential Biomarkers for Canine Atopic Dermatitis
Harm HogenEsch, DVM PhDS

 Lipids play a critical role in the skin as structural components of the epidermal barrier and as signaling and antimicrobial molecules. CAD is associated with changes in the lipid composition of the skin, but is not clear if these changes precede the onset of dermatitis or occur secondary to dermatitis.  Changes in lipid composition may serve as biomarkers to aid in the diagnosis and potential stratification of CAD into subtypes with different therapeutic outcomes, and to assess the response to treatment. They developed a mass-spectroscopy-based technique for rapid, non-targeted profiling of the lipids. This methodology was applied to lesional and non-lesional skin of dogs as well as dried blood spot lipids (dbs), and determined changes after CAD treatment. A total of 30 control dogs and 30 dogs with mild to moderate dermatitis were studied. Marked differences were seen in the control, lesional and non-lesional skin. Phosphatidylserines and cholesteryl esters contributed significantly to these differences. Seventeen dogs were treated with Apoquel and ten with Cytopoint which resulted in a significant decrease in the clinical severity after 4 and 8 weeks. Machine-learned classification was able to recognize samples as healthy control or CAD with very high accuracy (AUC=0.95), and DBS samples at 0.9. The data suggests that CAD is a systemic disease, supports the use of rapid, non-targeted lipid profiling to identify novel biomarkers and demonstrated the feasibility of computer-aided diagnostics in veterinary medicine.

CAD is influenced by genetics (40-50-% heritability, with more seen in terriers and retrievers), environment (more urban and rural), and nutrition. These together alter the microbiome, skin barrier function and immune response. The lipid composition differs in controls when compared to allergic dogs with or without current lesions. The blood lipids also follow these changes. Also noted that changes were improved when treated with Apoquel or Cytopoint. In addition, female and male dogs differ in their magnitude of lipid change. With more change noted in CAD male skin lipids and more in CAD female blood lipids.

The lipids of the skin are primarily located on the surface, stratum corneum. There are thousands of lipids on canine skin and there are changes in their profile and concentration in CAD. These biomarkers serve as fingerprints for disease. These changes in CAD were also found to be present in the blood, thus systemic. The lipid profile changes are currently not ready for clinical application for diagnosing, subtyping CAD and success of treatment.

Atopic Dermatitis- A One Health Approach
Domenico Santoro DVM MS DrSc PhD DECVD DACVD DACVM

 Atopic Dermatitis (AD) is a common, relapsing, chronic, pruritic, and inflammatory skin disease affecting 10% of dogs, 20% children and 10% adults worldwide. Because of the strong similarities between human and canine AD, the dog has been recognized as the best animal model for human AD. Furthermore, it has been hypothesized that environmental factors and inheritable epigenetics modifications of both genes and associated proteins play a role in the increased prevalence of AD in both species. In particular, a profound alteration of skin barrier and the host-microbe interaction has been shown in both. Such studies have shown the need of a One health approach for this common disease. In fact, dogs and people share the same environment and same cutaneous microbiome with a potential of transferring beneficial, but also pathogenic, microorganisms between dogs and their owners. The latter aspect is very important considering that bacterial infections are extremely common in atopic dogs and people. In addition, the recurrent presence of skin infections leads the prescription of more antibiotics favoring the increase in antimicrobial resistance in the community. For this reason, an increased number of studies have been focused on a better understanding of bacterial resistance and the testing of new antimicrobials. Because of the role that the environment plays in this highly complex disease and its effects on humans and dogs. AD represents a disease that very well defines One Health approach to medicine.

AD is influenced by the environment (pollution, lifestyle, cleaners and chemicals). The exposure can be dynamic in a lifetime and challenging to try and determine the exposome, the totality of exposure that affects health. One study on air pollution (fine particles, NO, O2, black carbon) looked at the short- and long-term effect on AD and asthma. They were shown to damage the barrier, allow inflammation, sensitize to future exposure, create oxidative stress, and decreased mucociliary clearance. The study of exposure to common airborne endotoxins (e.g., LPS, lipopolysaccharide) from farms, agriculture (plays a role in grain production) and households, at low dose, resulted in improved barrier and inflammatory mediators.  The influence of microbial exposure from natural products included good bacteria, unlike the bacteria found on plastics or manmade products had bad bacteria.

