A total of 35 tested animals exhibited clinical symptoms of CNS at the time of sample collection. The dogs tested in this study had never been vaccinated with any CDV vaccines. In 2020, peripheral blood samples were collected into sterile tubes containing etilendiamintetra-asetat (EDTA) from the antebrachial cephalic vein of 35 dogs. Blood samples with EDTA were centrifuged at 4°C at 1500 x g for 10 minutes for buffy coat separation. For 5 min, blood serum samples were centrifuged at 3000 rpm. Serum samples were transferred to sterile microtubes at a volume of 2 mL. Until testing, the samples were kept at -80°C. Before testing, all serum samples were inactivated for 30 min at 56°C. The dogs were suspected of CDV infection by determination of their pre-vaccination status in private veterinary clinics. During the sampling period, the individual features of all dogs were recorded according to their owners’ declarations and clinical observations. Anamnesis and clinical symptoms were taken into consideration as risk factors in this study.
CDV-specific IgG ELISA detection
Dogs with CNS symptoms were sampled for further study based on an abnormal neurologic examination and were diagnosed by ELISA-IgG for the initial time. An indirect ELISA was used to detect CDV-specific IgG antibodies in serum samples. CDV-specific IgG was detected using a commercially available kit (Agrolabo, Italy). The test was carried out in accordance with the manufacturer's recommendations.
Extraction and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted from the samples and commercial vaccine, using RiboExTM (GeneAll®, Korea) according to the manufacturer’s instructions. Extracted samples were used to amplify the targeted N protein-encoding gene by RT-PCR assay on a Thermo Cycler (Applied BiosystemsTM, USA). Primer sets and cycling conditions were used as previously described by Frisk et al. (1999), with only slight modifications. The PCR cycling conditions were cDNA synthesis at 50°C for 15 min, pre-denaturation at 94°C for 1 min, 35 cycles of 1 min at 94°C, 2 min at 59°C, and 1 min at 72°C, and a post-extension at 72°C for 7 min. For the H gene, RT-PCR cycling conditions were 5 min at 95°C pre-denaturation, 35 cycles each of 94°C for 1 min, 60°C for 1 min, and a post-extension of 72°C for 10 min (Lan et al. 2005). For the F gene, RT-PCR cycling conditions were 5 min at 95°C pre-denaturation, 35 cycles each of 94°C for 1 min, 45°C for 1 min, and a post-extension of 72°C for 10 min (Romanutti et al. 2016). All primers were shown in Table 1 together with their nucleotide positions and amplicon sizes. In all tests, cDNA was synthesised using the RevertaidTM First Strand cDNA Synthesis Kit (Thermo ScientificTM, Germany) according to the manufacturer’s instructions. The electrophoresis of 5 µl of PCR products was performed in 2% agarose gel. Sterile purified water and CDV RNA (extract from a commercial vaccine) were used as negative and positive controls, respectively.
Sequence and Phylogenetic Analyses
All positive PCR product samples with expected size of 1046 bp for the H gene and 797 bp for the F gene were sequenced in both directions by a commercial company (Macrogen; BMLabosis, Turkey). The obtained sequences of CDV partial H and F genes were subjected to BLASTn to compare for sequence identities/variations with other sequences of CDV strains around the world present in the GenBank (NCBI) database (Altschul et al. 1990; Benson et al. 2012). Further, amino acid sequences were subjected to multiple alignment with BioEdit software (v.7.2) using the Clustal-W method (Hall 1999; Thompson et al. 1994). The deduced amino acid sequences of the genes along with the other CDV strains from different geographical regions were used to construct the phylogenetic trees with bootstrap values calculated with 1000 replicates (Fig. 4-5) by using the maximum likelihood (ML) method in MEGA 11 software (Tamura et al. 2021). Additionally, positive sequences were submitted to GenBank through the Banklt interface to receive an accession number. Similarity and identity rates regarding bet sequences have been calculated in MatGAT 2.0 (Campanella et al. 2003).
Nucleotide sequence accession numbers
The GenBank accession numbers of the sequences used in this study are as follows
GQ214373 (Austria), KR002661 (Switzerland), AY386315 (USA), DQ494317 (Italy), AF478544 (Denmark), AF478550 (Denmark), DQ494318 (Italy), GQ214384 (Austria), Z47761 (Denmark), DQ889177 (Hungary), DQ494319 (Italy), AY093674 (Turkey), Z77673 (Germany), Z77671 (Germany), HM443723 (Italy), HM443718 (Italy), KT588923 (Turkey), DQ228166 (Italy), Z47759 (Denmark), AY498692 (USA), FJ392651 (Argentina), KT266736 (Mexico), JN215475 (Uruguay), KF835422 (Colombia), KF835411 (Colombia), EU098103 (Brazil), EU098104 (Brazil), EU098105 (Brazil), EU098102 (Brazil), AY649446 (USA), Z47762 (USA), Z54156 (USA), KC916714 (Tanzania), JN812976 (Tanzania), KC916716 (Tanzania), FJ461696 (SouthAfrica), FJ461713 (South Africa), FJ461711 (South Africa), FJ461717 (South Africa), FJ868166 (SouthKorea), EU716072 (SouthKorea), KU983521 (China), KU030831 (China), KU030832 (China) KU521345 (China), KF880678 (China), KP127966 (China) JX844220 (China) EU325730 (China), JQ732171 (China), JQ732170 (China), AB025271 (Japan), AB212965 (Japan), AB025270 (Japan), AB212730 (Japan), Z47760 (Greenlandic), AF172411 (China), FJ461709 (Nobivac PuppyDP), FJ461701 (Nobivac), FJ461702 (Vanguard Plus), FJ461710 (Canigen DHPPI), MN702774 (Canishot K5), FJ461708 (Galaxy), EU143737 (Onderstepoort), EF418782 (Lederle), GU810819 (Rockborn), AF378705 (Onderstepoort), Z35493 (Convac), AF259552 (SnyderHill) and GU266280 (Rockborn-Candur).
GQ214364 (Austria), AY386315 (USA), GQ214360 (Austria), MH430946 (Germany), AF355188 (Denmark), KT224732 (Argentina), KT224718 (Argentina), KT224719 (Argentina), AY466011 (USA), AY649446 (USA), EU716337 (USA), KP769803 (China), EF596902 (China), KJ848781 (China), JN896331 (China), KX371581 (China), KX371582 (China), KX371583 (China), EU192026 (Taiwan), AY964108 (USA), AY964112 (USA), AB476403 (Japan), AB476402 (Japan), AB475100 (Japan), AB475097 (Japan), AF378705 (Onderstepoort) and GU138403 (SnyderHill).