The Ehlers–Danlos syndromes (EDS) are a heterogeneous group of heritable disorders affecting connective tissues. The mutations causing the various forms of EDS in humans are well characterized, but the genetic mutations causing EDS-like clinical pathology in dogs are not known, thus hampering accurate clinical diagnosis. Clinical analysis of two independent cases of skin hyperextensibility and fragility, one with pronounced joint hypermobility was suggestive of EDS. Whole-genome sequencing revealed de novo mutations of COL5A1 in both cases, confirming the diagnosis of the classical form of EDS. The heterozygous COL5A1 p.Gly1013ValfsTer260 mutation characterized in case 1 introduced a premature termination codon and would be expected to result in α1(V) mRNA nonsense-mediated mRNA decay and collagen V haploinsufficiency. While mRNA was not available from this dog, ultrastructural analysis of the dermis demonstrated variability in collagen fibril diameter and the presence of collagen aggregates, termed ‘collagen cauliflowers’, consistent with COL5A1 mutations underlying classical EDS. In the second case, DNA sequencing demonstrated a p.Gly1571Arg missense variant in the COL5A1 gene. While samples were not available for further analysis, such a glycine substitution would be expected to destabilize the strict molecular structure of the collagen V triple helix and thus affect protein stability and/or integration of the mutant collagen into the collagen V/collagen I heterotypic dermal fibrils. This is the first report of genetic variants in the COL5A1 gene causing the clinical presentation of EDS in dogs. These data provided further evidence of the important role of collagen V in dermal collagen fibrillogenesis. Importantly, from the clinical perspective, we showed the utility of DNA sequencing, combined with the established clinical criteria, in the accurate diagnosis of EDS in dogs.

Background -Anecdotally, amikacin has been added to compounded topical preparations for the management of canine bacterial otitis externa. However, the stability of amikacin within these solutions is unknown.Hypothesis/Objectives -The purpose of this study was to determine the stability of amikacin at 10 and 30 mg/ mL concentrations in four topical solutions over a 56 day period. We hypothesised that amikacin would maintain chemical stability within the various solutions.Methods and materials -Amikacin was formulated to 10 and 30 mg/mL (1% and 3%) concentrations within four topical solutions: tris-EDTA (TrizEDTA Aqueous Flush) (TE); 0.15% chlorhexidine gluconate and tris-EDTA (TrizCHLOR Flush) (TC); 0.9% NaCl (NA); and 0.9% NaCl + 2 mg/mL dexamethasone (ND). Samples were made in duplicate and stored at room temperature (25°C) for 0, 7,14, 21, 28 and 56 days. Amikacin content was quantified, in triplicate, by ultrahigh-performance liquid chromatography tandem mass spectrometry.Results -The recovered amikacin concentrations for the 10 mg/mL solutions ranged from 10 to 13.5 mg/mL (mean 11.5 mg/mL) with the exception of NA sample 2 at Day (D)0 (9.4 mg/mL) and D7 (9.2 mg/mL). The recovered amikacin concentrations for the 30 mg/mL solutions ranged from 30 to 40.2 mg/mL (mean 35.7 mg/mL). No significant difference was seen between the amikacin concentrations at D0 compared to D56 for all solutions except 10 mg/mL TE (P < 0.001).Conclusions and clinical relevance -Amikacin maintained stability within TE, TC, NA and ND over 56 days except when formulated at 10 mg/mL within TE.

Topical compounded Timentin(®) diluted with an inactive vehicle has been reported to be effective in the treatment of otitis externa caused by Pseudomonas aeruginosa. The aims of this study were to determine the biological efficacy of Timentin(®) (ticarcillin and clavulanic acid) when diluted in the carrier vehicle Methopt(®) against P. aeruginosa and to determine the efficacy and stability of Timentin(®) aqueous stock concentrate solution. Timentin(®) stock concentrate was tested against four P. aeruginosa isolates on days 0, 7, 14, 21 and 28; then after 2, 3, 4, 5, 6, 9 and 12 months of storage at 4 or -20°C. The diluted Timentin(®)-Methopt(®) solutions were tested against all isolates after 0, 2, 4, 6, 8, 10, 12, 14, 17, 21, 24 and 28 days of storage at 24 or 4°C. Minimal inhibitory concentration (MIC) levels for all strains were determined using the broth microdilution method. The MIC of the stock solution remained relatively constant and acceptable throughout the study when stored at -20°C and was also acceptable for shorter time periods (6-9 months) when stored at 4°C. The MIC for the diluted Timentin(®)-Methopt(®) solution remained relatively constant and acceptable throughout the study for all four bacterial strains, with no difference between the solutions stored at 4 or 24°C. The results of this study indicate that storage of the Timentin(®) stock solution at -20°C does not compromise efficacy for at least 12 months and that Timentin(®) diluted in Methopt(®) was stable for 28 days when stored at either 4 or 24°C.

BACKGROUND:The Ehlers Danlos syndromes (EDS) are a heterogeneous group of heritable disorders affecting connective tissues. The mutations causing the various forms of EDS in humans are well characterized, but the genetic mutations causing EDS-like clinical pathology in dogs are not known, thus hampering accurate clinical diagnosis. RESULTS:Clinical analysis of two independent cases of skin hyperextensibility and fragility, one with pronounced joint hypermobility was suggestive of EDS. Whole genome sequencing revealed de novo mutations of COL5A1 in both cases, confirming the diagnosis of the classical form of EDS.The heterozygous COL5A1 p.Gly1013ValfsTer260 mutation characterized in case 1 introduced a premature termination codon and would be expected to result in 1(V) mRNA nonsensemediated mRNA decay and collagen V haploinsufficiency. While mRNA was not available from this dog, biochemical analysis of the dermis suggested reduced collagen V in the dermis and ultrastructural analysis demonstrated variability in collagen fibril diameter and the presence of collagen aggregates, termed 'collagen cauliflowers', consistent with COL5A1 mutations underlying classical EDS. In the second case DNA sequencing demonstrated a p.Gly1571Arg missense variant in the COL5A1 gene. While samples were not available for further analysis, such a glycine substitution would be expected to destabilize the strict molecular structure of DECLARATIONS Ethics approval and consent to participateAll procedures for case 1 (FB) were approved by the Murdoch Children's Research Institute Animal Ethics Committee (Approval # A815) . Blood and skin samples for cases 2 and 3 (RA) were collected as part of routine veterinary diagnostic assessment.