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Genetic Evolution and Development of Recombinant Vaccine Against Newcastle Disease for Chicken in Pakistan

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Genetic Evolution and Development of Recombinant Vaccine Against Newcastle Disease for Chicken in Pakistan GENETIC EVOLUTION AND DEVELOPMENT OF RECOMBINANT VACCINE AGAINST NEWCASTLE DISEASE FOR CHICKEN IN PAKISTAN ABDUL WAJID 2009-VA-705 A THESIS SUBMITTED IN THE PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN MOLECULAR BIOLOGY AND BIOTECHNOLOGY UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES, LAHORE 2017 i To, The Controller of Examination, University of Veterinary and Animal sciences, Lahore. We, the Supervisory Committee, certify that the contents and form of the thesis, submitted by Mr. Abdul Wajid, Regd. No. 2009-VA-705 have been found satisfactory and recommend that it be processed for the evaluation by the External Examiner (s) for award of the degree. Supervisor: ____________________________________ Dr. Muhammad Wasim Member: ____________________________________ Prof. Dr. Tahir Yaqub Member: ____________________________________ Dr. Muhammad Tayyab ii iii IN THE NAME OF ALLAH, THE MOST COMPASSIONATE, THE MOST MERCIFUL All praises and thanks are for Almighty Allah, The source of all knowledge and wisdom endowed to mankind, who guides us in darkness and helps us in difficulties And all respects are for His last Holy Prophet HAZRAT MUHAMMAD (Peace Be Upon Him) Who enabled us to recognize our creator. iv Dedicated To My Parents & My late Brother Abdul Raziq (Jaan) & My Supervisor & Dr. SF Rehmani Who always encouraged me to Achieve higher goals in life v ACKNOWLEDGEMENTS I would like to give all my praises and humblest thanks to the Most Gracious, Merciful and ALMIGHTY ALLAH, who guides us in darkness and thankful to my ALLAH, who has conferred me with potential and ability to complete this research study. I offer mu humblest thanks from the core of my heart to the Holy Prophet Muhammad (S.A.W.), who is forever a torch of guidance and knowledge for humanity as a whole. This thesis has been completed as a collaborative research project between South East Poultry Research Laboratory (SEPRL), USA and Quality Operations Lab, University of Veterinary and Animal Sciences, Pakistan, entitle “Molecular characterization of NDV and Development of approaches to vaccination”USDA-ARS-BEP CRDF Newcastle Disease Virus program #31063 sponsored by theUnited States Department of State. I am thankful to this donor agency for providing the financial support for the described work. I feel enormous intensity of obligation to my respected Supervisor, Dr. Muhammad Wasim, Associate Prof. IBBt-UVAS, Lahore for his valuable guidance, stimulating ideas and extreme patience with my work, which proved to be a panacea in the completion of this thesis. I have no word to thank Dr. Shafqat Fatima Rehmani, for not only support in studies, generous advice, inspiring guidance, and encouragement through my research, she taught me everything about life. I have deep sense of appreciation to the members of my Supervisory Committee, Prof. Dr. Tahir Yaqub, Department of Microbiology and Dr. Muhammad Tayyab, for their personal interest and cooperation. I would especially like to express my deep sense of gratitude to Prof. Dr. Claudio L Afonso, Newcastle disease Lead Scientist, South East Poultry Research Laboratory (SEPRL), USA. Thank you Dr. Patti Miller, Kiril Dimitrov and Poonam Sharma for your unconditional scientific support. I would especially like to thank Asma Basharat for her inspiring attitude, kindness and help during this research. I also would like to thank Saima Arif and Abdul Basit for helping and support through this research work. I am very much thankful to my friends Kamran Abbas, AhsanUllah, Zia Uddin, Asif Rahim and especial thanks to Dr. Andleeb Batool for their moral support. I am grateful to my parents and whole family for their support and encouragement. I would like to thank my late brother Abdul Raziq (Jaan), I know where I am today is due to your’s prays. Now I know why you always told me to be strong because you knew, you knew that one day I would need the strength to bear your loss. Thanks for making me laugh every time as you were joking, singing and I love you so so much. Abdul Wajid vi CONTENTS DEDICATION ------------------------------------------------------ i ACKNOWLEDGEMENTS---------------------------------------- ii TABLE OF CONTENTS ------------------------------------------ iii ABSTRACT --------------------------------------------------------- iv SR. NO. CHAPTER PAGE NO. 