Circulation of canine parvovirus among dogs living in human-wildlife interface in the Atlantic forest biome, Brazil

Vieira, Flávia V.; Hoffmann, Daniel J.; Fabri, Carolina U.F.; Bresciani, Katia D.S.; Gameiro, Roberto; Flores, Eduardo F.; Cardoso, Tereza C.

Faecal samples were collected per rectum from apparently healthy dogs and stored at −86°C prior to virus isolation. The studied region is localized 21°12′32′′S 50°25′58′′W and was divided into 4 different areas (50 Km 2 ). The collection was performed at the same period (one week) corresponding to each area, during 2014 to 2016 and corresponded to 100 different individuals. This study was approved by the ethical committee (CEEA) of Universidade Estadual Paulista "Júlio de Mesquita Filho". The study was performed under all applicable institutional guidelines for the care and ethical use of animals were followed as recommended by Animal Brazilian Experimentation Committee protocol number 2015/09754. Approximately 2 g of faeces was homogenized in 1 volume of sterile phosphatebuffered solution (PBS), clarified by centrifugation at 2,500 x g for 10 min. The supernatant was filtered through a 0.75 μm filter (Millipore TM ) and treated with amphotericin and penicillin (concentrated at 100X; Sigma-Aldrich ® , St. Louis, MO, USA). AF-72 cells (ATCC, CRL 1542) were cultured in MEM (Sigma-Aldrich ® ) supplemented with antimycotic/antibiotic 1X solution, 10% foetal calf serum (Sigma-Aldrich ® ), 2 mM L-glutamine (Sigma-Aldrich ® ), and non-essential amino acids (100x, Invitrogen ® , Life Technologies, Carlsbad, CA, USA). Cultures were incubated at 37°C in 5% CO 2 with 95% humidity. After AF-72 cells reached 80% confluence, 1 ml of faecal preparation was added to 4.7 × 10 5 cells/ml and the culture supernatant was submitted to three blind passages at 5-day intervals. Inoculated and control cells were monitored under phase-contrast using an Olympus IX-70 microscope for production of cytopathic effect (CPE) (Olympus ® , Tokyo, Japan). Approximately 10 fields were analysed in each condition, and photographs were taken at 200 x magnification by using cell Sens TM software (Olympus ® ). CPV VR-2016 TM (ATCC), Cornell strain, which is present in all Brazilian vaccines, were used as controls. Mock-infected AF-72 cells were used as negative controls nd cells inoculated with CPV VR-2016 TM , strain Cornell were used as positive controls. Other enteric viruses, e.g., canine adenoviruses 1 and 2 (CAV-1, CAV-2) and canine distemper virus (CDV), were not detected in the examined samples by molecular analysis (data not shown).