Targeted Next Generation Sequencing Panel for Comprehensive Testing for Reproductive and Neurologic Pathogens of Dogs

The large number of pathogens that contribute to infectious reproductive or neurologic disease and overlap of clinical signs makes using tests for multi-pathogen detection useful. Routinely, several individual or multiplex PCR assays are performed to identify active infection with these agents.

Determining which tests to use for any given case can be difficult. Therefore, investigators propose to validate a comprehensive targeted next generation sequencing (NGS) panel for detection of all known neurologic and reproductive pathogens that infect dogs, as well as other infectious pathogens of dogs, including vector-borne agents previously validated through our work funded by AKC CHF. This takes the guesswork out of proper test selection for infectious disease diagnostics.

The amount of data that can be generated with NGS allows for comprehensive testing, and use of a targeted method (adding primers to the protocol for amplification of targets of interest) reduces the cost to the point that this testing can be effectively used in a veterinary diagnostic lab. More and more veterinary laboratories are purchasing sequencing equipment, so this method has merit to be expanded to additional labs. With the comprehensive nature of the panel, epidemiological evaluation of infectious diseases in the dog population can also be improved.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 03137

RESEARCHERS

Rebecca Wilkes, DVM, PhD; Purdue University
Amount: $3,000

Evaluation of the Effects of Diluted Sodium Hypochlorite on Skin Microbiome in Dogs with Superficial Pyoderma and Atopic Dermatitis: a Single Blinded, Randomized, Controlled Clinical Trial

In canine allergies, recurrent skin infection is frequent, often requiring repeated antibiotic use. This leads to a financial burden on owners and higher risk for antibiotic resistance. Recent studies in people have shown that diluted bleach baths can help patients with allergies and skin infections. In dogs, bleach has shown antimicrobial and anti-inflammatory effects in vitro. However, no studies have been done to evaluate the effects of bleach in client-owned dogs skin infection secondary to allergies. The researchers hypothesize that compared with chlorhexidine spray, another commonly used topical antimicrobial, diluted bleach will be as effective to treat skin infections, but will also help with the allergic disease. The aim of this study is to evaluate if diluted bleach is a viable alternative to chlorhexidine and its ability to restore normal skin microflora in canine allergies. Results from this study may significantly change the way clinicians treat superficial skin infections and potentially reduce antibiotic usage. This will be the first study to demonstrate the benefits of diluted bleach in canine allergies.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 03166-A

RESEARCHERS

Domenico Santoro, DVM, MS, DrSc, PhD, University of Florida – Board of Trustees
Amount: $2,000

Canine Semen Microbiome

Insufficient male reproductive performance and subfertility are important problems for dog breeders, but the causes underlying acquired infertility cannot be identified in about 50% of dogs. In humans, recent studies have demonstrated an association between semen quality and bacterial communities in the ejaculate, but further research is needed to assess a causal relationship between semen microbiome and semen quality.

Dog semen is not sterile and contains up to 10^5 bacteria/mL. Although previous studies showed no influence of some bacterial species such as Escherichia coli, Staphylococcus aureus, Klebsiella spp., Mycoplasma canis, Pseudomonas aeruginosa, and Streptococcus spp. on conception rates in dogs, it has been suggested that a higher overall concentration of bacteria and the presence of potentially pathogenic microorganisms might be associated with lower semen quality. Furthermore, antimicrobials are often misused in canine reproduction to address infertility problems. Previous research on dog semen microbiome is limited and relied only on culture, potentially missing some pathogens. Next generation sequencing (NGS) allows to describe novel microbiomes without prior knowledge of sequencing information or specific culture requirements. These techniques have been recently used to investigate the seminal microbiome of humans and domestic animals.

The research team’s aim is to perform a comprehensive investigation on the seminal microbiome of male dogs, as such identifying possible links between the seminal bacterial population and different semen parameters, opening a new chapter in canine andrology.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 03165-A

RESEARCHERS

Penelope Banchi, DVM; Ghent University
Amount: $2,000

Characterization of hookworm resistance in dogs with a novel diagnostic test for early intervention

The dog hookworm is the most common worm parasite of dogs in the United States. It can cause disease, resulting in suffering and even death in both young and old dogs of all breeds. Dog hookworms can also infect humans who come into contact with hookworm larvae present in dog stool contaminated areas. Hookworm infections in people will cause cutaneous larval migrans, a painful skin condition. Dogs must be dewormed monthly to prevent the ill effects of acquiring hookworms. While these medications were very effective 20-30 years ago, recently they have stopped being fully effective in many cases. This is because hookworms have become resistant.