The result can be what is termed targeted hygiene. This involves exposing children to dirt and environmental good bacteria, parasites, viruses, and dust that produce good immunoregulation. The Hygiene hypothesis is that untargeted cleaning may decrease human exposure to good microbiome. Several UK and Finland studies were referenced, involving hundreds to nine thousand dogs. They showed less AD in rural vs urban dogs, less AD if living with another dog or walked in nature.

The amount and diversity of bacteria vary in the skin microbiome in AD dogs compared to normal dogs. Allergic dogs have less diversity, increased Staph (not causing infection), and more skin fungal. Healthy dogs are also resistant to yeast (Malassezia) and fungal growth. There is also sharing of the dermal microbiome between dogs and their owners. They can share both good and occasionally bad bacteria.

Panel Derm and Immunology Discussion with above presenters:

 Importance to identify beneficial vs pathogenic bacteria. For example, pseudomonas is found in normal skin but not associated with inflammation. Inflamed skin allows more bacteria to attach by releasing inflammatory cytokines which feed the bacteria. Normal skin continually produces antimicrobial peptides.

Multidrug resistance is present in both dogs and humans and it is important to minimize the use of oral antibiotics which promote further resistance. Since COVID19, as a result of using more antibiotics, there has been a significant increase in antimicrobial resistance.

Nanoparticles (metallic: iron, silver gold, sulfur) are commonly used in human medicine and recently looked at in dogs in a Florida study. Nanosulfur reduced the staph pseudointermedius biofilm within 48 hours. This may provide a good option for MDR bacteria.

Other natural options are being looked at such as manuka honey and other naturally occurring products. They have topical value in promoting a healthy microbiome. In addition, creams containing lactobacillus and enterococcus have shown a potential to prevent bad bacteria over growth in AD by inhibiting and competing with pathogens.

The Canine Health Information Center (CHIC) program Status Update
Eddie Dziuk, MBA

 The CHIC program, co-sponsored by OFA and AKC CHF, was first implemented in 2001 with 8 pilot breeds. The goal of the program is to provide a consolidated source for canine health test results, and recognize those dogs tested in accordance with a breed specific health testing protocol, as established by the breed’s parent club. Over 180 breeds now participate in the program and over 130,000 dogs have earned their CHIC numbers. This conference is an appropriate venue to remind clubs that their requirements are fluid and should be periodically reviewed and updated.

Another key element of the CHIC program is the CHIC DNA repository. The CHIC DNA repository stores canine DNA samples from blood (58%, partnered with U of MO) or buccal swabs (42% partnered with UC Davis) for future health research efforts. The bank has over 30,000 samples representing many different breeds. More than 3,000 samples have been distributed to a variety of research institutions all over the world. While all samples may be of potential use, the majority of the samples represent young, healthy dogs at the time of collection. However, most valuable samples are from affected dogs. Club members should periodically update the bank with any health updates of significance that may increase the value of a sample and likelihood of its use. CHIC is now working with AKC to hopefully include CHIC data or numbers on pedigrees.

Environmental Cancer Risks in Dogs and People
Lauren Trepanier, DVM PhD DACVIM DACVCP

 Dr. Trepamnier’s lab is interested in genetic and environmental risk factors for cancer in dogs with a focus on lymphoma and bladder cancer. She completed a case-control study of lymphoma in the high-risk Boxer breed and found a higher risk among dogs living within 10 miles of a nuclear power plant or within two miles of a chemical supplier, or an active crematorium. However, they were unable to find risk associations with genetic variants in canine glutathione-S-transferase genes, which detoxify chemicals. More recently in this same population, they demonstrated an association between lymphoma in Boxers and living in a county with higher airborne concentrations of ozone, 1,3 butadiene and formaldehyde. They are currently recruiting a new case-control study of Boxers with lymphoma to measure urinary and household concentrations of several environmental chemicals linked to lymphoma in humans.

Lymphoma accounts for 20% of all canine cancer and resembles non-Hodgkin’s lymphoma in humans. There is a breed predisposition (Golden, Boxer, Bullmastiff, and mixes). Lymphoma is linked to benzenes, trichloroethylene, and herbicides (e.g., Roundup).

