Development And Application Of In-Situ Hybridization For Detection Of Chicken Neoplastic Diseases

Avian viral neoplastic diseases including Marek's disease (MD), Avian leucosis(AL) and Reticuloendotheliosis(RE), caused by MDV, ALV and REV respectively, are responsible for economic loss due to both mortality and depressed performance. These diseases share similar symptoms and exist in some chicken farms in China to varying extent. In situ Hybridization techniques, allowing the demonstration of specific nucleic acid sequences in their cellular environment, is becoming a routine laboratory tool to detect infectious diseases. In this study, we examined whether the in situ hybridization technique could be used to differentially detect avian neoplastic diseases. At the same time, some relevant genes of these three oncogenic viral agents were sequenced and analyzed. The main study contents and results are followed as:1. Three pair PCR primers were designed and synthesized according to MDV, ALV-J and REV sequences deposited in GeneBank. The PCR amplified products were cloned and confirmed by sequencing. Subsequently, these products were subcloned into pSPT18 plasmid to generate DIG-labeled RNA probes targeting MDV, ALV-J and REV respectively.2. Thirty-two samples from different chicken flocks were processed as paraffin sections and probed with above RNA probes. Of these chicken flocks, 16 flocks were negative. Of the positive chicken flocks, MDV, ALV-J and REV infection were 25%, 18.5%, 3.13% respectively.3. MDV meq gene, ALV and REV env genes from the positive flocks were amplified by PCR and cloned into pMD18-T plasmid for further sequencing. The results indicated that MDV meq gene was composed of 1020 nucleotides encoding 340 amino acids. Compared to the corresponding sequences collected in GeneBank, MDV meq gene identity ranged from 98.6%～99.8% at the nucleotide level and 96.5%～99.4% at the amino acid level. Phylogenetic analysis revealed that the samples in this study were close to each other at the meq nucleotide level, its identity ranging from 99.5%～99.6%, close to MDV strain 0095 and far from GA, 648 and N.ALV-J gp85 gene was composed of 921 nucleotides encoding 307 amino acids. When compared with sequences of reference isolates, its gene identity ranged from 86.8%～97.5% at the nucleotide level and 84.0%～95.4% at the amino acid level. ALV-J gp37 gene was composed of 591 nucleotides encoding 197 amino acids. Its identity between those in this study and reference sequences in Genebank ranged from 93.7%～98.3% at the nucleotide level and 89.8%～98.0% at the amino acid level. Phylogenetic analysis revealed that the samples studied were close to each other at ALV-J gp37 gene, the nucleotide identity ranging from 94.4%～98.7, close to ALV-J strains GD06SL1 and GD06SL2 and far from ADOL-7501.The amplified REV env segment contained 477 nucleotides encoding 159 amino acids. REV env identity between those in this study and the reference in Genebank ranged from 94.3%～100% at the nucleotide level and 87.3%～100% at the amino acid level. The samples studied were close to REV strain APC-566 and far from SNV.To summarize, an in situ hybridization technique was developed and applied to detect chicken neoplastic diseases. Relevant genes from these oncogenic agents were studied for genetic variation. The results provided more data and diagnostic tool for diagnosis and control of these diseases.