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 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 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 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
iii


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

iv


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

v


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

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

min

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 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 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 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

1


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.

2


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

E. tenalla

Caeca

High to very high Low

E. mitis

Ileum

Low

Moderate

E. praecox

Duodenum, jejunum

Low

Moderate

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
3


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].

4


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 watersoluble 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
5


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

6


1.1.5.1 Eimeria Acervulina
a. Classification
Table 1.2 Scientific classification of E.acervulina

Domain:

Eukaryota

(unranked):

Sar

(unranked):

Alveolata

Phylum:

Apicomplexa

Class:

Conoidasida

Order:

Eucoccidiorida

Family:

Eimeriidae

Genus:

Eimeria

Species:

E. acervulina

b. Sequence of EaGAPDH

7


10
20
30
40
50
60
.... | .... | .... | .... | .... | .... | .... | .... | .... | .... | .... | .... | ....

EaGAPDH

1

ATGGTGTGCCGTATGGGAATCAACGGCTTCGGCCGCATCGGCCGTTTGGTCTTCCGCGCCGCTA
110
120
130
140
150
160
....|....|....|....|....|....|....|....|....|....|....|....|....

EaGAPDH

101

TGATGAGTGGTAACAGATAGAAACAGCACACAATGCTTTTCATTTACGCCCTTTTCCATTGAAT
210
220
230
240
250
260
.... | .... | .... | .... | .... | .... | .... | .... | .... | .... | .... | .... | ....

EaGAPDH

201

AATAGCACTACTCAGGCTACCCCCGCAGCGCTCTCTTTCGCACACACACACGCACACACACGCA
310
320
330

340
350
360
....|....|....|....|....|....|....|....|....|....|....|....|....

EaGAPDH

301

CTCAGGTGCAGAAGGTGAAAAGTCTTGTAAAATGTCCTGCGATTATGTATAAAGTGTGCAGATG
410
420
430
440
450
460
....|....|....|....|....|....|....|....|....|....|....|....|....

EaGAPDH

401

CAGTGAACGACCCGTTCATGGACGTGCAGTACATGGCCTACCAGCTGAAGTACGACTCTGTGCA
510
520
530
540
550
560
....|....|....|....|....|....|....|....|....|....|....|....|....

EaGAPDH

501

CAATTTGGTAGTGGAGGGGAAGACAATCCAAGTGTTTGCTGAGAAGGACCCCGCAGCCATTCCT
610
620
630
640
650
660
....|....|....|....|....|....|....|....|....|....|....|....|....

EaGAPDH

601

ACTGGTGTATTCACAAACAAGGAGAAGGCTGGTCTGCACATATCTGGCGGTGCTAAGAAGGTCA
710
720
730
740
750
760
.... | .... | .... | .... | .... | .... | .... | .... | .... | .... | .... | .... | ....

EaGAPDH

701

TCGTTATGGGTGTGAACCACGAGGAATACCAGCCCACTCTTCAGGTTGTTTCTAATGCTTCCTG
810
820
830
840
850
860
....|....|....|....|....|....|....|....|....|....|....|....|....

EaGAPDH

801

GCACGAGAAGTTCGGTATTGTTGAGGGTCTTATGACCACCGTGCACGCTATGACAGCTAACCAG
910
920
930
940
950
960
.... | .... | .... | .... | .... | .... | .... | .... | .... | .... | .... | .... | ....

EaGAPDH

901

TGGAGGGCTGGACGCTGTGCAGGCAGCAACATTATCCCTGCAAGCACAGGTGCAGCAAAGGCAG
1010
1020

1030
1040
1050
1060
....|....|....|....|....|....|....|....|....|....|....|....|....

EaGAPDH

1001 CAGGCATGGCTTTCCGTGTGCCCACCCCTGACGTCTCCGTCGTCGACCTCACATGCAGGCTGTC
1110
1120
1130
1140
1150
1160
....|....|....|....|....|....|....|....|....|....|....|....|....

EaGAPDH

1101 CCGTGCAGCTTCTGAGGGCCCCCTTAAGGGTATTTTGGGTGTCACAGAGGAGGAGGTTGTATCC
1210
1220
1230
1240
1250
1260
....|....|....|....|....|....|....|....|....|....|....|....|....