In a case-control study of bladder cancer in dogs, they found an association with household insecticide use and more recently with living in a county with higher ozone concentrations and higher tap water concentrations of trihalomethanes, which are chlorination byproducts. They further found that urinary exposure to the bladder carcinogen acrolein and arsenic, and the herbicide metabolite 4-chlorophenol, are shared between dogs and their owners, and creatinine-adjusted concentrations were 3-6-fold higher in dogs. Urinary concentrations of acrolein and arsenic metabolites were also correlated between dogs and their owners in the same household. This suggest that dogs might act as sentinels for human urinary exposures to some bladder carcinogens. They are now recruiting dogs with bladder cancer, and their owners, to measure urinary and household concentrations of these environmental chemicals, compared to dogs and owners living in control households of matched unaffected dogs.

Urothelial carcinoma is the number one bladder cancer in women affecting 1/89 older women and 1/27 men. There appears to be a strong environmental influence but since later in life, makes it challenging to know lifetime exposures. Risks factors for people include smoking, herbicides, and living in industrial areas. In dogs, it can be seen in any breed with some increase incidence in Beagles, Shelties, Scotty and Westies. Females are more commonly affected then males. There are similar associations to people with an association of being overweight, not unexpected since chemical can reside in fat.

The overall goal is to understand potential modifiable environmental risk factors for the cancers in dogs.

Tumor-permissive Collagen Signatures in Canine Mammary Gland Tumors: Development of Prognostic Markers and Targeted Therapies for Improved Outcomes
Susan Volk VMD PhD DACVS

 Dr. Volk has developed a research program focused on understanding reciprocity between cells and their surrounding extracellular matrix in both regenerative and tumor microenvironments. The success of her program capitalizes on the ability to lead cross-disciplinary collaborative teams capable of efficiently developing novel therapies for surgical and cancer patients, including her patented approach to reengineering the tumor microenvironment.

Mammary gland tumors are the most common malignancies in intact female dogs and the associated premature death and morbidity in canine mammary tumor (CMT) patients represent a significant health problem. Two major obstacles limit the care of CMT: 1) accurate identification of dogs at risk for local recurrence and/or metastasis and 2) effective therapies for at risk individuals. The lack of prognostic indicators results in increased morbidity and mortality due to both over treatment of patients bearing malignant tumors with low metastatic potential and inadequate therapies for those requiring early, aggressive intervention. Thus, defining superior prognostic indicators to accurately predict CMT reoccurrence and development of safer, targeted therapies for dogs at high-risk for reoccurrence will have a profound impact on a large number of dogs, their owners, and veterinarians. Until recently, investigators of cancer development and progression have focused on mechanisms within neoplastic cells that drive uncontrolled growth and metastasis. In a recent paradigm shift, she now realizes that growth and spread of cancer depends on the support of non-malignant cells and the extracellular matrix, particularly collagen, in the surrounding tumor microenvironment (TME). Overall collagen abundance correlates with both an increased breast cancer risk and poor prognosis, and the organization and stiffness of the collagen matrix are key mediators of mammary tumor growth and invasion. Notably, her recent study was the first to define the role of collagen in regulating clinical outcome in dogs with malignant CMT. Subsequently published and ongoing studies define collagen architecture and type, as well as extent of collagen cross-linking as critical regulators of the biomechanical and biophysical properties of the TME which contain (tumor-restrictive) or facilitate (tumor-permissive) malignant behaviors including escape from dormancy, metastasis, and therapeutic resistance, and apply this knowledge to improve clinical care.

Mammary cancer is the third most common canine cancer with 50% of canine cancers malignant and 80% of feline cancers malignant. In humans, type III collagen is associated with decreased growth and decreased metastasis and LH 2, a collagen crosslinking enzyme, is associated the tumor permissive behavior. The tumor collage organization which wraps tightly around the tumor is tumor restrictive verses collagen loosely and spread out like spokes is associated with tumor permissive behavior. The stiffness of collage (measured by YAP) shows with increased YAP, is less dense and tumor permissive.

