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

Discovery of public tumorspecific neoantigens (pTSNAs) and pTSNAreactive T cells arisen from hotspot mutations in canine hemangiosarcoma

Researchers will build on their discovery of “hotspot” mutations (tumor mutations shared between patients with the same cancer) to see if they can identify public tumor-specific neoantigens (pTSNAs) that are thought to play a role in immune response to cancer. Researchers hope findings will be a step toward a new immunotherapy that can be used in many different dogs suffering from hemangiosarcoma.

Co-sponsored with the Morris Animal Foundation, Grant Number: D24CA-536

RESEARCHERS

Shaying Zhao, PhD, University of Georgia
Amount: $10,000

Using a heritable immunostimulatory bacterium to block heartworm transmission by mosquitoes

Summary: Researchers will investigate the use of a bacteria to help block transmission of heartworm parasites by mosquitoes.

Description: More than 150,000 cases of canine heartworm disease are reported annually in the United States alone. Monthly preventive treatments for dogs have been widely available since the mid-19805. However, drug-resistant strains are on the rise, highlighting the need for novel disease control strategies. Researchers will investigate how infection by certain bacteria decreases the capacity of mosquitoes to transmit infectious larvae of Dirofilaria immitis, the agent of heartworm disease. Bacteria-infected mosquitoes (and control groups) will be exposed to both drug-sensitive and drug-resistant parasites. The team hopes findings will provide a new approach to block the spread of the heartworm disease by targeting parasites in their mosquito host.

Co-sponsored with the Morris Animal Foundation, Grant Number: DZ2CA·015

RESEARCHERS

Michael Povelones; University of Pennsylvania
Amount: $10,000

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

Understanding the Genetic Basis of Addison’s Disease in PWDs

Addison’s disease is a common, life-threatening disorder in dogs characterized by the immune-mediated destruction of portions of the adrenal gland. This damage prevents the adrenal gland from synthesizing hormones that are necessary for normal cell metabolism, kidney function, and maintenance of the immune system. Dogs with Addison’s disease are also highly predisposed to succumbing to a life-threatening adrenal crisis. Addison’s disease is most common in Portuguese Water Dogs (PWDs), which have a 29-fold greater risk of developing the disease compared to other dog breeds, indicating a strong genetic component. To date, no genetic variants have been associated with Addison’s disease in PWDs.

This lack of knowledge has prevented the development of a genetic test that would allow for prediction of a dog’s disease risk and the development of informed breeding practices related to Addison’s disease. In this study, we will use state-of-the-art scientific tools to understand the genetic basis of Addison’s disease in PWDs. The data generated here will provide the foundation for the development of a genetic test for Addison’s disease in PWDs, enabling early diagnosis and treatment, as well as maintenance of genetic diversity within the breed while helping to decrease disease incidence.

Research funded by the Portuguese Water Dog Foundation, Inc.

For additional details including eligibility criteria and how to enroll, please visit Dr. Friedenberg’s page for the Addison’s Disease – Genetics Study in Portuguese Water Dogs Study.

Related News

Announcement: Understanding the Genetics of Addison’s Disease in PWDs
FAQs Addison’s Disease Research in PWDs

Published Research

A case-control survey study of environmental risk factors for primary hypoadrenocorticism in dogs.
Authors: Amy E. Treeful, Kelly M. Searle, Dana M. Carroll, Kathleen J. Yost, Anna L. Hedger, Steven G. Friedenberg
Published: 13 October 2023, Journal of Veterinary Internal Medicine

Webinar

Dr. Friedenberg’s on March 28, 2023 on Addison’s disease and his investigation into the genetic component of the disease. This research made possible through your generous support of the Portuguese Water Dog Foundation.

RESEARCHERS

Steven Friedenberg, DVM, PhD, University of Minnesota
Leigh Anne Clark, PhD, Clemson University
Amount: $226,000
Administrative Amount: $2,300