Abstract
The present study was conducted for the isolation, identification, biological and molecular characterization of Newcastle disease virus (NDV) from recent outbreaks in layer chickens during the year 2012 and 2014 and for the development of inexpensive killed ND vaccines. A total of 9 isolates were isolated from field samples. All these 9 isolate caused death of chicken embryo within 72 hours of inoculation and revealed positive result in HA, HI, AGIDT and RT-PCR. RT-PCR performed using primer for 'F' gene (356 bp amplicon) which was confirmed by PCR using the cDNA to amplify a larger sized 'F' gene specific amplicon (532 bp) and nested PCR using primers to amplify 216 bp internal sequence of the cleavage activation site of 'F' gene. Matrix (M) protein gene specific primers were designed to amplify 232 bp amplicon of that gene. Isolates BSN-NDV/2012 and BMS-NDV/2013 were sequenced and submitted to GenBank (Ac. No. KU341110 and KU341111 respectively). BMS-NDV/2013 was adapted in BHK-21 cell culture which produced clear CPE: syncytia formation, rounding of cell and multinucleated giant cell. MDT, ICPI, IVPI of that isolate was 54.34, 1.55, and 2.13 respectively which revealed that the isolate was velogenic. Then binary ethyleneimine (BEI) inactivated oil-based vaccines were developed using NDV isolate which was previously titrated as log 109.5 EID50/ml and log 107.7 TCID50/ml (HA titer: log 212 and log 210) respectively. Experimental killed ND-AF vaccine from allantoic fluid and experimental killed ND- TF vaccine from tissue cultured fluid were prepared. Equal volume of inactivated virus suspension and oil-base were homogenized . A total of 240 layer chickens used for immune response study of experimental killed ND vaccines along with commercial killed ND vaccine (Newcavac®, Intervet). Three different trials (Trial 1, Trial
D)
2 and Trial 3) conducted using 80 layer chicks (subdivided group A, B, C and with the dose variation of vaccines. Chickens were immunized with the dose of 0.5 ml/bird in Trial 1, 0.25 ml/bird in Trial 2 and again 0.5 ml/bird in Trial 3 with
commercial
experimental killed ND-AF and killed ND-TF vaccines at 120 days of age, where as killed ND vaccine were administered at the rate of 0.25 ml/bird as recommended by the manufacturer for each trial). During Trial 1, HI antibody titers were 160±83.79, 144±45.25, 168±76.02 and 28±7.40 of group A, B, C and D
respectively, whereas ELISA titers were 13360.7±799.70, 12454.61±1156.06, 14815.2±573.18 and 377.814±50.96 respectively. The HI titers were 136±53.40, 120±22.62, 152±67.88, 32±14.81 and ELISA titers 11501.76±1352.36, 10999.63±963.24,
26±8.28
13318.58±1657.38 and 446.412±50.70 at Trial 2. 152±67.88, 144±45.25, 160±59.25,
338.312±75.83
HI titers and 10883.58±1677.21, 9487.75±3272.58, 14255.03±1701.62 and ELISA titers were obtained at Trial 3. BEI inactivated vaccine showed a
satisfactory antibody titer along with commercial vaccine at each trial. At protection test, experimental vaccines conferred 100% protection along with commercial
experimentally
(Newcavac®, Intervet) vaccine. From the present study it may be concluded that