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

DO THI THANH TRA TOPIC TITLE: DEVELOPMENT OF ORAL VACCINE FOR COCCIDIOSIS PROTECTION IN CHICKEN: CLONING OF GAPDH GENE FROM... UNIVERSITY OF AGRICULTURAL AND FORESTRY DO THI THANH TRA

DO THI THANH TRA

TOPIC TITLE: DEVELOPMENT OF ORAL VACCINE FOR COCCIDIOSIS PROTECTION IN CHICKEN: CLONING OF GAPDH GENE FROM

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

DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry

Student name Tra Thi Thanh Do

Thesis title Development of oral vaccine for Coccidiosis protection in

chicken: cloning of GAPDH gene from coccidia species

Supervisors 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 1 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 anti-

protozoan 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

Number of pages 35

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

CONTENTS

ACKNOWLEDGMENTS ii

CONTENTS iii

LIST OF TABLE v

LIST OF FIGURES vi

LIST OF ABBREVIATION vii

CHAPTER 1 1

INTRODUCTION 1

1.1 Research rationale 1

1.1.1 Chicken coccidiosis 2

1.1.2 Characteristic of chickens coccidia 3

1.1.3 Life cycle of Eimeria 3

1.1.4 Coccidian oocyst wall 5

1.1.5 Glyceraldehyde 3-Phosphate dehydrogenase (GAPDH) 6

1.2 Objectives 12

1.3 Scope of work 12

CHAPTER 2 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

2.2.2 Preparation total RNA from Coccidian oocysts 17

2.2.3 First strand cDNA Synthesis and PCR Amplification 21

CHAPTER 3 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 4 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

LIST OF TABLE

Table 1.1 Site of development, relative pathogenicity and relative immunogenicity of the

seven species of Eimeria parasitic in chickens 3

Table 1.2 Scientific classification of E.acervulina 7

Table 1.3 Scientific classification of E.maxima 9

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

LIST OF FIGURES

Figure 1.1 Life cycle of coccidia (Eimeria spp.) 5

Figure 1.2 Sequence of EaGAPDH 8

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

LIST OF ABBREVIATION

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

GAPDH Glyceraldehyde 3-Phosphate dehydrogenase

RT-PCR Reverse transcription polymerase chain reaction

CHAPTER 1 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 sub-clinical 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 6 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

subunit vaccines requires new types of antigen delivery vehicles and adjuvants for optimal immune responses

GAPDH is one of the immunogenic common antigens among Eimeria tenella,

E acervulina , and E.maxima GAPDH is 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 anti-protozoan drugs.[7]

This study was conducted by cloning of GAPDH gene from the Coccidia species total RNA extracted using the RT-PCR technique to amplify the cDNA sequence of the GAPDH gene Thus, the title of this study is 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 is a common protozoan disease in domestic birds and other fowl, characterized by enteritis and bloody diarrhoea The intestinal tract is affected, with the exception of the renal coccidiosis in geese Clinically, bloody faeces, ruffled feathers, anaemia, reduced head size and somnolence are observed Depending on the localization of lesions in intestines, the coccidioses are divided into caecal,

induced by E tenella, and small intestinal, induced by E acervulina, E brunetti, E

maxima , E mitis, E mivati, E necatrix, E praecox and E nagani In caecal

coccidiosis, a marked typhlitis is present and haemorrhages are seen through the intestinal wall Each species has its own characteristic, site of infection, pathogenicity and immunogenicity as shown in Table 1 [7][8]

Symptoms of coccidiosis in chickens include droopiness and listlessness, loss of appetite, loss of yellow color in shanks, pale combs and wattles, ruffled, unthrifty feathers, huddling or acting chilled, blood or mucus in the feces, diarrhea, dehydration, and even death[1] Other signs include poor feed digestion, poor weight gain, and poor feed efficiency Some symptoms can be confused with other diseases For example, necrotic enteritis is a gut disease that also causes bloody diarrhea

1.1.2 Characteristic of chickens coccidia

Coccidia are microscopic, spore-forming, single-celled protozoan parasite of the

phylum Apicomplexan and Sporozoasida class [9] At present, species of Eimeria

may be differentiated by the dimensions and morphology of the oocysts host- and site- specificity the morphology of the endogenous stages pathogenic effects immunological specificity (cross-immunity) the timing of the pre-patent and patent periods in experimental infections[9] The species of a given genus can rarely be differentiated by a balance of characters which may vary in significance for particular species

Table 1.1 Site of development, relative pathogenicity and relative immunogenicity of the

seven species of Eimeria parasitic in chickens[10]

Eimeria

species Site of development Pathogenicity Immunogenicity

E brunetti Small intestine Moderate to high High

E maxima Jejunum, ileum Moderate to high High to very high

E acervulina Duodenum, ileum Low to moderate Moderate

E necatrix Jejunum, ileum, caeca High to very high Low

1.1.3 Life cycle of Eimeria

The life cycle of Eimeria is complex but can be conveniently viewed as occurring in

three distinct stages – sporogony, schizogony and gametogony [11][12] as shown in