The Life and Times of the Hero Dogs of 9/11
Cynthia Otto, DVM, PhD, DACVECC, DACVSMR, Keynote speaker and 2021 Asa Mays DVM Award Recipient)

Twenty years ago, America experienced the worst act of terrorism on home soil in history. As part of the response to the attacks on the World Trade Center and the Pentagon, thousands of first responders risked their lives to attempt to rescue survivors and recover victims. A critical part of that response included search and rescue dogs. Although the exact numbers are still unknown, it was estimated that approximately 300 dogs responded to the World Trade Center and at least 80 to the Pentagon. Since that day that changed the world, reports of physical and mental illness in first responders and residents of lower Manhattan have been reported. Monitoring programs continue to determine the long-term effects in the human responders. Through the vision and support of CHF, Dr. Otto was able to follow the health and behavior of the dogs that responded to 9/11 throughout the course of their lives. Comparing their results to those dogs that did not deploy, we have been able to conclude that dogs did not suffer the same kind of morbidities that their human counterparts experienced. In fact, on average search and rescue dogs live longer than the reported breed averages and no detectable adverse events on health or behavior were detected.

When tracking canine morbidity, cuts were by far the most common but most were superficial and only 4 required sutures, followed by weight loss and change in appetite which quickly resolved. Unlike the human counterparts, lacerations were also the most common, but respiratory disease was second. This is likely due to the massive amount of particulate material (asbestos, metals, dioxins, polychlorinated biphenyls, volatile organic compounds, smoke). Theories as to why the canines did not suffer from respiratory disease include: long noses, dog lungs are made for athleticism, Asthma rare in dogs, frequent eye flushing in the dogs, and small number of dogs compared to humans.

As a legacy to these invaluable partners, the Penn Vet Working Dog Center was established to continue to investigate new strategies to improve their overall health and welfare.  In addition, canine fitness has been studied, and open access articles are available.

Precision Medicine in Canine Lymphoma
Nicola Mason BVetMed PhD DACVIM

The clinical response of dogs with lymphoma to multi-agent chemotherapy is highly variable. Although up to 85% of dogs respond initially, some relapse within weeks, while others enjoy remission of two years or more. The heterogeneity in clinical response is in part explained by the recognition that “lymphoma” is not a signal disease entity but consists of different subtypes that can be characterized on a molecular level by mutations in specific genes. As in human medicine, it follows that different lymphoma subtypes, driven by different molecular mechanisms, may respond better to therapies that are specifically selected to inhibit the driver mechanism within that patient’s tumor. Recent work using sophisticated genetic sequencing tools (NGS: next-generation sequencing) has begun to shed light on the different molecular subtypes of canine B cell lymphoma and specific therapies aimed at targeting patient specific driver genes and pathways are being developed. To enable targeted therapies to move into the clinic, a personalized diagnostic tool must be developed that can rapidly and cost-effectively determine the mutation profile of a patient’s cancer allowing selection of the most effective drug for that patient. With support from CHF, Dr. Mason has developed a comprehensive, targeted NGS panel to map mutation profiles within canine B cell lymphoma.  The panel consists of 283 common cancer genes that are recurrently mutated in lymphoma and other common cancer types.  NGS performed on standard biopsy samples using this panel aims to identify the molecular drivers of a patient’s lymphoma, enabling the most appropriate, targeted therapy to be selected for that patient. Furthermore, a specific mutation profile within canine lymphoma are predictive of outcome. Together, this personalized approach aims to provide diagnostic and prognostic information that will improve standard of care for canine cancer patients.

Currently Lymphoma is divided into B cell lymphoma (more common in dogs) or T cell by flow cytometry and/or PARR testing. T cell lymphoma is typically associated with a worse prognosis. The most common canine lymphoma is DLBCL (diffuse large B cell lymphoma). Within DLBCL there are subtypes.

Dr. Mason looked at the 283 gene mutations commonly associated with a variety of cancer and then looked at DLBCL and specifically 36 genes (thus far 51 samples completed, but waiting for 100 samples total). She has also looked at hemangiosarcoma and specifically 20 genes. This has a potential clinical value, for example a dog with pericardial effusion is typically either hemangiosarcoma or idiopathic, with a much better prognosis. If the fluid could be evaluated genetically to determine the cause proper treatment can done.

Precision medicine is an emerging approach to human and dog cancer directing treatment against the genetic drivers with immunotherapy (using the patient’s own immune system) and potentially many other factors. For example, human lung carcinoma has double survival time with precision medicine, and now available with other cancers. Recently Palladia in dogs, treats 30-50% of mast cell tumors that have the ckit mutation.  Other immunotherapy options are approved in dogs for malignant melanoma, B cell lymphoma, osteosarcoma and brain cancer. This field continues to grow.

