An outbreak of AI in a duck farms (N=700) was reported with a history of higher rate of morbidity (50%) and morbidity to mortality (85%). Following on farm investigation, the clinical signs observed were torticollis, tremor of neck, head pressing on ground, diarrhea, dizziness and respiratory distresses. Out of 300 dead ducks, five were examined, collected representative samples to identify the pathology, pathotype and subtype of AI viruses by means of pathological investigation and genomic analyses of M, HA and NA genes of AIV.
Following necropsy pathological changes in lungs, liver, kidney, pancreas, intestine, heart, gizzard and skeletal muscles were recorded. The tissues from these organs were collected in 10% neutral buffered formalin for histopathological investigation. Portion of lungs and trachea were snap frozen and preserved at -80°C for molecular characterization. The formalin fixed tissues were processed, sectioned and stained with routine hematoxylin and eosin stain and Goldner’s trichrome staining.
Portion of trachea and lungs were used for viral RNA extraction and detection of specific genes of AIV by using RT-PCR. Briefly 0.5 gm frozen tissues were crushed in liquid nitrogen and extracted viral RNA using commercial kit (Viral nucleic acid extraction kit II, Geneaid, USA). The quality and quantity of the extracted RNA was measured by using agarose gel electrophoresis and spectrophotometry (A260/A280).
The partial M, HA and NA genes of AIV were amplified using SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA polymerase (Life Technologies, USA ) kit. Primers for RT-PCR protocols were designed from sequences available in GenBank, synthesized from commercial source (1st base, Singapore) and fragments of M, HA and NA genes were amplified by RT-PCR. The RT-PCR was carried out using One-Step RT-PCR System (SuperScript® III One-Step RT-PCR System, USA). The reaction was carried out in 50μl volume consisting of 2x master Mix, 1μl of each primer (20pmol/μl), 1μl of SuperScript III RT/Platinum Taq Mix, 1μl Rnase inhibitor, 5μl template RNA (50ng/reaction) and 17μl Nuclease free water. As negative control 5μl nuclease free H2O was used instead of template RNA. The reaction was performed in an oil-free thermal cycler (Master Cycler Gradient, Eppendorf, Germany). RT-PCR for the amplification of selected genomes of AI virus (designed unified cycling parameter) was started with the reverse transcription at 45°C for 45mins. Then the initial denaturation was carried out at 95°C for 2mins followed by 40cycles of amplification reaction consisting of denaturation at 94°C for 30secs, annealing at 54°C for 30secs, elongation at 72°C for 3mins and final elongation at 72°C for 10mins. The RT-PCR was held at 4°C, the RT-PCR amplicons were analyzed by electrophoresis in 1.5% agarose gel containing ethidium bromide (0.5 μg/ml) and images were captured.
Following gel cleaning of RT-PCR products, the cDNA was sequenced directly from the both direction using forward and reverse primers. Sequencing was done from commercial laboratory (1st Base, Singapore). The quality of raw sequence data were analyzed, edited and assembled with the programmes Chromas Lite, EditSeq and MegAlign for molecular and phylogenetic analyses.