EaGAPDH

1201 GACGTCAAGGCAGGTATTCAGCTTAACGACTCCTTCGTTAAGCTCGTCTCCTGGTATGACAACG
1310
1320
....|....|....|....|....|....

EaGAPDH

1301 TCTACATGTCTAAGAAGGACGGCAACTAA

Figure 1.2 Sequence of EaGAPDH

8


1.1.5.2 Eimeria Maxima
a. Classification
Table 1.3 Scientific classification of E.maxima

Domain:

Eukaryota

(unranked):

SAR

(unranked):

Alveolata

Phylum:

Apicomplexa

Class:

Conoidasida

Order:

Eucoccidiorida

Family:

Eimeriidae

Genus:

Eimeria

Species:

E. maxima

b. Sequence of EmGAPDH

9


10

20
30
40
50
60
....|....|....|....|....|....|....|....|....|....|....|....|....

EmGAPDH

1

ATGGTTTGCCGCATGGGCATCAACGGCTTCGGCCGCATTGGCCGCCTGGTGTTTCGCGCTGCCA
110
120
130
140
150
160
....|....|....|....|....|....|....|....|....|....|....|....|....

EmGAPDH

101

TTTGTTGTGTATTCCCTCAGGAACAGGAAATAGGCAGTAGTATAGCCAGTAAATAACGCACCAC
210
220
230
240
250

260
....|....|....|....|....|....|....|....|....|....|....|....|....

EmGAPDH

201

GTCGTTCGGGGGAGCTGACTCTAACCGGCAACCCCCGCAGGAGCAGCAGCACCAACACTAGAAG
310
320
330
340
350
360
.... | .... | .... | .... | .... | .... | .... | .... | .... | .... | .... | .... | ....

EmGAPDH

301

CGAGAACATTCATTGGCCGTGCGTGAAGTGCATGCAATCTTCTTTGTTTGGGGCGCCTTTATCT
410
420
430
440
450
460
....|....|....|....|....|....|....|....|....|....|....|....|....

EmGAPDH

401

AGGAGGTCGTTGCAGTGAACGACCCTTTCATGGACGTGCAGTACATGGCCTACCAGCTTAAGTA
510
520
530
540
550
560
....|....|....|....|....|....|....|....|....|....|....|....|....

EmGAPDH

501

CGTTAAGGACGGAAACTTGGTTGTCAACGGCAAGACCATCAATGTGTTCGCCGAGAAGGAGCCC
610
620
630
640
650
660
.... | .... | .... | .... | .... | .... | .... | .... | .... | .... | .... | .... | ....

EmGAPDH

601

GTGTGCGAGTCCACCGGTGTCTTCACCAACAAGGAGAAGGCCGGGCTCCACATCGGTGGTGGTG

710
720
730
740
750
760
....|....|....|....|....|....|....|....|....|....|....|....|....

EmGAPDH

701

ACACCCCCATGTTCGTCATGGGTGTGAACCACGAGGAGTACCAGTCCTCTCTCCAGGTGGTCTC
810
820
830
840
850
860
....|....|....|....|....|....|....|....|....|....|....|....|....

EmGAPDH

801

TGCAAAGGTCGTTCACGAGAAGTTCGGCATTGTTGAGGGTCTCATGACAACTGTGCACGCTATG
910
920
930
940

950
960
....|....|....|....|....|....|....|....|....|....|....|....|....

EmGAPDH

901

GGTGGCAAGGACTGGAGAGCTGGACGTTGCGCAGGCAGCAACATCATCCCCGCAAGCACCGGAG
1010
1020
1030
1040
1050
1060
.... | .... | .... | .... | .... | .... | .... | .... | .... | .... | .... | .... | ....

EmGAPDH

1001 ACGGCAAGCTCACTGGTATGGCCTTCCGTGTGCCCACACCCGACGTCTCCGTCGTCGACCTTAC
1110
1120
1130
1140
1150
1160
....|....|....|....|....|....|....|....|....|....|....|....|....

EmGAPDH

1101 CGTCGCTGCCATCCGCGCTGCCTCTGAGGGTCCCCTCAAGGGCATCCTCGGCGTCACCGATGAG
1210
1220
1230
1240
1250
1260
....|....|....|....|....|....|....|....|....|....|....|....|....

EmGAPDH

1201 TCCTCCATCTTCGACGTCAAGGCCGGTATCCAGCTCAACGACACCTTCGTTAAGCTCGTCTCCT
1310
1320
1330
1340
.... | .... | .... | .... | .... | .... | .... | .... | .

EmGAPDH

1301 TTGACCTCGCCATCTACATGTCCAAGAAGGACGGCAACTGA

Figure 1.3 Sequence of EmGAPDH.

10


1
1

2

3

4

5

6

69.7

68.4

69.7

68.8

69.1

1

ChGAPDH.pro

87.7

95.0

92.6

94.1

2

EaGAPDH.pro

88.7

86.5

87.4

3

EbGAPDH.pro

91.4

92.6

4

EmGAPDH.pro

97.3

5

EnGAPDH.pro

6

EtGAPDH.pro

2

38.8

3

41.0

13.4

4

38.8

4.3

12.3

5

40.3

7.8

15.0

9.1

6

39.8

6.2

13.8

7.8

2.7

1

2

3

4

5

6

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
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 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.

