Complete and Active AKC/CHF Grants

Grant 01894-A Documentation of disease-causing ticks

Final Report to Grant Sponsor from Investigator:

Many species of Ehrlichia exist, but the most detrimental of these affect canines. Unfortunately, Ehrlichia incidence is increasing in the southeastern United States. The primary approach to Ehrlichiosis prevention is through tick control, which includes application of acaricides to hosts and the habitat. We identified three Ehrlichia species in two species of ticks (A. americanum and I. scapularis) and two Anaplasma species in two tick species (A. americanum and I. scapularis). The most frequently collected ticks on canines, D. variabilis (American dog tick), did not have any Ehrlichia. We then screened all of the collected ticks for Rickettsia and did not identify any pathogenic Rickettsia within those ticks. There was no spatial clustering of Ehrlichia or Rickettsia positive ticks. The identification of pathogenic Ehrlichia and non-pathogenic Rickettsia within two different tick species warrants further investigations to understand the etiological agents and interactions of the vectors responsible for the high incidence of Ehrlichiosis and Rickettsiosis in the region. Such research will improve surveillance and diagnostic protocols, provide insights into improving management practices, and inform the public on how to protect their dogs from ticks and their associated pathogens.

Grant: 01708-A: Validation of a general method for enrichment of canine cancer stem cells

Principal Investigator: Dr. Timothy D. O'Brien, DVM PhD

Research Institution: University of Minnesota

Report to Grant Sponsor from Investigator:
Osteosarcoma, hemangiosarcoma, and glioblastoma multiforme are three types of incurable cancers that are responsible for reduced quality of life and significant mortality in dogs. The processes that control the clinical behavior of these tumors is not well understood, but recent research suggests that a specialized group of cells called "cancer stem cells" (or SCs) might play important roles. CSCs are important in the development, growth and spread of various malignant tumors of humans, so it would not be surprising if they also contributed to canine cancers. However, the definitive existence of CSCs in canine cancers remained to be proven. Defining these cells and understanding their basic biology will be necessary to develop cures for cancer, so we developed a unique, widely applicable method to enrich for cells that behave like CSCs from canine tumors. This method allowed us to make direct comparisons among CSCs from osteosarcoma, hemangiosarcoma, and glioblastoma to find unique or shared targets for prevention and therapy. Our objective was to show that the presumed CSCs are indeed uniquely capable of forming tumors using the gold standard assay of "xenotransplantation at limiting dilution". We confirmed the utility of our method and demonstrated the enrichment of CSC within our culture system. Additionally, we demonstrate that our system will enrich for CSC in tumors where they are infrequent, but provide minimal enrichment for those (aggressive) tumors where a CSC has already become enriched. This provides a new and intriguing possibility to use our culture system to evaluate tumors for the presence of CSC and potentially make therapeutic decisions based on the result. This work also helped provide critical support for further groundbreaking genetic analyses of CSCs that will help us reach the promise of targeted therapies to reduce morbidity and mortality and improve outcomes for dogs with these otherwise deadly cancers.

Grant: 01670-A: Pinpointing the causal mutation(s) underlying a genome-wide association signal for hereditary cataract in Northern breeds

Principal Investigator: Dr. Sally L Ricketts, PhD

Research Institution: Animal Health Trust  

Report to Grant Sponsor from Investigator:  
We have conducted a study to resequence a region of the genome on canine chromosome 18 that we previously found to be statistically associated with hereditary cataract (HC) in Northern, or Arctic, breeds in a genome-wide association study, in order to identify causative mutation(s) for HC in these breeds. We defined HC in these breeds as dogs with cataracts in both eyes located within the posterior polar subcapsular region of the lens. We have used molecular targeted sequencing technology to obtain data for approximately 3.8 million letters of DNA spanning the HC-associated region of the genome in ten Siberian Huskies (five HC cases and five control dogs with clear eyes over the age of 6 years). In this dataset we found over 35,000 variants among the ten dogs and the Boxer reference genome sequence. We devised a computer program to examine the segregation of each variant with HC using both a dominant and recessive genetic model and we found 200 variants that showed perfect segregation with HC in our ten dogs. We excluded variants where the potential 'risk' allele was the same as the Boxer reference genome sequence, as it is unlikely that these would represent causal mutations. This left us with 78 variants that we checked manually against online databases to assess which were within, or very close to, gene coding regions. This confirmed that ten variants were located in gene coding regions. Two of these coding variants were predicted to cause a change in the protein sequence of the gene and were found to be potentially damaging to protein function by analysis of online databases. Furthermore, the non-risk allele was highly conserved among many different animal species, which is additional evidence to suggest that it is important for normal function of the protein. We assessed these two variants in 200 Siberian Huskies, 200 Alaskan Malamutes and 130 Samoyeds and found that one of the variants showed strong statistical association with HC in two of the breeds. However, this variant is very common in both dogs with HC and in older dogs with clear eyes, and doesn't completely account for HC in our samples. It may be that this is a susceptibility variant and there is an environmental component to HC that affects how the genetic susceptibility is expressed, however, before we explore this possibility we are continuing with our analysis of the resequencing data to ensure we have completed a fully comprehensive investigation of the region.