Canine Cancer Panel Discussion

University of Florida is studying the association of dilated cardiomyopathy and grain free diets, legume rich diets.  Dogs fed grain free diet for one year have increased troponin (cardiac enzyme), increased BNP (another cardiac enzyme), and decreased heart function determined by echo.  Dogs in this study had a normal troponin once returned to a grain diet. There may be a predisposition in Goldens, Dobermans and schnauzer. More studies and the role of taurine are currently being studied.

Is Cannabidiol the cure for Canine Epilepsy?
Stephanie McGrath, DVM MS DACVIM

Cannabis-based therapies have been used for centuries for various medicinal purposes. They have recently gained publicity as an effective medication for use in human medicine and, as such, awareness is increasing among veterinarians and pet owners. However, there are still many unknowns regarding side effects, pharmacokinetics and efficacy in dogs. Colorado State University have successfully performed tolerability and pharmacokinetic studies assessing cannabidiol (CBD) in healthy dogs. The results of these studies showed that CBD seemed to be well tolerated in dogs and that exposure was dose proportional to blood level. These results provided framework for clinical studies in canine epilepsy. The objective of the clinical trials was to compare CBD with placebo for the treatment for naturally occurring canine idiopathic epilepsy.  The studies were double blinded, placebo-controlled, randomized clinical trials using client owned dogs with naturally occurring disease. Idiopathic epilepsy can be a devastating disease in our veterinary patients and, to date, does not have an ideal treatment. If these current and future studies can demonstrate that dogs attain sufficient blood exposure with oral dosing of CBD to be efficacious in treating idiopathic epilepsy, it has the potential to improve the quality of life of this population of dogs, as well as decrease the rate of euthanasia.

Idiopathic epilepsy is the number one neurologic disease in dogs and is frequently (1/3 of affected dogs) not well controlled with current therapies available or with such bad side effects to produce a poor quality of life.  Dogs are commonly euthanized due to poor seizure control or undesirable side effects.  There are state and federal ramifications in the use of hemp and CBD.  The studies thus far used two different dosages in 50 epileptic dogs, continued on their current therapy and were consider responders if there was at least 50% reduction in seizures. There was some reduction at 2.5mg/kg but no change at 4.5 mg/kg (in fact worse than placebo), although a 3-year data analysis has started.  Dr. McGrath feels CBD may help some dogs but in no miracle cure. The newest study will look at 2.5mg/ml, 5mg/ml and 10mg/ml every 12 hours.

The other concern with CBD is that it is an unregulated market. The best that can be done is looking for third party testing at a couple different labs. The FDA approved human product is very expensive. Side note: CBD may be a p-glycoprotein substrate, potentially affecting the drug and its side effects in mdr1 mutation affected dogs.

Lysosomal Storage Diseases (LSD) in Dogs
Gary Johnson, DVM PhD

LSD are a diverse group of heritable diseases characterized by the accumulation of partially degraded complex biological molecules within a subcellular organelle known as the lysosome. Almost all LSD are inherited as recessive traits and thus inherit a mutant gene from both parents (93% of canine LSD are autosomal recessive). LSD are associated with a wide range of clinical manifestations affecting multiple organs and causing visceral, ocular, hematologic, skeletal and neurologic signs. Various types of human LSD have been attributed to mutations in over 70 different genes. The earliest identified genes that cause LSD encoded enzymes that are directly responsible for degrading complex biological molecules. Enzymatic assays were used to discover the responsible diseases and to diagnose these diseases in individual cases. More recently, genes encoding proteins that lack enzymatic activity but still interfere with lysosomal function have been shown to cause LSD. Most of these genes were discovered by whole-genome sequencing or by related next-generation sequencing technologies. DNA tests are now used to diagnose these diseases in individual cases. LSD have been characterized into subtypes, mostly defined by the biochemical nature of the partially degraded complex biological molecule that accumulate.

Mutations in the canine versions of many of the same genes responsible for human LSD diseases have been implicated a cause for canine LSD. Examples of canine LSD with known mutations will be discussed so that breeders can use DNA tests to avoid LSD in their breeding program. In addition, educate dog breeders and owners to recognize cases as of yet uncharacterized LSD and engage research labs to study these cases and develop DNA tests.

