The petiole was placed in a test tube, one leaf per tube, containing either 5 mM allantoic
acid or 5 mM allantoic
acid + 50 [micro]M MnS[O.
For this purpose 25 l of fresh allantoic
fluid was added in 1st well of microtiter plate and 2 fold serial dilutions were made in the PBS up to 11th well.
Molecular identification was done using reverse transcription polymerase chain reaction (RT-PCR) performed using viral RNA extracted from infective allantoic
The presence of virus in the harvested allantoic
fluid was confirmed by amplification of complete S1 sub-unit gene by Reverse Transcription- Polymerase Chain Reaction (RT-PCR).
The virus presence in allantoic
fluid was checked by rapid HA (haemagglutination) test (Allan and Gough, 1974) using 5% RBCs suspension.
Fresh infective allantoic
fluid obtained after passaging the AIV H9N2 in embryonated eggs with a HA titer greater than 1/16 was diluted 1/10 in sterile isotonic saline.
Virus stocks were propagated in specific pathogen-free chicken eggs, and the allantoic
fluids were harvested and stored at -70degC.
The end of 7 days post inoculation the allantoic
fluid of dead embryos was harvested and the 50% embryo-infective dose (EID50) was determined using the Reed and Muench method (Reed and Muench 1938).
1 mL of a 1:10 dilution of the H5N2qd14 allantoic
Each of the samples was inoculated in the embryos through allantoic
cavity, incubated for 48 hours and allanto-amniotic fluid (AAF) was harvested according to the method as described by (Hitchner et al.
Before inactivation Aamnio allantoic
Fluid (AAF) centrifuged at 4000 rpm (Hettich Zentrifugen Germany) for 10 minutes and supernatant was separated and treated with formalin (37.
The tracheal aspirate specimen was inoculated on monolayers of MDCK cells and into the allantoic
cavities of 10-day-old embryonated chicken eggs.
2 ml) was inoculated into the allantoic
cavity of 9 days old chicken embryos.
We attempted to isolate the virus by injecting the chorioallantoic membranes (CAM) and allantoic
cavities of 9-day-old specific pathogen free (SPF) chicken embryos with the scab specimens that were positive for FWPV.
20 um porosity and inoculated in 10 days old chicken embryos (200 (mu) l) through allantoic
cavity route in triplicate (Khan et al.