Nineteen dogs (4–12 years; 11 males and 8 females) diagnosed with CB were included in this study. Between April 2005 and March 2010, these dogs had been referred to the Animal Medical Center of Nihon University for diagnoses and consultations for long-term therapy for their clinical signs. The breeds included Miniature Dachshund (n = 12), Shih Tzu (n = 2), and 1 each of Pug, Maltese, Papillon, Shetland sheepdog, and mixed breed. CB was diagnosed based on normal ranges of blood test results, thickened bronchial walls on chest X-ray and computed tomography, mucosal secretions, and patterned indented surface in bronchoscopy. Total nucleated cell counts were determined and the results were used, provided the dogs did not present with other nasal, throat, cardiac, infectious and neoplastic diseases. Either an elevated total nucleated cell count (> = 400 cells/μL) with macrophage dominance (> = 50%) or normal total nucleated cell count (<400 cells/μL) with neutrophil dominance (> = 15%) in the BALF samples was used as one of the parameters for the inclusion criteria. All examinations were performed after informed consent of the dogs’ owners under the guideline for the care and use of laboratory animals by The College of Bioresource Sciences, Nihon University.
Blood samples were collected from jugular veins as in routine clinical examinations. Serum was isolated by centrifugation and stored at −20°C until used for cotinine measurements. Serum cotinine was determined using a Cotinine Passive Smoking ELISA Kit (Cosmic Corporation, Tokyo, Japan). BALF samples from the dogs were obtained by gentle aspiration through a biopsy channel of a bronchoscope after infusing sterile saline (0.9% NaCl) solution (40 mL, divided into 2 aliquots) under general anesthesia with isoflurane. BALF sample supernatants were isolated by centrifugation (200 × g for 10 min) and immediately stored at −20°C until DNA extraction. Free-floating DNA in 200 μL of BALF supernatant was concentrated to 20 μL using a DNA Extractor Kit (Wako, Japan). Concentrations of the extracted DNA in sera and BALF samples were determined using a Nanodrop Spectrophotometer (NanoDrop Technologies, USA) and corrected by urea concentrations (QuantiChromTM Urea Assay Kit, BioAssay Systems, Hayward, CA, USA).
DNA methylation rates were determined with a Methylamp Global DNA Methylation Quantification Ultra Kit (Epigentek, USA) according to the manufacturer’s instructions. The capture antibody in this kit binds to 5-methylcytosine. The total DNA methylation level was determined as a percentage of the total DNA in a given sample. This technique measured the total DNA fragments in the samples but not specific sites, patterns, or methylation types. Samples were run in duplicate and points for the standard curve were run in triplicate.
Medians with the range of minimum and maximum values were used to express results. Group results were compared by the Mann–Whitney rank sum test for non-normally distributed variables. P values < 0.05 were considered significant. All statistical analyses were performed using SigmaPlot for Windows, Version12.0 (Systat Software, Inc., San Jose, CA, USA).