Breeds currently known to have LSD include Am Staff, Cattle dog (2), Aussie (2), Dachshund (3), English Setter, Boston, Irish Setter, Plot hound, PWD, Saluki, Germ Shep, Swedish Lapphund, Bulldog, four non-AKC breeds, and many more. Many remain unpublished.

The four major subtypes:

1. Mucopolysaccharides that are necessary to protect structures around cells
2. Sphingolipidosis necessary for the ceremide on cell surface
3. Glycoproteinosis includes thousands necessary to process glucose and simple carbohydrates
4. Neuronal ceroid lipofuscinosis which accumulates ceroid in the brain (differentiated from lipofuscin in the brain and muscle during aging).

There are multiple diseases and different organs affected. How long a patient will live depends on the disease, but many live less then 36 months. There are many articles available on Pubmed NIH search.

Genetic and Therapeutic Explorations on Canine Degenerative Myelopathy (DM)
Joan Coates, DVM MS DACVIM

DM was first described by Averill in 1973 as an insidious, progressive disease-causing loss of coordination and spastic weakness of the pelvic limbs, progressing to the front legs within a year, and ultimately leading to paraplegia and necessitating euthanasia. In 2009, Awano et al. identified the SOD1 mutation which underlies most cases of DM. Since not all SOD1:c118A homozygous develop clinical signs, DM initially appeared to be an autosomal recessive disease with incomplete penetrance. Thus, homozygosity for the mutation is a major risk factor in dogs for developing DM. However, DM has also been histopathologically confirmed in a few heterozygous dogs. The occurrence of DM in a heterozygote seems plausible since most human SOD1 mutations cause dominant amyotrophic lateral sclerosis (ALS: Lou Gehrig’s disease). Nonetheless, the age at onset for many dogs has exceeded the mean life expectancy of dogs indicating that DM has an age related, incomplete penetrant mode of inheritance. A second SOD1 missense mutation (c.52A>T) appears to be restricted to the Bernese Mountain Dog breed where it is less common than the c.118A allele.

Homozygous (at risk), DM dogs if they live to seven or greater have a 60% of becoming affected. Heterozygous breeds that may become affected include Chesapeake Bay Retriever, German Shepard, PW Corgi, Rhodesian Ridgeback, PWD, and Shilo Shepard but are at lower risk. Even if they live to 11 years old, less then 5% develop clinical disease.

After discovery of the mutation in SOD1, DM is now recognized as a naturally-occurring progressive adult-onset neurodegenerative disease that has many similarities to human ALS and may serve as an important disease in therapy development. Nucleic acid-based therapies (i.e., Antisense oligonucleotide) offer the promise of modifying or arresting the course of neurodegenerative disease in which down-regulation (knock-down) of a protein is a treatment objective. Target engagement, cerebrospinal penetration, and delivery methods are areas that are undergoing transformation in treatment in neurodegenerative diseases. Dr. Coates believe therapies that decrease the amount of aggregated SOD1 in neurons are likely to reverse or slow the disease in canine DM and studies continue to investigate.

Meningoencephalomyelitis of Unknown Origin in Dogs
Nicholas Jeffery, Johnson McGrath

Meningoencephalomyelitits is the term for inflammation of the brain, spinal cord, and the surrounding membranes (the meninges) and has many different causes. Sometimes caused by an infection but much more commonly, it is caused by inappropriate activation of the immune system, often referred to as meningioencephalomyelitis of unknown origin (MUO), and previously referred to as GME, MUE, MUA.

MUO can cause clinical signs associated with abnormal function of the brain or spinal cord, but seizures, loss of balance and limb coordination are especially common. Some breeds of dog, especially smaller breeds, including Pugs, Dachshunds, Malteses, and Yorkie, appear to be particular susceptible to MUO, although any dog can be affected. The disease often has rapid onset, over a period of several days.

There are many possible causes for the typical collection of clinical signs, so many other diseases must be ruled out, usually by MRI of the brain, analysis of cerebrospinal fluid that surrounds the brain and spinal cord, and analysis of blood to look for evidence of infection. Biopsy is the only confirmatory test but is not commonly done in dogs.

