Detection and differentiation of field and vaccine strains of canine distemper virus using reverse transcription followed by nested real time PCR (RT-nqPCR) and RFLP analysis.
Canine distemper virus (CDV) is the cause of a severe and highly contagious disease in dogs. Practical diagnosis of canine distemper based on clinical signs and laboratory tests are required to confirm CDV infection. The present study aimed to develop a molecular assay to detect and differentiate field and vaccine CDV strains. Reverse transcription followed by nested real time polymerase chain reaction (RT-nqPCR) was developed, which exhibited analytical specificity (all the samples from healthy dogs and other canine infectious agents were not incorrectly detected) and sensitivity (all replicates of a vaccine strain were positive up to the 3125-fold dilution - 10(0.7) TCID50). RT-nqPCR was validated for CDV detection on different clinical samples (blood, urine, rectal and conjunctival swabs) of 103 animals suspected to have distemper. A total of 53 animals were found to be positive based on RT-nqPCR in at least one clinical sample. Blood resulted in more positive samples (50 out of 53, 94.3%), followed by urine (44/53, 83.0%), rectal (38/53, 71%) and conjunctival (27/53, 50.9%) swabs. A commercial immunochromatography (IC) assay had detected CDV in only 30 conjunctival samples of these positive dogs. Nucleoprotein (NC) gene sequencing of 25 samples demonstrated that 23 of them were closer to other Brazilian field strains and the remaining two to vaccine strains. A single nucleotide sequences difference, which creates an Msp I restriction enzyme digestion, was used to differentiate between field and vaccine CDV strains by restriction fragment length polymorphism (RFLP) analysis. The complete assay was more sensitive than was IC for the detection of CDV. Blood was the more frequently positive specimen and the addition of a restriction enzyme step allowed the differentiation of vaccine and Brazilian field strains.
Laboratório de Diagnóstico Molecular, Universidade Luterana do Brasil, Av. Farroupilha, 8001, 92425-900, Canoas, Rio Grande do Sul, Brazil. Electronic address: tinevet@terra.com.br.
Cristine Dossin Bastos
CD
Fischer
Nilo
N
Ikuta
Cláudio Wageck
CW
Canal
Aline
A
Makiejczuk
Mariangela da Costa
Mda C
Allgayer
Cristine Hoffmeister
CH
Cardoso
Fernanda Kieling
FK
Lehmann
André Salvador Kazantzi
AS
Fonseca
Vagner Ricardo
VR
Lunge
D000818:Animals; D001938:Brazil; D004216:Distemper / Q000175:diagnosis / Q000821:virology; D004217:Distemper Virus, Canine / Q000145:classification* / Q000302:isolation & purification*; D004283:Dog Diseases / Q000175:diagnosis / Q000821:virology; D004285:Dogs; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D048348:Reverse Transcription; D012680:Sensitivity and Specificity; D014730:Veterinary Medicine / Q000379:methods; D014765:Viral Vaccines / Q000302:isolation & purification*; D014773:Virology / Q000379:methods
D014765:Viral Vaccines
Canine distemper virus; Detection; Diagnosis; Dog; RT-PCR