ABSTRACT
The clone library method using PCR amplification of the 16S ribosomal RNA (rRNA) gene was used to identify pathogens from corneal scrapings of C57BL/6-corneal opacity (B6-Co) mice with bacterial keratitis. All 10 samples from the eyes with bacterial keratitis showed positive PCR results. All 10 samples from the normal cornea showed negative PCR results. In all 10 PCR-positive samples, the predominant and second most predominant species accounted for 20.9 to 40.6% and 14.7 to 26.1%, respectively, of each clone library. The predominant species were Staphylococcus lentus, Pseudomonas aeruginosa, and Staphylococcus epidermidis. The microbiota analysis detected a diverse group of microbiota in the eyes of B6-Co mice with bacterial keratitis and showed that the causative pathogens could be determined based on percentages of bacterial species in the clone libraries. The bacterial species detected in this study were mostly in accordance with results of studies on clinical bacterial keratitis in human eyes. Based on the results of our previous studies and this study, the B6-Co mouse should be considered a favorable model for studying bacterial keratitis.
Subject(s)
Disease Models, Animal , Gene Library , Keratitis/microbiology , Nucleic Acid Amplification Techniques/methods , Polymerase Chain Reaction/methods , Pseudomonas Infections , Pseudomonas aeruginosa/isolation & purification , Pseudomonas aeruginosa/pathogenicity , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Sequence Analysis/methods , Staphylococcal Infections , Staphylococcus/isolation & purification , Animals , Female , Male , Mice, Inbred C57BL , Mice, Mutant Strains , Staphylococcus epidermidis/isolation & purification , Staphylococcus epidermidis/pathogenicityABSTRACT
To examine the phylogenetic information regarding the gene pool of AIV in domestic ducks in eastern China, the NA genes of twenty-six viruses isolated during 2002-2006, including two H1N1 strains, tenH3N1 strains and fourteen HSN1 strains, which reflected the predominant N1 subtype viruses were subjected to phylogenetic analysis. The results indicated that AIVs of N1 subtype circulating in domestic ducks in eastern China were undergoing a gradual evolution. Analysis of the deduced amino acid sequences revealed that NAs from all isolated H5N1 viruses had a 20-aa deletion in the stalk region (residues 49-68), whereas no deletion was seen in the NAs from other HA subtype viruses. The viruses of H3N1 and H1N1 might have a propensity for reassortment of NA genes, whereas no direct evidence of reassortment of NA gene was obtained in H5N1 viruses.
Subject(s)
Influenza A Virus, H1N1 Subtype/classification , Influenza A Virus, H3N2 Subtype/classification , Influenza A Virus, H5N1 Subtype/classification , Influenza A virus/classification , Neuraminidase/genetics , Sequence Deletion , Animals , Birds , China , DNA, Viral/analysis , Evolution, Molecular , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H3N2 Subtype/genetics , Influenza A Virus, H5N1 Subtype/enzymology , Influenza A Virus, H5N1 Subtype/genetics , Influenza A virus/enzymology , Influenza A virus/genetics , Influenza in Birds/virology , Influenza, Human/virology , Phylogeny , Poultry Diseases/virology , Sequence AlignmentABSTRACT
To date, nine neuraminidase (NA) subtypes of avian influenza viruses have been identified. In order to differentiate the NA of avian influenza viruses rapidly, a reverse transcription PCR (RT-PCR) was developed. Nine pairs of NA-specific primers for the RT-PCR were designed based on the analysis of 509 complete NA sequences in GenBank. The primers were designed to amplify partial NA genes and each pair is unique to a single NA subtype (N1-N9). By nine RT-PCRs simultaneously in a set of separate tubes, the subtype of NA was determined by subsequent agarose gel electrophoresis and ethidium bromide staining, since only one of the nine RT-PCRs would give a product of expected size for each virus strain. In comparison with the established method of sequence analysis of 101 reference strains or isolates of avian influenza viruses, the RT-PCR method had a sensitivity of 97.3% and a specificity of 91.1% in subtyping avian influenza viruses. These results indicate that the RT-PCR method described below provides a specific and sensitive alternative to conventional NA-subtyping methods.