Once Dr. Jeffery is reasonably sure of MUO as the diagnosis, the treatment is with medications that suppress the immune system, such as corticosteroids (e.g., prednisone), cyclosporin, cytarabine, and many other agents. Most dogs respond well (75%), although there is a tendency for the disease to progress over months to years after the diagnosis. In some dogs, the disease progresses rapidly and some individuals may die (20-25%). Because it is known that MUO is associated with abnormal immune function, and that the bacteria in the gut (microbiome) can affect the immune system, Dr. Jeffery has been analyzing whether there is an imbalance in the microbiome of dogs with MUO, which might provide an opportunity to improve treatment of the disease. In human MS there has been an association that affected human have lower levels or a couple common bacteria in the gut microbiome. This study is currently recruiting dogs for microbiome transplants.

An Update on Genetic Evaluation of Labrador Tricuspid Valve Dysplasia and Boxer ARVC.
Kathryn Meurs, DVM, PhD, DACVIM

 Tricuspid valve dysplasia (TVD) is a congenital heart defect, observed most commonly, in the Labrador Retriever. Some dogs have only a mild form of the defect and can live comfortably, while others can have significant valvular abnormalities which can lead to the development of congestive heart failure. The defect has been shown to be an inheritable trait in Labs, and is suggested to be consistent with a single gene defect. Dr. Muers hypothesis that the genetic marker for the trait can be identified by performing whole genome sequencing on 10 affected Labs, and use this information to begin to develop a strategy to gradually reduce the prevalence of TVD.

Thus far a gene has been found associated with TVD (P=0.0001). This gene is associated with prostaglandin production. There are 3-4 prostaglandins known to be associated with development of normal heart structure. The current gene has been associated with 60% penetrance and a variety of expression of severity. Research is looking for another gene as a single genetic mutation cannot explain all the dogs affected.

Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is an inherited heart disease diagnosed most commonly in the Boxer dogs. The disease most commonly affects dogs 6 to 8 years of age by replacing normal heart muscle with fat. This prevents normal electrical function and is diagnosed by electrocardiogram (ECG).  The straitin gene had been previously identified but does not explain all affected cases. To identify a second causative variant, DNA samples from affected Boxer dogs that are negative for the striatin mutation are being studied. Dr. Meurs is proposing a whole genome sequencing approach to find the second cause.  Dr. Meurs previously used this approach to successfully identify a second causative mutation for dilated cardiomyopathy in the Doberman Pinscher. Ultimately this information can be used to improve the understanding of pathophysiology of ARVC, to help improve treatment modalities, and develop strategies to reduce the prevalence of the additional variant.

The Role of Echocardiographic Exam in Screening for Myxomatous Mitral Valve Disease in Cavalier King Charles Spaniels
Michele Borgarelli, DVM, PhD, DECVIM-CA

 Myxomatous mitral valve disease (MMVD) represents the most common cardiovascular disease in dogs and one of the leading causes of morbidity and mortality in this species. In Cavalier King Charles Spaniel (CKCS) the disease is considered to be influenced by genetic factors (polygenetic). More, in CKCS the prevalence of the disease is reported to be higher compared to similarly aged dogs of other breeds. Current screening recommendation of MMVD in CKCS include careful cardiac auscultation for identification of mid-systolic click and/or murmurs, however, recent data demonstrates that the echocardiograph represents a more sensitive diagnostic for identifying early onset MMVD.  Dr. Borgarelli has recently reported that 79% of CKCS older then 5 years demonstrate echo evidence of more than trivial mitral valve regurgitation, despite the fact that they did not have a murmur, suggesting that the echo exam should represent the recommend diagnostic screening. Using 3-dimensional echo technology, Dr. Borgarelli’s group has recently demonstrated that the morphology of the mitral valve in some CKCS is different from the mitral valve in other breeds. The mitral valve in dogs with MMVD is flatter and the mitral valve annulus is more rounded, suggesting that anatomy of the mitral valve plays an important role in the pathogenesis of the disease. They are currently enrolling apparently healthy CKCS in order to determine whether the morphology of the mitral valve, assessed with 3-dimensional echo, can be used to identify dogs with high risk of early onset of the disease. At this time the only breeder recommendation is to screen with echo (starting young) and only breed those dogs that develop disease late in life, which is less likely to produce early disease in their offspring.