Figure 1.1 Under suitable environmental conditions of oxygen supply, humidity

and temperature, the free-living stage of the organism – the oocyst – undergoes

sporogony to form a sporulated oocyst Sporulated oocysts of Eimeria contain four

sporocysts, each of these containing two sporozoites Following ingestion of the sporulated oocyst by the host, the microenvironment of the host’s digestive tract stimulates excystation of the oocyst, resulting in the release of motile sporozoites [10] Sporocysts and then sporozoites are released in the gut from the sporulated

oocyst by excystation, a process facilitated by the physical grinding effect and the presence of digestive enzymes and bile salts The sporozoites penetrate the gut cells

to initiate development of asexual intracellular schizonts Schizonts divide many times producing large numbers of a second invasive stage, called merozoites that penetrate other gut cells to produce a further generation of schizonts [12][13] The number of asexual generations varies from two to four depending on the species of Coccidia [13][14] Asexual multiplication results in an exponential increase in parasite numbers Following the asexual lifecycle, a sexual lifecycle begins during which male and female gametes form The male and female gametes fuse to form a zygote which develops into an immature, unsporulated oocyst that is shed onto the litter in the feces With each successive cycle, the number of oocysts in the environment increases Unless immunity has developed or an anticoccidial is used, when the environmental conditions are favourable for sporulation of this built-up threat, the birds will not be able to cope with this sudden, massive exposure in the number of infective sporulated oocysts [12] [13]

Figure 1.1 Life cycle of coccidia (Eimeria spp.)

www.immucox.com/Coccidiosis/Lifecycle

1.1.4 Coccidian oocyst wall

The oocyst wall is extremely robust It is resistant to mechanical and chemical damage; breaking oocysts for laboratory studies requires prolonged, high-speed vortexing with glass beads and oocysts are routinely cleaned with bleach and stored

in the harsh oxidant, potassium dichromate, or the mineral acid, sulphuric acid[14][15] The wall is also resistant to proteolysis and impermeable to water-soluble substances, including many detergents and disinfectants[15][16]

The oocyst wall is essentially consistent in structure across different species of

coccidian parasites[16] but it is the oocyst wall of Eimeria that has been best

studied, largely because of the relative ease of acquiring large numbers of oocysts

of the parasites of this genera

The first serious microscopic and chemical examination of the oocyst wall (of E

maxima) was conducted by Monné and HQnig (1954), who used a number of destructive treatments that led them to conclude that the outer layer of the oocyst

wall contained mainly quinone-tanned proteins without lipids, since the outer layer reacted with ammoniacal silver nitrate solution (an indication of quinones) They also noted that the outer layer was stripped off upon exposure to sodium hypochlorite, whereas the structure of the inner layer remained unchanged, leading them to conclude that the inner layer consisted of a lipid-protein matrix; they believed that lipids were bound firmly to proteins, thus protecting the inner layer against disintegration by sodium hypochlorite

The first true biochemical examination of the oocyst wall was carried out by Ryley

in 1973 using E tenella Ryley (1973) also noted that the outer layer was removed

by sodium hypochlorite and found that it contained carbohydrates and proteins, with high proline content, whereas the inner layer consisted of 1.5% carbohydrates, 30% lipids and 70% proteins The lipid in the inner layer was extractable in chloroform and appeared to be a mixture of "waxes" containing very small amounts of nitrogen and phosphorus However, there are some limitations in this report: first, it did not show detailed analyses of the experimental work and, second, it did not document the metabolites detected in the inner wall

1.1.5 Glyceraldehyde 3-Phosphate dehydrogenase (GAPDH)

The immunogenic common antigens among E tenella, E acervulina and E maxima

is glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a key glycolytic enzyme that catalyzes the glyceraldehydes-3-phosphate to 1,3-diphosphoglycerate and generates NADP+ that can enter the respiratory chain and generates an ATP in the process of glycolysis Owing its role in the glycolysis pathway, GAPDH was regard merely as a housekeeping gene[17][18]

In recent years, more and more researchers found that GAPDH contains many isomers, the function is not only involved in energy metabolism but also related with many cellular processes of life, such as apoptosis, neuronal disorders, viralpathogenesis, phosphotransferase activity, membrane fusion, cell endocytosis regulating, microtubule binding, RNA output, DNA replication and DNA repair [18, 19]

In this report, two species are used for studying is Eimeria Acervulina and Eimeria

Maxima

Figure 1.2 Sequence of EaGAPDH

EaGAPDH 1301 T T C T T T AA G AA GG A GG C AA C AA

Figure 1.3 Sequence of EmGAPDH

Figure 1.4 Amino acid similarities of GAPDH between Eimeria acervulina, E

maxima , E tenella, E necatrix and E brunetti ChGAPDH=GAPDH of Chichken;

EtGAPDH=GAPDH of E tenella; EaGAPDH=GAPDH of E acervulina; EbGAPDH=GAPDH of E.brunetti; EmGAPDH=GAPDH of E.maxima;

EnGAPDH=GAPDH of E necatrix[20]

BLAST analysis revealed that EaGAPDH and EmGAPDH shared similarities of 99% in nucleotides and amino acid sequences with the genes in NCBI (Gene ID: EaGAPDH 25337292; EmGAPDH 25268815), respectively GAPDH shared similarities of more than 86% in amino acid among five species of chicken coccidia

ChGAPDH.pro EaGAPDH.pro EbGAPDH.pro EmGAPDH.pro EnGAPDH.pro EtGAPDH.pro

1.2 Objectives

1 Isolation Coccidian oocysts from coccivac®- D

2 Isolating and cloning GAPDH gene

1.3 Scope of work

1 Isolation and purification Coccidian oocysts from coccivac

2 Breaking Coccidian oocysts wall

3 Isolation total RNA from Coccidian oocysts

4 First strand cDNA Synthesis

5. PCR Amplification of First Strand cDNA of E.acervulina and E.maxima

Development oral vaccine using BLPs for Coccidosis in Chicken

Lactococcus lactis culture,

growth in MRS broth medium

and harvesting cells

L.Lactis treatment to make

Isolation and purification Coccidian oocysts from

Preparation of GAPDH– protein anchor fusions