Development of oral vaccine for coccidiosis protection in chicken cloning of gapdh gene from coccidia species

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURAL AND FORESTRY DO THI THANH TRA TOPIC TITLE: DEVELOPMENT OF ORAL VACCINE FOR COCCIDIOSIS PROTECTION IN CHICKEN: CLONING OF GAPDH GENE FROM COCCIDIA SPECIES INTERNSHIP DIARY Study Mode : Full-time Major : Biotechnology Faculty : Biotechnology and Food Technology Batch : 2014 – 2018 Thai Nguyen, 6/2018 THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURAL AND FORESTRY DO THI THANH TRA TOPIC TITLE: DEVELOPMENT OF ORAL VACCINE FOR COCCIDIOSIS PROTECTION IN CHICKEN: CLONING OF GAPDH GENE FROM COCCIDIA SPECIES BACHELOR THESIS Study Mode : Full-time Major : Biotechnology Faculty : Biotechnology and Food Technology Batch : 2014 – 2018 Thai Nguyen, 6/2018 DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Major Biotechnology Student name Tra Thi Thanh Do Student ID DTN1453150027 Thesis title Supervisors Development of oral vaccine for Coccidiosis protection in chicken: cloning of GAPDH gene from coccidia species Asst Prof Dr Kanokwan Poomputsa Assoc Prof Dr Duong Van Cuong Abstract: Coccidiosis is one of the most important diseases in poultry and often causes by simultaneous infections of several Eimeria species Every chicken in a production systemis considered to be infected with one or more Eimeria species and economic losses are estimated to be over billion dollars annually Control of avian coccidiosis is currently accomplished by either medication of feed with anti-coccidial drugs or administration of live vaccines composed of low doses of Eimeria oocysts The increasing incidence of drug-resistance and cost of live vaccines is prompting alternative control strategies, such as immunization of chickens with recombinant Eimeria proteins GAPDH is one of the immunogenic common antigens among Eimeria tenella, E acervulina, and E.maxima and a key glycolytic enzyme in the process of metabolism of coccidian, as several pathogenic protozoa entirely depend on glycolysis as the source of ATP in the host Thus, protozoan GAPDHs are considered potential targets for antiprotozoan drugs The genes of GAPDH cloned from E.acervulina and E.maxima were named as EaGAPDH and EmGAPDH, respectively Total RNA from Coccidian oocyst were extracted by using three method to compare RNA concentration cDNAs were synthesized by reverse transcription reaction with primers specific to EaGAPDH and EmGAPDH The first strand cDNA synthesis was amplified by PCR The PCR product will be ligated into pGEM-TA vector Keywords Coccidia – oocyst- Eimeria – GAPDH Number of pages 35 i ACKNOWLEDGMENTS Firstly, I would like to express my sincere gratitude to my main advisor Asst Prof Dr Kanokwan Poomputsa for the continuous support of my internship and related research, for her patience, motivation, and immense knowledge Her guidance helped me in all the time of research and writing of this thesis I am also gratefully thank to Assoc Prof Dr Duong Van Cuong, my co-advisor who always providing useful advice for the improvement of this work I thank my fellow labmates at Animal Cell Culture (ACC) laboratory, for their advises, kind motivation, and warm friendship during my internship I would also like to acknowledge my teachers at TUAF, Assoc Prof Dr Duong Van Cuong, MSc Trinh Thi Chung, Dr Nguyen Xuan Vu that contributed to making this work and had an enjoyable and fulfilling experience Last but not the least, I would like to thank my family: my parents and to my brothers and sister for supporting me spiritually throughout writing this thesis and my life in general Many thank you and best regards Student Do Thi Thanh Tra ii CONTENTS ACKNOWLEDGMENTS ii CONTENTS iii LIST OF TABLE v LIST OF FIGURES vi LIST OF ABBREVIATION .vii CHAPTER INTRODUCTION 1.1 Research rationale 1.1.1 Chicken coccidiosis 1.1.2 Characteristic of chickens coccidia 1.1.3 Life cycle of Eimeria 1.1.4 Coccidian oocyst wall 1.1.5 Glyceraldehyde 3-Phosphate dehydrogenase (GAPDH) 1.2 Objectives 12 1.3 Scope of work 12 CHAPTER 14 MATERIALS AND METHODS 14 2.1 Instruments and materials 14 2.1.1 Types of instruments 14 2.1.2 Chemicals and materials 15 2.2 Methods 15 2.2.1 Preparation of Coccidia oocysts 15 iii 2.2.2 Preparation total RNA from Coccidian oocysts 17 2.2.3 First strand cDNA Synthesis and PCR Amplification 21 CHAPTER 24 RESULTS AND DISCUSSIONS 24 3.1 Coccidian oocysts isolation 24 3.2 Breaking Coccidian oocysts 26 3.3 Total RNA isolation 27 3.4 cDNA synthesis and RT-PCR 29 CHAPTER 32 CONCLUSION 32 4.1 Isolation Coccidian oocysts from coccivac®- D 32 4.2 Isolating and cloning GAPDH gene 32 4.3 Recommendations 32 REFERENCES 33 APPENDIX 35 iv LIST OF TABLE Table 1.1 Site of development, relative pathogenicity and relative immunogenicity