PREPARATION AND FIELD EVALUATION OF CELL CULTURE ADAPTED LYOPHILIZED THERMOSTABLE NEWCASTLE DISEASE VACCINE

Abstract

Newcastle disease is one of the nastiest disease of chicken throughout the world, particularly in developing countries like Pakistan. The present study was aimed to prepare a cell culture adapted vaccine for propagation and adaptation of avirulent thermostable NDV I -2 strain on Vero cell line, molecular confirmation and characterization of cell culture adapted thermostable NDV I-2 strain and experimental and field evaluation of the cell culture adapted NDV I-2 vaccine (Thermo-Vac) in broiler birds. A well characterized avirulent, Australian origin thermostable I-2 ND Virus strain was used for vaccine production. In the current study, Vero cell line was used for production of thermostable I-2 NDV vaccine. Vero cells were grown at the rate of 3×107 cells per ml in MEM199 medium. The morphological alterations such as roundening, aggregation of cells and detachment of cells were observed after the growing of virus, called cytopathic effect (CPE). The first clear CPE was observed in passage no. 10 following of 120 hours post infection. Haemagglutination test for each passage showed that supernatant contained the virus. Consistent CPE was observed at passage no. 13th which conferred the adaptation of NDV I-2 of Vero cells. Thermostablity was evaluated after each passage of virus and results showed that thermostability of NDV I-2 strain was retained after adaptation on Vero cell line. Biological titration of that adapted passage produced 106 pfu/ml and log10 8 per ml tissue culture infective dose fifty (TCID50). Reverse transcription polymerase chain reaction (RT-PCR) was used for molecular detection of Vero cell adapted virus. Specific primer amplified fusion protein cleavage site and produced 204 bp DNA fragment. The accession number i.e. KM043779 was obtained from Genbank. Phylogenetic analysis revealed that 80-90% homology of in nucleotides amino acids was existed among the other reported thermostable NDV isolates. The Vero cell adapted virus was used for preparation of Thermo-Vac (cell culture adapted lyophilized NDV I-2) vaccine. Thermostability of vaccine and sterility of prepared vaccine was checked by inoculating culture media. An experimental chicken NDV infection experiments in group 1, maximum HI and ELISA mean antibody titers Log2 and standard deviation was achieved at days 14 e.g. 7.5±0.79a, 6812±0.654a as followed 2±0.41a,, 1633±0.341a, 6.0±0.13a, 4899±0.546a, 6.8±0.49a, 5899±0.879a, 6.75±0.64a, 5716±0.546a and 4.5±0.53a, 3480±0.347a at day 0, 7, 21, 28 and 35 following Thermo-Vac vaccination via drinking water method as compared to commercially 12 available NDV LaSota vaccine (Group 2) HI and ELISA mean antibody titer Log2 and standard deviation i.e. 2±0.41b, 1633±0.632a, 2.95±0.29b, 2300±0.231b, 4.04± 0.62b, 4520±0.234b, 3.09±0.73b, 3400±0.543b, 2.20±0.11b, 2372±0.653b and 2 ± 0.65b, 1500±0.436b at day 0, 7, 14, 21, 28 and 35 respectively. The antibody titers of Thermo-Vac vs NDV LaSota were vaccinated birds significantly different from one another except at day 0. In negative control (group 3) no protective antibodies were produced. After NDV challenge infection, we observed any morbidity and mortality in chicks in all groups. The results showed that administration of Thermo-Vac vaccine in broiler birds was feasible and was found to induce more protective antibody response i.e. 90% against challenge infection in group 1 as compared with group 2 NDV LaSota 60% protection after challenge viral infection. In group 3, all birds died after challenge infection within 2-3 days. Cellular immune response was examined through spleenic cell migration inhibition assay. The results showed that in group 1, Thermo-Vac vaccine start producing % inhibition migration at day 3 (40%) and reached optimized level at day 6 (50%) as gradually decreased at day 9, 12, 15 and 18 i.e. 38%, 26%, 14%, 13% respectively. In group 2, LaSota ND vaccine shown % migration inhibition at day 3, 6, 9, 12, 15, 18 i.e. 32%, 43%, 34%, 19%, 10%, 12% respectively. The % migration inhibition was less than 15% in control group throughout the experiment. The % migration inhibition with ND I -2 antigen in group 1 was significantly higher as compared with Group 2 LaSota ND vaccine. In field conditions, maximum geometric mean anti-NDV-HI (Log27.83) and anti-NDV-ELISA (6017) antibodies titers were observed, respectively on 14th day post vaccination. In conclusion, Thermo-Vac vaccine produced protective cellular and humoral immunity against NDV, So, Thermostable NDV I-2 strain can be a preferred choice against NDV in developing countries like Pakistan.