To know if a dog has resistant hookworms, veterinarians have to use tests performed at a diagnostic lab. The test that is currently used takes 14 days to complete. In this project, we will create a new diagnostic test that can be performed in less than a day. This new test will let the veterinarian know if the medication that they are using against the hookworms will work or not. This project will add to our knowledge about how much resistance we should expect to see in hookworms from dogs that live in the U.S. Knowing how much resistance to expect in a geographical area and knowing how many hookworms within a dog patient are specifically resistant will help veterinarians make important treatment decisions quickly.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 03169

RESEARCHERS

Jeba Jesudoss Chelladurai, BVSc, PhD
Kansas State University
Amount: $3,000

Evaluating Reproductive Diseases in vitro with a 3D Canine Endometrial Organoid Model

Pyometra is a life-threatening disease that affects over 66% of intact older bitches. Bitches are pre-disposed to this disease due to a number of unique features of their reproductive cycle. Endometritis is an underdiagnosed condition in bitches that results in reduced fertility and can lead to the development of pyometra.

These diseases have mostly been studied in live dogs resulting in welfare concerns and limitations in our understanding of the disease onset and progression and importantly, prevention of these conditions. Organoids are miniature organs in a culture dish that can be grown long-term while maintaining the characteristics and function of the original organ.

This unique 3-dimensional (3D) structure facilitates the study of disease processes such as endometritis and pyometra in canines, potential treatments, and assessment of preventative therapeutics such as novel vaccines. Optimization of canine uterine organoids has the potential to: a) improve the health and welfare of intact middle-aged and older female dogs; b) enhance fertility, genetic gain and health in working dog programs; and c) allow the study of female reproductive diseases without the use of research dogs.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 03055

RESEARCHERS

Fiona Hollinshead, BVSc, PhD; Colorado State University
Amount: $1,000

Artificial Intelligence and Machine Learning for Diagnosis of Dog Sperm Morphology

Sperm morphology evaluation is an important component of dog fertility analysis. However, visual evaluation of sperm morphology is difficult to teach, and results are largely dependent on the proficiency and experience of the evaluator, leading to large variability in results within and across evaluators.

The objective of this pilot study is to explore recent advances in artificial intelligence for image pattern recognition, similar to that used by the likes of Google and Facebook, to create an automated method for sperm morphology evaluation. A large database with 10,000 dog sperm images will be created. The images will be evaluated by expert veterinarians, then used to train a Convoluted Neural Network (CNN) using deep learning methods. Investigators expect to generate an algorithm capable of classifying sperm morphology with greater than 90% accuracy and precision.

Employing such an algorithm could allow veterinarians to establish more reliable reference values and guidelines for prospective stud dogs and for semen to be used for artificial insemination, improve veterinarian’s abilities to formulate diagnoses and prognoses for infertility problems related to specific sperm defects, and to establish the reproductive safety of drugs and compounds.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 03045-A

RESEARCHERS

Leonardo Brito, DVM, PhD; University of Pennsylvania
Amount: $1,000

Genome-wide Association Study to Investigate Genetic Markers for Antimicrobial Resistance in Mycoplasma cynos Associated with Canine Infectious Respiratory Disease

Mycoplasma cynos is an emerging respiratory bacterium in dogs. It is associated with kennel cough and may lead to fatal pneumonia. This bacterium is very complex, and the lack of standardized diagnostic and knowledge on antibiotic therapy makes clinical management difficult. Since no vaccine is available, current treatment of M. cynos-associated outbreaks relies on antibiotics. However, the in vitro activity of antibiotics against M. cynos has never been studied and the effectiveness of antibiotic therapy for M. cynos disease is completely unknown. This may lead to unsuccessful treatment and antibiotic resistance.

In vitro testing of antibiotics is an urgent need to provide appropriate treatment, and to avoid inappropriate use of antibiotics. Unfortunately, these tests are very time-consuming for mycoplasmas, partially explaining the absence of information on M. cynos.