1658: Urinary Protein and Gene Expression Characterization and Comparison with Renal Biopsy Findings and Clinical Data in Dogs with Proteinuric Renal Diseases

Grant Status: Open

Grant Amount: $80,000

Dr. Mary B Nabity, DVM, PhD, Texas A&M University

January 1, 2012 - December 31, 2014

Sponsor(s): Papillon Club of America, Soft Coated Wheaten Terrier Club of America, Inc.

Disease(s): Protein-Losing Nephropathy

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Abstract
Primary glomerular diseases are very common causes of chronic kidney disease (CKD), which is a significant source of illness and death in dogs, affecting up to 15% of elderly individuals. Early treatment generally prolongs the lives of dogs with CKD, but timely detection can be difficult and the outcome for each patient based on current, early non-invasive testing is unpredictable. While glomeruli are often the initiating site of kidney disease in dogs, collateral damage to the tubulointerstitium (TI) is important in determining disease severity and progression because of the extensive interplay between these 2 kidney compartments. Evaluations of urinary proteins have enhanced the early identification of TI and glomerular damage and demonstrated prognostic utility in people with CKD. In addition, particular urinary cells (called podocytes) that wash down from damaged glomeruli have been evaluated, with increased podocyte detection indicating greater severity of glomerular damage and thus poorer prognosis for glomerular disease in people. The purpose of this study is to evaluate promising indicators of kidney injury that might improve detection and/or assessment of progression or prognosis in dogs with CKD. We plan to use urine samples from dogs with various kidney diseases to measure: 1) urinary proteins indicating tubular and glomerular damage, and 2) gene expression profiles indicating podocyte loss. Results will be correlated with conventional measures of tubular and glomerular function, kidney biopsy findings, and information regarding disease outcome to determine the utility of the novel tests to non-invasively detect and accurately assess kidney damage in dogs.

Funded Grant 01502-A: Myxoma Virus Oncolytic Therapy in Mouse Models of Canine Cancer  

Amy McNeill DVM PhD  

Problem: Novel cancer treatments are needed to minimize patient discomfort caused by cancer therapy and eliminate failure of current treatment modalities. The use of oncolytic viral vectors is an exciting therapeutic option that deserves further study.  

This project is designed to determine if the poxvirus, myxoma virus (MYXV), can be used to eliminate cancer cells and prevent the spread of tumors throughout the body. Poxviruses have several characteristics that make them ideal cancer therapeutics: 1) they target neoplastic lesions where leaky vessels are formed; 2) they are easily engineered to express antitumor agents; 3) they elicit a strong immune response which can be used to target abnormal cells; and 4) they are effectively cleared from the body by antibodies, preventing latent or recurrent infection.  

MYXV is a poxvirus that does not cause disease in any animal except the rabbit. However, MYXV does grow in some cancer cells. Since MYXV only grows in abnormal cells, virus replication in healthy tissue should not occur. The lab has tested several canine tumor cell cultures and many support growth of MXYV. We also created a genetically altered MYXV that no longer causes disease in rabbits, but causes increased numbers of canine cancer cells to die as compared to wild-type MYXV infection.  

This project aims to evaluate the anticancer effects of MYXV in mouse models of canine cancer. If the tumors can be reduced in size or eliminated, these studies may lead to the use of genetically modified poxviruses as cancer treatments in companion animals.