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 6 3 EXPERIMENT 1 53 4 EXPERIMENT 2 73 5 EXPERIMENT 3 96 6 EXPERIMENT 4 113 7 EXPERIMENT 5 118 8 SUMMARY 140 vii ABSTRACT Newcastle disease (ND) is one of the most contagious diseases of poultry worldwide. The disease is endemic in Pakistan and recurrent outbreaks have been reported in commercial poultry flocks, domestic pet and migratory birds since 1963 an inception of commercial poultry farming in the country. Disease surveillance is necessary to determine the incidence of the disease as well as to identify the etiological agent of the disease status in the region. The analysis of the field data provides a clue for the higher authorities to take steps for the remedy of the devastating outbreak. A virulent strain (or variant) of Newcastle disease virus caused an outbreak in the northern region of Pakistan during the mid of 2011. The virus was identified as a virulent viscerotropic vvNDV and characterized, belonging to the sub genotype VIIi. However, the virus of this genotype is still circulating in the field though the intensity of the strain to succumb the chickens to cause mortality does not exist. The particular thing in this genotype was its susceptibility to other avian species like pheasants, peafowls, ducks turkeys, peacocks, sparrows and parakeets. As this genotype is circulating since 2011, until 2016 and occasionally still spill over in these avian species. Thus for the last five years (2011-16), 3500 healthy, diseased and dead chickens, pheasants, peacocks, turkeys, peafowls, ducks, sparrows, exotic parakeets, rosy- faced parrots, pigeons, and partridges from 750 different locations were monitored. Samples were collected from the Northern region of the country including Punjab, Khyber Pakhtoonkhawa, Azad Kashmir, as well as Gilgit Baltitssan and from Southern region, Karachi, Hyderabad, Mirpursakro and other small cities where the poultry farms are located. The samples were collected by the local veterinarians, poultry assistants and animal health practitioners who participated during the surveillance program. Samples were also collected from the farmers who brought their birds for inspection in the lab with the details of the farm locations. Mostly, sampling was done where there were reports of NDV outbreak, tissues were collected usually the trachea, spleen and brain. In addition, the pharyngeal and cloacal swabs were also collected the healthy birds living amongst the infected bird in order to assess the virus shedding in the flock. Blood samples were also collected (1% of the birds at farm), and the sera were used to assess the immune status of the flock using Haemagglutination Inhibition (HI) test and Enzyme linked immunosorbant assay (ELISA). The Survey Form met the international standard was filled for each farm for recording the information required to find the diagnostic clue as well as the viii molecular characterization of the isolates. Pool of five pharyngeal swabs were processed after the passage into 9-day old chicken embryonated eggs and confirming the positive HA test and then confirmed by real time PCR (RT-PCR). In addition, sera were tested against NDV by HI and ELISA tests. The targeted samples were sequenced by complete fusion gene and whole genome, using 22 pairs of overlapping primers. The observations indicated that the commercial broiler industry is highly susceptible to virulent NDV and confirmed by data available in the laboratory in the survey form. Contrary to that a little is known regarding the maintenance and enzootic trends of vNDV infection level in domestic and wild birds. Poor strategy of the use of vaccines and vaccination as well as the existence of virulent form of NDV in the domestic and pet birds indicate a possibility of the root cause of the ND eruption in the developing countries. A continuous isolation of virulent viruses of the panzootic Newcastle disease virus of sub-genotype VIIi since (2011-2016) from commercial chickens and from various other avian species in the country provide an evidence for the existence of epidemiological links intermingling of the strain among them. Therefore, to avoid the huge economical losses in the commercial poultry, the second largest industry in Pakistan, their close proximity should be strictly avoided. The mass vaccination of the poultry flocks is a common practice in all commercial poultry farms in Pakistan. However, the use and availability of a reliable and standard vaccine, as well as the correct usage of vaccine dose of the live attenuated LaSota vaccine are the key factors to improve their efficacy in the field. Minor outbreaks have been occurring in the field even though a severe outbreak has occurred in 2011-12, that almost collapsed the poultry industry with other pet and wild birds. To minimize the continuity of these minor outbreaks in the field for a long time period, there is a need for more effective vaccine to control the particular genotype of the ND virus. In the present study, DNA vaccine was developed using the SFR-55 NDV strain as an antigens, in the form of fusion (F) and hemagglutinin-neuraminidase (HN), namely pcDNA3.1-F and pcDNA3.1-HN. In vitro expression of both genes construct was assessed by reverse- transcriptase-PCR (RT-PCR) and western blotting. In the trial an inactivated oil-based emulsion vaccine was prepared using the field strain SFR-55 and compare with the commercial ND vaccine (LaSota strain) commonly used by the poultry industry. Birds were divided into six groups, the first two groups were immunized with pcDNA3.1-F and pcDNA3.1-HN alone respectively and third group was vaccinated with both antigens pcDNA3.1-F+HN. The other two groups were immunized with inactivated (wvSFR-55) and LaSota vaccines as described above, ix the last group was injected with empty vector as control.
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