In this project, researchers will investigate the in vitro activity of antimicrobials against M. cynos isolated from dogs with respiratory disease, and implement the first genome-wide association study to identify genetic mutations linked to antibiotic resistance in M. cynos, which has never been investigated before. This will help develop genetic-based diagnostic assays for the rapid detection of resistant Mycoplasma isolates to better allow the results to inform clinical therapy. Results from this project will direct effective diagnosis and therapeutic interventions for an emerging respiratory disease in dogs.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 03053-A

RESEARCHERS

Grazieli Maboni, DVM, MSc, PhD; University of Guelph
Amount: $1,000

Luteinizing Hormone Receptor Activation Induces Migration and Adhesion in Neoplastic Canine Lymphocytes

Luteinizing hormone (LH) is secreted from the brain (pituitary) in sexually intact dogs to stimulate synthesis of estrogen and testosterone in females and males, respectively. However, LH is secreted at concentrations up to 20 times higher following gonad removal with spaying or castration because hormonal negative feedback is lost. Although LH is considered to be a reproductive hormone, there are dozens of non-reproductive tissues in dogs that contain receptors for LH including immune system cells, specifically lymphocytes.

Lymphoma is a common malignant cancer of dogs involving lymphocytes, and spayed/castrated dogs are reportedly 3-4 times more likely to develop lymphoma. Conventional chemotherapy results in remission in approximately 60-90% of cases with a median survival time of 6-12 months. Preliminary work has identified LH receptors in canine lymphoma tissue and demonstrated LH- receptor-induced proliferation of neoplastic lymphocytes in vitro.

This study aims to determine if LH receptor activation induces adhesion and migration of neoplastic lymphocytes in vitro. Characterizing the role of LH receptor in neoplastic lymphocyte proliferation may help guide future lymphoma treatment options.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 02751-A

RESEARCHERS

Michelle Kutzler, DVM, PhD; Oregon State University
Amount: $3,000

Identifying Early Stage Ultra-rare Mutations as Predictive Biomarkers of Lymphoma in High-risk versus Low-risk Breeds Within the Dog Aging Project

The most common type of cancer in dogs is lymphoma, with ~80,000 cases diagnosed annually in the United States. Breeds vary in their risk of lymphoma, but it is unclear why there is variation despite considerable effort to identify the genetics of cancer risk and progression in dogs. Cancer typically arises from the accumulation of non-inherited (i.e. somatic) mutations. However, variation among breeds in cancer risk could be due to breed-specific variation in the types of mutations, the rate of accumulation of mutations, or the downstream effects of mutations in healthy dogs.

This study will use novel sequencing technology to test the hypothesis that breed-specific lymphoma risk is due to variation in the frequency and type of rare precancerous mutations. Normally, measuring these low-frequency mutations has been beyond the range of standard sequencing technology, which is limited to detecting mutations present in >1% of cells. The new technology applied here represents a >10,000-fold improvement in accuracy, enabling the investigators to accurately detect a precancerous mutation present at a single site at a frequency of just one out of every 10 million DNA base pairs. By determining if mutation frequency in blood of healthy high-risk and low-risk dogs can predict lymphoma risk, this work could lead to the development of novel tests for the early diagnosis and prognosis of canine lymphoma.

This work has the potential to shed light on the mechanisms that underlie breed-specific variation in lymphoma risk, and in the long term, could lead to the development of novel tests for the early diagnosis and prognosis of canine lymphoma.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 2772

RESEARCHERS

Daniel Promislow, PhD; University of Washington
Amount: $10,000

Reprogramming the Tumor Immune Niche in Canine Hemangiosarcoma

Hemangiosarcoma (HSA) is a common, devastating disease of dogs. The malignant tumor is seen frequently in older Golden Retrievers, German Shepherd Dogs, Portuguese Water Dogs, Labrador Retrievers, and Schnauzers, but it can occur in any dog of any breed at any age. Survival times of dogs with the tumor are short, even with surgical removal and standard of care treatment. Inflammation within the tumor tissue is common in canine HSA, and the immune response may contribute to tumor heterogeneity and prognosis for the dog. Yet, the immunological features in the context of the HSA niche are virtually unknown. The investigators have found that HSA cells have a strong capacity to promote proliferation and differentiation of hematopoietic stem and progenitor cells, with increased inflammatory cytokines, suggesting a niche regulatory function of HSA cells.

This study will focus on understanding the functional relationships between HSA cells and immune cells that contribute to the tumor niche to identify molecular mechanisms that regulate critical signaling pathways in canine HSA. This approach will improve our understanding of the tumor immunity and heterogeneity, as well as aid in patient selection for novel immunotherapies.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 2759

RESEARCHERS

Jong Hyuk Kim, DVM, PhD; University of Minnesota
Amount: $5,000