Grant 01480: Leptospirosis an emerging concern

Final Report to Grant Sponsor from Investigator:

Leptospirosis is a re-emerging infectious disease of dogs, people, and wildlife. Infected dogs may develop acute renal failure but also multiple-organ failure and even death. This is one of the most commonly diagnosed infectious diseases in dogs at UC Davis. We now have vital information about hot spots for risk of canine leptospirosis as well as epidemiological information including breed and vaccination status for affected dogs, exposure routes, and serovars. Importantly, we demonstrate that small dog breeds are indeed vulnerable to leptospirosis and thus vaccine protocols need to include them. We have ongoing surveillance in these hot spots to detect cases in order to obtain bacterial isolates and critical data on patterns of leptospiral infection in blood and urine. One promising aspect of ongoing research is the interaction of canine and wildlife leptospirosis, and whether some wild animals that may enter yards and suburban landscape could be a source of infection to dogs. Our data to date implicate squirrels and skunks, although other wildlife species or domestic farm animal species may be important sources to dogs even in their home yards. Further molecular characterization of infection will help us understand this linkage. The most important jobs in our to-do for Leptospira research at present are to use molecular techniques to understand dog-wildlife risk and to continue surveillance and use molecular approaches to determine the pathophysiology of infection by west-coast strains of Leptospira in dogs.

GRANT PROGRESS REPORT REVIEW  

Grant 00972: Identification of Mutations Associated with Hereditary Cataracts in Northern Breeds

Principal Investigator: Dr. Cathryn Mellersh, Ph.D.

Research Institution: Animal Health Trust


Report to Grant Sponsor from Investigator:
Our study aimed to investigate the genetic cause of hereditary cataract (HC) in Northern breeds. We have collected samples from 228 dogs affected with bilateral cataract (both eyes) and 300 dogs with a clear eye examination from 15 Northern breeds, initially screening known human cataract-causing genes in 66 dogs from two of these breeds. We used a panel of 39 markers near to 20 genes involved in human inherited cataract and found no evidence for association of any markers with HC in the two breeds. Using genome-wide association (GWA) study (whole genome scanning) approaches we conducted an initial analysis of HC in the Finnish Lapphund, Icelandic Sheepdog, Samoyed and Siberian Husky. For the Samoyed we found evidence for an association with HC on chromosome 7, but no associations were identified in the other three breeds. These initial results suggested that HC in Northern breeds is not caused by a single gene mutation and may have a more complex genetic background. This finding is not entirely surprising when it is considered that to date only a single gene, HSF4, has been implicated in the development of HC in the dog despite the fact that close to 100 breeds are known to be affected by the condition. To detect mutations in numerous genes requires analysis of a larger number of samples using a rigorous definition for affected dogs (‘cases’) and unaffected dogs (‘controls’). Therefore for our final investigation we concentrated our efforts on four breeds that display a clinically similar form of HC and for which we have sufficient numbers of cases and controls for analysis. We have conducted a GWA scan of HC in these four breeds—the Alaskan Malamute, Siberian Husky, Karelian Bear Dog and Samoyed—using an updated high-density genome scanning array comprising around 174,000 markers spanning the DNA. Our initial analyses of these data are promising and we have found evidence of HC-associated regions for at least three of the breeds. We are currently investigating these association signals and we aim to design and conduct further studies to attempt to pinpoint the causal mutations.

Grant no: 386
Report rec'd 10/3/2008 (due 6/30/08)  
Title of Grant: Elucidation of the Genetic Defect in Familial Renal Disease in the Norwegian Elkhound
Principal Investigator: Peter Leegwater
Research Institution: University of Utrecht
Start Date : 7/1/2005

Performance summary:
Association mapping identified 10 MB genomic region containing 153 genes. The PI has not been able to fine map this region down. Until they can be get more samples and narrow this region down, a mutation is not able to be detected in such a large region.

Only one of the objectives was able to be completed because the other two relied on the success of the first.

The PI plans to continue the research when more samples and employee resources are available.

