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Development of Bhk-21 Cell Line Adapted Inactivated Newcastle Disease Vaccine.

Md. Enamul Haque
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Marzia Rahman
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Md. Tanvir Rahman
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Sukumar Saha
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Md. Bahanur Rahman
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Mohammad Ferdousur Rahman Khan
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Abstract/ Executive Summary:

The present study was undertaken to isolate and characterize Newcastle disease virus (NDV) from the outbreak of layer farms for the development of BHK-21 cell adapted inactivated vaccine. A total of 6 dead birds were brought to the laboratory from the outbreak area from which 18 samples (trachea, liver and brain from each) were collected. Among them 3 samples were found positive for NDV through chicken embryo inoculation followed by HA test. The MDT, ICPI, IVPI of all isolates was 54.4, 1.56, and 2.20 respectively which revealed that the field isolates were velogenic. The isolated viruses were confirmed as NDV by reverse transcription-polymerase chain reaction (RT-PCR) using fusion gene-specific primers. The isolated virus was used to infect the BHK-21 cell line. Later the BHK-21 cell adapted viruses were used to develop oil adjuvanted inactivated vaccine and were inoculated into chickens according to vaccination schedule. The MDA was very high (112±29.62) during BCRDV vaccination, which declined quickly (88±33.12). Before vaccination with experimental vaccine, the level of antibody titre (HI) was very low (9±4.65). After vaccination at 65th day through IM, ELD50=109.7/ml with experimental vaccine, the highest HI titre in RDV vaccinated group was 160±59.25 whereas, experimental vaccinated group was 128±59.25, and control group was 7±4.65. ELISA antibody titres of all the groups were 2549.71 (RDV, LRI@0.5 ml/bird), 2450.37 (experimental@ 0.25ml/ bird), 2218.579 (experimental@ 0.50ml/bird), 2152.352 (experimental@1ml/bird) 1125.846 (control) respectively. The present study indicated that, BHK-21 cell adapted ND inactivated vaccine produced a satisfactory antibody titre along with conventional live RDV vaccine.

Keywords: NDV, RT-PCR, BHK-21 cell line, Inactivated Vaccine

Location: Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Poultry

Objectives:

1. The present study was undertaken for the production and to compare the level of antibody titres of experimental BHK-21 cell adapted inactivated ND vaccine with the conventional live virus vaccines (RDV, LRI).

Methodology:

Isolation and propagation of viruses in avian embryos - A total of 6 dead birds died from suspected cases of NDA were brought to the laboratory from Mymensingh and the surrounding areas from which a total of 18 samples (trachea, liver and brain from each dead bird) were collected aseptically and inoculum were prepared. Initially virus was propagated in chicken embry. Briefly, the inoculum (0.2ml) of each sample was inoculated into 10-day-old embryonated chicken egg through allantoic cavity route. After inoculation the eggs were incubated at 37⁰C with a humidified condition (50-60%) observed twice daily for mortality of the embryo. The embryos died due to ND virus were chilled followed by harvesting of the allantoic fluids and then preserved at -20⁰C until further use. The presence of virus in allantoic fluid was confirmed by slide HA test using 2 % chicken RBC suspension. The lesions on the body of the embryos were examined and recorded. Adaptation of virus in BHK-21 cell line - The cells those formed complete and confluent monolayer in the culture bottle within 24-72 hours were selected for infection with viruses. The BHK-21 cell line was infected with 1 ml of inoculum and was spread over the cell sheet by tilting for about 45-60 minutes for the establishment of better interaction. Then 5-10 ml of the maintenance medium (MEM supplemented with 2% fetal bovine calf serum) was added and followed by incubation of the bottle at 370C. The cells were examined twice daily under DIC microscope until cytopathic effect (CPE) was formed by inoculated virus. Primers and RT-PCR for detection of NDV - In this study two primers forward: 5'-GCA GCT GCA GGG ATT GTG GT-3' nucleotide position (158-177) and reverse: 5’-TCT TTG GAG CAG GAG GAT GTT G-3’ nucleotide position 493-513) were used. Total RNA from the allantoic fluid was extracted with Invisorb® Spin Virus RNA Mini kit as per manufacturer’s instructions. RT-PCR was carried out using Access RT- PCR system (Promega, USA). DNA amplifications were performed in a total volume of 50 μl containing 10μl 5X reaction buffer, 1μl dNTP mix (10mM), 10 pmol of each primer 1μl, 2μl 25mM MgSO4, 1μl AMV Reverse Transcriptase (5u/μl), 1μl Tfl DNA polymerase (5u/μl), 4μl RNA sample and nuclease-free water was added to a final volume of 50μl reaction mixture. For first Strand cDNA synthesis was conducted at 45°C for 45 minutes for reverse transcription (1 cycle), 94°C for 2 minutes for AMV RT inactivation and RNA/cDNA/primer denaturation (1cycle). The reaction mixture were thermocycled 40 times beginning with an initial denaturation step of 4min at 94 ºC. The temperature and time profile of each cycle was as follows: 94°C for 30 seconds (denaturation), 60°C for 1 minute (annealing), and 68°C for 2 minutes (extension). PCRs were finished with a final extension step of 68°C for 7 minutes and the products were stored at 4°. The PCR products were separated by electrophoresis in 1.5% agarose gel. The PCR products were visualized by UV transillumination after staining with 0.5 μg/ml ethidium bromide. Determination of antibody titer by HI and ELISA tests - The sera samples were collected from all vaccinated and nonvaccinated birds and subjected to HI test to determine the antibody titer. To perform HI test the sera samples were heated into water bath maintaining 56⁰C for 30 minutes. The AF was used as test virus after determination of. At first two fold dilutions of the sera were prepared using PBS which were mixed with 4 HA unit virus and the plate was left for one hour at room temperature followed by addition of 1% (v/v) cRBCs to each well for 45 minutes at room temperature. The result of hemagglutination inhibition was recorded by observing the formation of button at the bottom of the plates. The antibody titer in the vaccinated and unvaccinated birds was determined by indirect ELISA test using commercially available NDV virus coated ELISA kit (as per manufacturer’s protocol). The test was performed according to the instruction of the manufacturer company and the absorbance of the test samples and control in the micro-titer plate was recorded at 405 nm using Multiscan Ex (Thermo, USA). Interpretation of results - For the test result to be valid the mean negative control absorbance should be ?0.30 and the difference between the mean negative control and the mean positive control should be ?0.15. The NDV positive control has been carefully standardized to represent significant amounts of antibody to NDV in chicken serum. The relative amounts of antibodies in chicken serum can then be calculated by reference to the positive control. This relationship is expressed as Sample to Positive (S/P) Ratio.

Source of Information: Microbes and Health, June 2014. 3(1): 1-4; ISSN: 2226-0153 (Print) 2305-3542 (Online);

Article Link (Online Journal): http://journal.bsvmph.org/

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In the ELISA test, it was found that the OD value of maternal antibody was 1.04±0.11 which was much higher than normal titer. But after BCRDV administration the OD value was not satisfactory (0.84±0.10) which may due to higher maternal antibody. Before experimental vaccination the OD value was 0.64±0.17. After experimental vaccination the OD value of all groups were (0.94±0.13, 0.90±0.117, 0.82±0.15, 0.76±0.11, 0.45±0.19) respectively. From the OD value, S/P value of five groups were calculated as 0.77, 0.74, 0.67, 0.65, 0.34 and antibody titres were determined as 2549.71, 2450.37, 2218.579, 2152.352, 1125.846 respectively.

Knowledge Management: Journal