Evaluation of a fibrillin 2 gene haplotype associated with hip dysplasia and incipient osteoarthritis in dogs.

OBJECTIVE: To determine whether a mutation in the fibrillin 2 gene (FBN2) is associated with canine hip dysplasia (CHD) and osteoarthritis in dogs. ANIMALS: 1,551 dogs. Procedures-Hip conformation was measured radiographically. The FBN2 was sequenced from genomic DNA of 21 Labrador Retrievers and 2 Greyhounds, and a haplotype in intron 30 of FBN2 was sequenced in 90 additional Labrador Retrievers and 143 dogs of 6 other breeds. Steady-state values of FBN2 mRNA and control genes were measured in hip joint tissues of fourteen 8-month-old Labrador Retriever-Greyhound crossbreeds. RESULTS: The Labrador Retrievers homozygous for a 10-bp deletion haplotype in intron 30 of FBN2 had significantly worse CHD as measured via higher distraction index and extended-hip joint radiograph score and a lower Norberg angle and dorsolateral subluxation score. Among 143 dogs of 6 other breeds, those homozygous for the same deletion haplotype also had significantly worse radiographic CHD. Among the 14 crossbred dogs, as the dorsolateral subluxation score decreased, the capsular FBN2 mRNA increased significantly. Those dogs with incipient hip joint osteoarthritis had significantly increased capsular FBN2 mRNA, compared with those dogs without osteoarthritis. Dogs homozygous for the FBN2 deletion haplotype had significantly less FBN2 mRNA in their femoral head articular cartilage. CONCLUSIONS AND CLINICAL RELEVANCE: The FBN2 deletion haplotype was associated with CHD. Capsular gene expression of FBN2 was confounded by incipient secondary osteoarthritis in dysplastic hip joints. Genes influencing complex traits in dogs can be identified by genome-wide screening, fine mapping, and candidate gene screening.

Evaluation of quantitative trait loci for hip dysplasia in Labrador Retrievers.

OBJECTIVE: To identify the quantitative trait loci (QTL) that contribute to hip dysplasia in dogs. ANIMALS: 192 Labrador Retrievers. PROCEDURES: Hip dysplasia was measured by use of the Norberg angle (NA), dorsolateral subluxation (DLS) score, and distraction index (DI). Genome-wide screening was conducted by use of 276 unique microsatellites. Linkage analysis was performed with a variance-based linear model. Logarithm of the odds (LOD) scores were reported when values were > 2.0. RESULTS: Canis familiaris autosomes (CFAs) 01, 02, 10, 20, 22, and 32 harbored significant QTL at LOD scores > 2.0. Among the 6 QTL, the QTL on CFA02 had not been reported to harbor QTL for hip dysplasia. The highest LOD score of 3.32 on CFA20 contributed to the second principal component of the DLS score and NA of the right hip joint. The QTL that was mapped on CFA01 (LOD score of 3.13 at 55 centimorgans) was located on the same chromosome reported to harbor a QTL for hip dysplasia in Portuguese Water Dogs and German Shepherd Dogs. In this study, CFAs 10, 20, 22, and 32 harbored QTL for hip dysplasia that have been identified in a Labrador Retriever-Greyhound pedigree and in German Shepherd Dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple QTL were clearly involved with hip dysplasia. Identification of these QTL will enable fine-resolution mapping and subsequent assessment of candidate genes within the refined intervals to enable researchers to develop genetic screening tests and preventative and novel therapeutic regimens.

The long (and winding) road to gene discovery for canine hip dysplasia.

Hip dysplasia is a common inherited trait of dogs that results in secondary osteoarthritis. In this article the methods used to uncover the mutations contributing to this condition are reviewed, beginning with hip phenotyping. Coarse, genome-wide, microsatellite-based screens of pedigrees of greyhounds and dysplastic Labrador retrievers were used to identify linked quantitative trait loci (QTL). Fine-mapping across two chromosomes (CFA11 and 29) was employed using single nucleotide polymorphism (SNP) genotyping. Power analyses and preferential selection of dogs for ongoing SNP-based genotyping is described with the aim of refining the QTL intervals to 1-2 megabases on these and several additional chromosomes prior to candidate gene screening. The review considers how a mutation or a genetic marker such as a SNP or haplotype of SNPs might be combined with pedigree and phenotype information to create a 'breeding value' that could improve the accuracy of predicting a dog's hip conformation.

In vitro analysis of nonthermal plasma as a disinfecting agent.

OBJECTIVE: To determine the effect of nonthermal plasma on Staphylococcus aureus, fibroblasts in monolayer culture, and clean and contaminated skin explants. SAMPLE POPULATION: Normal skin from euthanized horses. PROCEDURES: S aureus organisms were plated and treated with nonthermal plasma followed by bacterial culture to assess viability. Fibroblasts in monolayer culture and the epidermal and dermal surfaces of clean and S aureus-contaminated skin explants were treated. The effects of distance and duration on the response to treatment were compared. RESULTS: Compared with controls, treatment with nonthermal plasma resulted in significantly decreased bacterial growth and significantly inhibited survival of fibroblasts in monolayer culture. When epidermal and dermal surfaces of skin explants were treated, there was no effect on production of normal fibroblasts during explant culture, except when extended exposure times of >or= 2 minutes were used. Treatment with nonthermal plasma resulted in significantly lower bacterial counts after 24 hours of culture of S aureus-contaminated epidermis but not of dermis. CONCLUSIONS AND CLINICAL RELEVANCE: Nonthermal plasma resulted in bacterial decontamination of agar and epithelium; negative effects on fibroblasts in monolayer; and no negative effects on skin explants, except at long exposure times. Use of nonthermal plasma appears safe for treatment of epithelialized surfaces, may be safe for granulating wounds, and results in decontamination of S aureus. Investigations on the effects that nonthermal plasma may have on patient tissues are indicated with a clinically applicable delivery device.