Completed Grant No: 1663 — Identifying the Genetic Casue of Canine Progressive Rod-cone Dysplasia (PRCD Type of PRA)

Researcher: George J. Brewer, MD – University of Michigan (University)

Researcher: George J. Brewer, MD – University of Michigan (University) Abstract: Using both cloning and DNA sequencing, researchers ruled out five genes (of an identified 31 candidate genes) as the causative gene for Progressive Rod-Cone Disease (PRCD), a form of Progressive Retinal Atrophy, an inherited disease that leads to blindness. With continued research, the cloning and sequencing of the other 26 candidate genes can be completed. Once the genetic cause and mutation are found, direct DNA tests, which are more accurate than linkage tests, can be developed to help breeders make breeding decisions in order to reduce or eliminate this disease. All 31 candidate genes were known to be in the region of the chromosome linked to PRCD by researchers at Cornell.

Completed Grant No: 2012 — Development of PCR Multiplexed Canine Marker Panels for the Purposes of Genome Screening and Linkage Analysis

Researcher: Marcia Eggleston, PhD – University of California, Davis (University)

Abstract: A number of tests have been developed to screen for affected or carrier animals with genetic diseases. The development of many of these tests was possible due to the availability of information regarding similar disorders in humans. The majority of disorders affecting purebred dogs have no known counterpart in humans and therefore no candidate gene. However, an indirect test that screens for a strong association of a microsatellite DNA marker with a disease phenotype can be incorporated in a simple diagnostic test for carriers of that trait. The ultimate goal of the research is to design marker panels containing a minimum of 300 microsatellite markers covering all the linkage groups or chromosomes of the canine genome. The Veterinary Genetics Laboratory has completed panels containing 100 markers with known map locations. This project will incorporate at least 200 additional markers that will then represent the entire canine genome and allow effective screening for linkage or markers to disease gene(s).

Closed Grant No: 2434 — Recombinant Thyrotropin (TSH): Standard for the Next Generation of Canine TSH Immunoassays with Improved Sensitivity

Researcher: Duncan Ferguson, DVM, PhD – University of Georgia (University)

Abstract: Hypothyroidism, a failure of the thyroid gland, is the most common hormonal abnormality in dogs, causing a variety of medical problems in many breeds, including hair loss and skin infections. The measurement of serum levels of the pituitary hormone thyrotropin (TSH) has been used as a reliable and sensitive screening test for ghyroid glandular insufficiency in human medicine for many years, but the first generation assays for canine TSH (cTSH) are missing as many as one out of four cases by hypothyroidism, resulting in no improvement in diagnostic sensitivity compared to total T4 measurement. Furthermore, the available assays have not been sensitive enough to distinguish low values of cTSH from those in the normal range. Towards the goal of improving current and future immunoassay sensitivity based upon a pure recombinant canine TSH(cTSH) hormone standard, our laboratory has succeeded in cloning and sequencing the two peptide subunits of canine TSH and have expressed them in small quantities. Using techniques recently developed in our parallel work on equine TSH, we plan to express and purify recombinant canine TSH in high quantities and validate its use as a pure immunoassay standard to facilitate its worldwide use.

Completed Grant No: 2616 — Molecular Analysis of Contributory Factors of Osteoarthritis in Canine Hip Dysplasia

Researcher: Alpana Ray, PHD – University of Missouri, Columbia (University)

Abstract: Hip Dysplasia is a common disease of dogs that ultimately leads to osteoarthritis (OA), a serious debilitating condition, which at present, is treated by symptomatic management of pain. Accidental injuries also lead to the development of OA. Cartilage degeneration is fundamental to the pathogenesis of OA. We propose to study the transcriptional control of MMP-1, a major enzyme involved in the degradation of articular cartilage. Expression of MMP-1 gene and the corresponding protein is markedly increased under osteoarthritic condition. Because cytokines like IL-1 and TNF increase expression of MMP-1 and biomechanical factors also influence its expression in osteoarthritic, unstable joints, the objectives are to understand what components of the promoter region of canine MMP-1 gene are influenced by these factors. At present no data is available on canine MMP-1 gene regulation. This proposal is aimed towards understanding the regulation of canine MMP-1 gene expression in response to biomechanical stress and cytokines by isolating canine MMP-1 gene, identifying the regulatory elements in the promoter responsive to biomechanical stress and cytokines, and analyzing MMP-1 expression in chondrocytes of articular cartilage form normal and osteoarthritic dogs with the intent to develop novel therapeutic drugs to combat this disease.