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.

Amblyomma dissimile (Acari: Ixodidae) em Hydrodynastes gigas (Squamata: Colubridae) no estado Mato Grosso do Sul, Brasil – Nota Prévia / Amblyomma dissimile (Acari: Ixodidae) in Hydrodynastes gigas (Squamata: Colubridae) in Mato Grosso do Sul State, Brasil – Short Communication

A Hydrodynastes giga, (Duméril, Bibron & Duméril, 1854) vulgarmente conhecida como surucucu-do-pantanal, é uma serpente de grande porte, que ocorre no norte, centro-oeste, sudeste e sul do Brasil e que pode ser parasitada por ectoparasitos como os carrapatos do gênero Amblyomma. Em dezembro de 2007 foram coletados manualmente três carrapatos de uma serpente H. gigas no pantanal de Miranda, MS,Brasil (19º51’-19º58’S; 56º17’-56º24’W). Os carrapatos foram armazenados e enviados para o Laboratório de Parasitologia do Hospital Veterinário da ULBRA, Canoas, RS, onde foi realizada a identificação de três machos da espécie Amblyomma dissimile. A presente nota faz o primeiro relato de A. dissimile parasitando serpentes da espécie H. gigas no pantanal de Miranda, MS, Brasil

Hydrodynastes giga (Duméril, Bibron & Duméril, 1854) best known as false water cobra, is a big-sized snake, which lives in Northern, Middle western, Southeastern and Southern Brazil and might be infested by ectoparasites, such as ticks from the Amblyomma genus. In December 2007 three ticks were manually collected from a H. gigas in the Miranda wetlands, MS, Brazil (19º51’-19º58’S; 56º17’-56º24’W). All ticks were placed in identified bottles and then sent to the Laboratório de Parasitologia of ULBRA Veterinary Hospital, Canoas, RS, where the identification of three males of the Amblyomma dissimile species was carried out. This note is the first report of A. dissimile parasitizing snakes of the H. gigas species in the Miranda wetlands, MS, Brazil