11


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

12


Development oral vaccine using
BLPs for Coccidosis in Chicken

Lactococcus lactis culture,

Isolation and purification

growth in MRS broth medium

Coccidian oocysts from

and harvesting cells
L.Lactis treatment to make

Binding of GAPDH- PA to
BLP

Breaking Coccidian

Isolation total RNA from
Coccidian oocysts

First strand cDNA
My work

Synthesis and

ACC lab’s work

Cloning of Eimeria
GAPDH gene
Preparation of GAPDH–
protein anchor fusions

Overexpression of
GAPDH- PA fusions

13


CHAPTER 2
MATERIALS AND METHODS
2.1 Instruments and materials
2.1.1 Types of instruments

Table 2.1 The instruments are used in study

No.
1

Name

Company

Holten MS-2010 1.2 Laminar Flow Thermo Scientific

Country
USA

Cabinet
2

Microscope D70

Olympus

US

3

Centrifuge MIKRO 200 Zenfuge

Hettich

Germany

4

Vortex genie 2

Scientific

USA

Industries Inc.
5

Accublock Digital Dry Bath

Labnet

USA

International
6

Sorvall Legend (centrifuge machine) Thermo Scientific

USA

7

Autoclave HVE-50

Hirayama

Japan

8

Water Bath WB7 bain-marie

Memmert

Germany

9

T100 Thermal Cycler

Bio-Rad

USA

10

E1101

Accuris

MyGel

Mini Benchmark

USA

Electrophoresis System

Scientific

11

UV InGenius3

SYNGENE

USA

12

Autopipette

Labnet

USA

International
13

Microscopic slide HAD

Yancheng Huida

China

medical
instruments co.,
Ltd
14

Glassbeads 0.5mm

BioSpec

US

15

Nanodrop 1000

Thermo Scientific

USA

14


2.1.2 Chemicals and materials
Table 2.2 Chemicals are used in study

No.

Name

Company

Country

Schering-Plough

USA

1

Coccivac®-D

2

Sucrose (C12H22O11)

Ajax Finechem Pty Ltd.

New Zealand

3

Glycerol

Ajax Finechem Pty Ltd.

New Zealand

Ajax Finechem Pty Ltd.

New Zealand

MERCK

Germany

Signma Aldrich

Germany

Carlo Erba Reagent

Italy

4
5

Disodium hydrogen phosphate
(Na2HPO4)
Phenol
Disodium hydrogen phosphate

6

dodecahydrate
(Na2HPO4.12H2O)

7

Sodium Chloride(NaCl)

8

Chloroform

Thermo Scientific

USA

9

Ethanol

Thermo Scientific

USA

10 TRIzol

Invitrogen

USA

11 Agarose

Thermo Scientific

USA

Signma Aldrich

Germany

LiChrosolv®

Germany

Qiagen

Germany

Bioneer

Korea

12 Trichloroacetic acid(C2HCl3O2)
13 Isopropanol
14 Rneasy Protect Mini Kit 50
15

MagListo™ 5M Tissue Total
RNA Extraction Kit

2.2 Methods
2.2.1 Preparation of Coccidia oocysts
Coccivac®-D, a commercial coccidiosis vaccine from Schering-Plough company
(USA), was used as unsporulated oocysts resource. This vaccine contains live
oocysts of E.acervulina, E.mivati, E.maxima, E. tenella, E.necatrix, E.praecox,
E.brunetti and E.hagani. Purified Coccidia oocysts by Gradient and Floatation
technique using Modified Sheather solution (500 grams of sucrose, 5 mL of Phenol
and 350 mL of distilled water)[21]. This simple technique is used for separating

15


oocysts from large quantities of fecal material. By allowing the heavier particles to
sediment from a suspension of fecal material, the lighter oocysts could be collected
along with the supernatant. Unsporulated oocysts were mixed with MSS and vortex
then carefully overlay the sucrose suspension with distilled water. Next, centrifuge
5,000 rpm for 5 min at room temperature. Carefully collect the top water and
transfer to new tube. Repeat this step several times. Add full tube with distilled
water then centrifuge 14,000 rpm for 20 min. Throw supernatant then wash pellet
with PBS several times. Finally, resuspend pellet in distilled water. Impurity check
was performed under microscope. The extracted coccidia was kept at -20ºC until
used.

Figure 2.1 Coccivac®-D

Firgure 2.2 Step of oocysts purification[21].

16