Quantitative trait loci for hip dysplasia in a cross-breed canine pedigree.

Canine hip dysplasia is a common developmental inherited trait characterized by hip laxity, subluxation or incongruity of the femoral head and acetabulum in affected hips. The inheritance pattern is complex and the mutations contributing to trait expression are unknown. In the study reported here, 240 microsatellite markers distributed in 38 autosomes and the X chromosome were genotyped on 152 dogs from three generations of a crossbred pedigree based on trait-free Greyhound and dysplastic Labrador Retriever founders. Interval mapping was undertaken to map the QTL underlying the quantitative dysplastic traits of maximum passive hip laxity (the distraction index), the dorsolateral subluxation score, and the Norberg angle. Permutation testing was used to derive the chromosome-wide level of significance at p<0.05 for each QTL. Chromosomes 4, 9, 10, 11 (p<0.01), 16, 20, 22, 25, 29 (p<0.01), 30, 35, and 37 harbor putative QTL for one or more traits. Successful detection of QTL was due to the cross-breed pedigree, multiple-trait measurements, control of environmental background, and marked advancement in canine mapping tools.

The potential and limitations of cartilage-specific (V+C)(-) fibronectin and cartilage oligomeric matrix protein as osteoarthritis biomarkers in canine synovial fluid.

OBJECTIVE: To determine if levels of the cartilage-specific (V+C)(-) fibronectin isoform in the synovial fluid is associated with cartilage change during osteoarthritis. DESIGN: Synovial fluid was collected from 26 healthy dogs presenting to the Orthopedic Surgery Clinic with unilateral cranial cruciate rupture, 22 control dogs, and 13 dogs from a colony maintained for the study of canine hip dysplasia. Total fibronectin, (V+C)(-) fibronectin, and cartilage oligomeric matrix protein (COMP) were quantitated by ELISA assays. Statistical analysis used Wilcoxon's signed-rank and rank-sum tests and Spearman's rank correlation. RESULTS: The concentration of total fibronectin was increased in affected (P<0.0001) and contralateral (P=0.0005) knees of the clinic population (compared to unaffected knees in colony controls). Both (V+C)(-) fibronectin and COMP concentrations were elevated in the contralateral knees in clinical patients relative to unaffected knees in the colony controls (P=0.03 and P=0.04, respectively), and relative to the affected knees (P=0.003); however, corrections for joint effusions suggest elevated totals in the affected knees. (V+C)(-) fibronectin and COMP concentrations were correlated (r(sp)=0.74; P<0.0001) in 30 unaffected knees of patients and colony controls. Total fibronectin was correlated negatively with months since the initial injury (r(sp)=-0.44; P=0.03) in the affected joints. The intraoperative lesion severity score did not correlate with total fibronectin or (V+C)(-) fibronectin (P>or=0.35). CONCLUSIONS: Concentration of total fibronectin in synovial fluid might be a useful biomarker for cross-sectional studies in osteoarthritis, but only (V+C)(-) fibronectin provides information specifically about cartilage damage. Elevated concentrations of (V+C)(-) fibronectin and COMP seen in the contralateral knees of patients with cranial cruciate rupture might indicate cartilage changes early in the disease process (pre-clinical). However, the wide range of values obtained limits the diagnostic value for any one individual. Joint effusions obscure the total amount of biomarkers in affected synovial joints.

Evaluation of multiple radiographic predictors of cartilage lesions in the hip joints of eight-month-old dogs.

OBJECTIVE: To determine the radiographic methods that best predict the development of osteoarthritis in the hip joints of a cohort of dogs with hip dysplasia and unaffected dogs. ANIMALS: 205 Labrador Retrievers, Greyhounds, and Labrador Retriever-Greyhound crossbred dogs. PROCEDURE: Pelvic radiography was performed when the dogs were 8 months old. Ventrodorsal extended-hip, distraction, and dorsolateral subluxation (DLS) radiographs were obtained. An Orthopedic Foundation for Animals-like hip score, distraction index, dorsolateral subluxation score, and Norberg angle were derived from examination of radiographs. Osteoarthritis was diagnosed at the time of necropsy in dogs > or = 8 months of age on the basis of detection of articular cartilage lesions. Multiple logistic regression was used to determine the radiographic technique or techniques that best predicted development of osteoarthritis. RESULTS: A combination of 2 radiographic methods was better than any single method in predicting a cartilage lesion or a normal joint, but adding a third radiographic method did not improve that prediction. A combination of the DLS score and Norberg angle best predicted osteoarthritis of the hip joint or an unaffected hip joint. All models that excluded the DLS score were inferior to those that included it. CONCLUSIONS AND CLINICAL RELEVANCE: A combination of the DLS score and Norberg angle was the best predictor of radiographic measures in 8-month-old dogs to determine whether a dog would have normal or osteoarthritic hip joints.