Congenital Heart Disease in Dogs
Samantha Kovacs, BS DVM PhD Candidate
Mentor Joshua Stern, DVM PhD DACCIM

Two of the most common congenital heart defects in dog are Subaortic stenosis and Pulmonic stenosis. Multiple breeds are over-represented. Subaortic Stenosis (SAS) is a narrowing of heart near the aorta which creates increase work on the left ventricle, turbulence endothelial damage, increasing risk of endocarditis, aortic insufficiency and post stenosis dilation. Breeds most commonly associated are large working dogs. Including their past grants that looked at SAS in Goldens, Rotts, Bullmastiff, and Newfy. The inheritance is complex and varies depending on the breed. A known gene in Newfoundland dog does not explain all the cases but is dominant or codominant. In Bullmastiff, Golden Retriever, Rottie, it’s recessive genetics. The Bouvier whole genome sequencing has currently found promising gene to explain their dominant clinical disease, and in addition, GWAS is being conducted on many breeds with promising similar peak found on GWAS.

Pulmonic stenosis (PS) is due to a valvular leaf fusion producing right ventricle overload, most commonly found in terriers and brachiocephalic breeds (e.g., Bulldog, French Bulldog). It can cause syncope (fainting), sudden death, right congestive heart failure, arrhythmias, and cyanosis. The prognosis varies depending on the severity of the defect.  The treatment involves treating the underlying heart disease and arrhythmia, and some dogs are balloon valvuloplasty candidates. They are currently enrolling affected French Bulldogs. Current results in Bulldogs are still being studied by GWAS followed by whole genome sequencing.

Their current AKC CHF grant is investigating diet-associated dilated cardiomyopathy in dogs.

Chagas Cardiomyopathy in Dogs Infected with Trypanosoma cruzi
Ashley Saunders, DVM, DACVIM

 The protozoal parasite, Trypanosoma, is transmitted by triatomine insect vectors and causes myocarditis and cardiac morbidity including heart failure and sudden death in humans and dogs. As awareness of Chagas disease grows, testing for T. cruzi has increased particularly in the southern United States, and Central and South America, where the insect vectors are encountered. Risk factor for T. cruzi infection include living in or travel history in an area with the vector, kissing bugs. Infected dogs with travel history or translocation from the south have been diagnosed in northern areas of the United States.  Current recommendations regarding screening tests, monitoring, and prognosis in asymptomatic dogs are limited by lack of awareness and knowledge about the natural history of this disease in dogs. A better understanding of the course of disease, particularly the frequency of cardiac abnormalities is crucial to improve care for infected dogs and allow owners to make informed decisions.

T. cruzi affects 6-7 million people worldwide and endemic in many Latin America countries with 30% of infected humans suffering heart disease. Screening shelter dogs in Texas found 6-13% seropositive (similar the level of heartworm disease found in shelter dogs) but as many as 50% in some kennels of hunting dogs. Awareness and disease prevention are critical since the disease is difficult to diagnose, lifelong and difficult to effectively treat. The T. cruzi parasite encysts into multiple organs, especially the heart, initially creating a massive inflammatory response, conduction and muscle abnormalities of the heart, followed by chronic scar tissue. The treatment involves vector control, antiparasitic (Benznidazole, nifurtimox, amiodarone/itraconazole) and managing the heart arrythmias and heart disease.

The AKC CHF study involved 50 asymptomatic dogs with natural T. cruzi infection followed by lab work (including PCR, flow cytometry and IFA for T cruzi, heart Troponin levels) and ECG every 6-12 months over 2 years. Halter ECG monitors and echo were abnormal in 43/50 dogs including diastolic and systolic problem similar to those found in humans. Sixteen percent died or were euthanized, many sudden deaths. Current research continues and focusing on vector control (primarily needed May through September), preventing infection, treating to disease (and now to monitor successfully treatment), using dogs as a model for human disease, and expanding awareness (especially due to pet movement). New studies are now looking into prophylactics has enrolled 38 controls and 40 affected seropositive dogs with treatment with an undisclosed drug.

Many thanks to Collie Health Foundation for supporting my attendance to the virtual 2021 AKC CHF Parent Conference.

Nancy Kelso, D.V.M.