ABSTRACT
This study aimed to investigate the occurrence of coronaviruses (CoVs) in captive birds placed inside a zoological park in Brazil. The role of captive birds in the epidemiology of CoVs in the tropics is poorly understood. A total of 25 (n=25) different species were tested for viral RNA using individual fecal samples collected from healthy birds. Reverse transcription-polymerase chain reaction targeting the 3' untranslated region was used to detect CoV RNA, and positive samples were submitted for sequence analysis. The phylogenetic search revealed nine mutations in the black shouldered peafowl (Pavus cristatus) CoV sequence, which clustered separately from samples previously described in England. This is the first report on the detection of the CoV genome in captive birds in Brazil.
Subject(s)
Animals, Zoo/virology , Bird Diseases/virology , Coronavirus Infections/veterinary , Coronavirus/genetics , Genetic Variation , Animals , Base Sequence , Birds , Brazil , Coronavirus/classification , Coronavirus Infections/virology , Cross-Sectional Studies , Molecular Sequence Data , Phylogeny , Sequence AlignmentABSTRACT
An in situ polymerase chain reaction (IS-PCR) hybridisation assay was carried out on the brains of 20 cattle infected naturally with bovine herpesvirus type 5 (BoHV-5). Sections from the olfactory bulb and the frontal cortex of each sample were analysed using IS-PCR followed by hybridisation targeting the BoHV-5 US9 gene using a biotinylated primer. Each of the IS-PCR and hybridisation steps was optimised, and three different methods for detecting the virus were used. No false positive signals were observed in any negative control sample (n=20), resulting in a specificity of 100%. The results of IS-PCR hybridisation analysis of the olfactory bulb and the frontal cortex be compared directly with the results obtained using virus isolation, and the specificity and sensitivity were calculated. The most suitable method of visualisation was the peroxidase/3'-3-diaminobenzidine (DAB) detection system coupled with the use of the fluorescent dye Cy3. Using either of these methods, 80% of the positive samples (16 out of 20 samples) were identified using olfactory bulb sections. This is the first report using IS-PCR hybridisation for the direct detection of BoHV-5 DNA in clinical samples, and it provides an additional method for veterinary virology.
Subject(s)
Cattle Diseases/virology , Encephalitis, Viral/veterinary , Herpesviridae Infections/veterinary , Herpesvirus 5, Bovine/isolation & purification , In Situ Hybridization/methods , Meningoencephalitis/veterinary , Polymerase Chain Reaction/methods , Animals , Brain/virology , Cattle , DNA Primers/genetics , Encephalitis, Viral/virology , Fixatives/pharmacology , Formaldehyde/pharmacology , Herpesviridae Infections/virology , Herpesvirus 5, Bovine/genetics , Meningoencephalitis/virology , Paraffin Embedding , Pathology, Molecular/methods , Sensitivity and Specificity , Tissue FixationABSTRACT
Infectious bronchitis virus (IBV) is produced in vitro using specific pathogen free chicken embryos, primary chicken kidney cells (CKC) and/or tracheal organ culture (TOC). Regulatory authorities in Europe (EMEA) and in the United States (FDA) have encouraged biological manufactures to reduce or eliminate the use of live animals and/or products of animal origin in biological manufacturing processes. In this paper, a stable chicken embryo-related (CER) cell line was adapted and maintained in serum free (SF) or animal protein free (APF) medium after a direct switch of the medium. The most suitable media were Ex Cell 520 and Ex Cell 302. CER monolayers adapted to SF or APF were infected with IBV (M41 strain) in agitated suspended culture and the IBV titre obtained 48 h post infection was 2.8 x 104 PFU/ml in both cases. Thus, propagation of CER cells and culture of IBV can be performed without the use of animal serum or animal protein.