of the seven species of Eimeria parasitic in chickens Table 1.2 Scientific classification of E.acervulina Table 1.3 Scientific classification of E.maxima Table 2.1 The instruments are used in study 14 Table 2.2 Chemicals are used in study 15 Table 2.3 The specific primer of E.acervulina and E.maxima 23 Table 3.1Evaluation of quality and quantity parameters of RNA samples extracted from Coccidian oocysts by Nanodrop 28 v LIST OF FIGURES Figure 1.1 Life cycle of coccidia (Eimeria spp.) Figure 1.2 Sequence of EaGAPDH Figure 1.3 Sequence of EmGAPDH 10 Figure 1.4 Amino acid similarities of GAPDH between Eimeria acervulina, E maxima, E tenella, E necatrix and E brunetti 11 Figure 2.1 Coccivac®-D 16 Figure 2.2 Step of oocysts purification 16 Figure 2.3 Extraction RNA using TRIzol method 17 Figure 2.4 Principle of MagListoTM5M Tissue Total RNA Extraction Kit 20 Figure 3.1 Steps of oocysts isolation from coccivac using density of sucrose: 24 Figure 3.2 Isolation Coccidian oocysts from Coccivac D under microscope 25 Figure 3.3 Breaking Coccidian oocysts 27 Figure 3.4 RNA concentrations from different RNA isolation 29 Figure 3.5 PCR product from cDNA synthesis using oligodT with taq polymerase on 1% agarose gel………………………………………………………………………………30 Figure 3.6 PCR product using normal taq polymerase on 1% agarose gel 31 vi LIST OF ABBREVIATION ºC Degree Celsius µg microgram µL microliter ATP Adenosine triphosphate BLP Bacteria- like- Particles dNTP deoxynucleoside triphosphates E.acervulina Eimeria acervulina E.maxima Eimeria maxima E.tenella Eimeria tenella EaGAPDH Eimeria acervulina Glyceraldehyde 3-Phosphate dehydrogenase EmGAPDH Eimeria maxima Glyceraldehyde 3-Phosphate dehydrogenase FDA Food and Drug Administration GAPDH Glyceraldehyde 3-Phosphate dehydrogenase GEM Gram-positive enhancer matrix GRAS Generally Regarded As Safe LysM Lysin Motif minute mL mililiter mm milimeter mM miliMolar ng Nanogram PBS Phosphate buffered saline pH Potential of hydrogen rpm Revolutions per minute RT-PCR Reverse transcription polymerase chain reaction sec second U Unit vii CHAPTER INTRODUCTION 1.1 Research rationale Coccidiosis is one of the most important diseases in poultry and often causes by simultaneous infections of several Eimeria species Coccidiosis inflicts the birds in both clinical and sub-clinical forms The clinical form of the disease are recorded through some remarkable signs like mortality, morbidity, diarrhea or bloody feces, and subclinical coccidiosis manifests mainly by poor weight gain and reduced efficiency of feed conversion and gives rise to highest proportion of the total economic losses [1] Nowday the methods for control of coccidiosis are incorporation of anticoccidial agent into feed or water, and use of live vaccines [2] The first commercial live anticoccidial vaccine, CocciVac, was introduced to the US market in 1952 It comprised a mixture of wild-type strains of E tenella oocysts, and conferred a homologous protection against those strains included in the mixture Therefore, the vaccine went through a number of reformulations over the past decades and variants of the original product, CocciVac-B®, CocciVacD® and Immucox®, are still in use today in more than 40 countries [3, 4] In parallel live oocysts vaccines proved to be efficient in turkeys[5] However, drawbacks of live vaccines include safety concern, short shelf-life and difficulties of large-scale production Since the live vaccines against coccidia are costly to produce given further that these vaccines are strain- and species-specific, a cocktail of antigens may be requires in order to raise protective immunity effectively Therefore, there is continued interest in devising new vaccines using defined recombinant antigens Despite that oral vaccines are safe and easy to use and convenient for all ages, all objects Induction of mucosal immunity is essential to stop person-to-person transmission of pathogenic microorganisms and to limit their multiplication within the mucosal tissue Vaccination through a mucosal route is shown to offer advantages for enhanced mucosal immune responses that result in better local protection [6] Mucosal immunization with ... UNIVERSITY UNIVERSITY OF AGRICULTURAL AND FORESTRY DO THI THANH TRA TOPIC TITLE: DEVELOPMENT OF ORAL VACCINE FOR COCCIDIOSIS PROTECTION IN CHICKEN: CLONING OF GAPDH GENE FROM COCCIDIA SPECIES BACHELOR... changed to ? ?Cloning of GAPDH gene from Coccidia species for Coccidia oral vaccine production” which is the first important step for development oral vaccine 1.1.1 Chicken coccidiosis Coccidiosis... necatrix and E brunetti ChGAPDH =GAPDH of Chichken; EtGAPDH =GAPDH EbGAPDH =GAPDH of of E tenella; E.brunetti; EaGAPDH =GAPDH of EmGAPDH =GAPDH E acervulina; of E.maxima; EnGAPDH =GAPDH of E necatrix[20] BLAST