nur amirah abdul latifpsasir.upm.edu.my/id/eprint/65892/1/fpv 2015 20 ir.pdfzoonotik bisul nodus...
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Reproductive physiology of caseous lymphadenitis in Katjang does following intradermal infection with Corynebacterium
pseudotuberculosis
NUR AMIRAH ABDUL LATIF
FPV 2015 20
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REPRODUCTIVE PHYSIOLOGY OF CASEOUS LYMPHADENITIS
IN KATJANG DOES FOLLOWING INTRADERMAL
INFECTION WITH
Corynebacterium pseudotuberculosis
By
NUR AMIRAH ABDUL LATIF
Thesis Submitted to the School of Graduate Studies,
Universiti Putra Malaysia, in Fulfillment of the Requirements for the Degree of
Master of Science
December 2015
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All material contained within the thesis, including without limitation text, logos, icons,
photographs and all other artwork, is copyright material of Universiti Putra Malaysia
unless otherwise stated. Use may be made of any material contained within the thesis for
non-commercial purposes from the copyright holder. Commercial use of material may
only be made with the express, prior, written permission of Universiti Putra Malaysia.
Copyright © Universiti Putra Malaysia
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DEDICATION
This thesis is dedicated to Allah s.w.t, family, thesis committee members and friends.
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of
the requirement for the degree of Master of Science.
REPRODUCTIVE PHYSIOLOGY OF CASEOUS LYMPHADENITIS IN
KATJANG DOES FOLLOWING INTRADERMAL INFECTION WITH
Corynebacterium pseudotuberculosis
By
NUR AMIRAH ABDUL LATIF
December 2015
Chairman : Faez Firdaus Jesse Abdullah, PhD
Faculty : Veterinary Medicine
Corynebacterium pseudotuberculosis is the etiological agent for zoonotic caseous
lymphadenitis (CLA). CLA is a zoonotic disease with high prevalence in sheep and
goats worldwide in chronic and subclinical forms. CLA cause high financial loss to
CLA endemic countries due to the condemnation of affected carcasses, reproductive
problems, decreased in milk and meat production, poor wool growth and mortality in
goats, sheep and other small ruminants. Therefore, the present study aims to examine
the presence of C. pseudotuberculosis in reproductive organs and iliac lymph node of
non-pregnant does in respect to cellular, hormone and cytokine changes inoculated
intradermally in chronic form. This is the first study to review the chronicity of the
infection in the reproductive organs and associated lymph node of non-pregnant does.
In this study, eighteen non-pregnant healthy Katjang does aged 2 years old were
divided randomly into two groups. The first and second group consisted of nine does
each and the two groups were subdivided into three subgroups. The first group was
experimentally inoculated with 1 ml of 107 cfu of live C. pseudotuberculosis by
intradermal route while the second group was given 1 ml of PBS (pH 7) intradermally.
The first group was further subdivided into three subgroups where the first subgroup
(B1) was kept for 30 days post infection, the second subgroup (B2) was kept for 60
days post infection and the third subgroup (B3) was kept for 90 days. The second group
was further subdivided into three subgroups (C1, C2, C3) where they were kept for 30,
60 and 90 days post infection respectively. During the post challenged periods, the
goats were observed for clinical signs and blood samples were collected at
predetermined intervals for three consecutive months for further analyses. Three goats
from the treatment group were sacrificed monthly and post mortem examination was
performed and reproductive organs and associated lymph nodes were collected for
histopathology. Isolation and identification of C. pseudotuberculosis and PCR were
conducted. Results showed there was successful isolation of C. pseudotuberculosis
from reproductive organs and iliac lymph nodes of the experimental does after the
second month from one of the infected does whereas the other two does only showed
positive isolation from the ovary. For the second and third month of post-infection, all
infected does showed positive C. pseudotuberculosis in the iliac lymph nodes and all
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reproductive organs. The concentration of IL-1β and IL-6 increased after three month
post-inoculation. Whereas, the hormonal response in the challenged does showed a
decrement pattern in progesterone concentration and increment pattern for estradiol
hormone throughout three month post-infection. The changes in the hormonal
concentration were further supported with the changes in the tissues of reproductive
organs in the challenged does. The histopathology of exposed group showed mild to
severe inflammatory cells, congestion, degeneration and necrosis in all the reproductive
organs and iliac lymph nodes in all treated groups. Iliac lymph node showed moderate
inflammatory cells (1.6±0.00), mild congestion (1.0±0.45), moderate degeneration and
necrosis (1.5±0.00) for one month post-infection group. In the second month post-
infection group, iliac lymph node revealed the presence of moderate inflammatory cells
(1.6±0.21), mild congestion (1.2±0.18) and moderate degeneration and necrosis
(1.5±0.22). For the treatment group of three month post-infection, the iliac lymph node
exhibited the presence of moderate inflammatory cells (1.6±0.22), moderate congestion
(1.5±0.11), severe lesion degeneration and necrosis (2.6±0.22). In the treated group of
one month post-infection showed mild infiltration of inflammatory cells (1.4±0.00),
mild congestion (1.4±0.55), mild degeneration and necrosis (1.4±0.55) of the ovary. In
the treated group of second month post-infection, the ovary exhibited mild
inflammatory cells (1.4±0.18), mild congestion (1.4±0.26) and mild degeneration and
necrosis (1.4±0.18). The third month post-infection group, ovary showed with moderate
degeneration and necrosis (2.4±0.15), moderate inflammatory cells (2.3±0.17) and mild
congestion (1.4±0.19). In the first groups of one month post-infection, the vagina
showed mild inflammatory cells (0.6±0.45), moderate congestion (1.5±0.55) and severe
degeneration and necrosis (2.6±0.45). The second month post-infection group revealed
vagina with moderate congestion (1.5±0.21), severe degeneration and necrosis
(2.5±0.22) and mild inflammatory cells (0.9±0.08). The third month group, the vagina
exhibited moderate inflammatory cells (1.5±0.22), severe congestion (2.6±0.22), and
severe degeneration and necrosis (2.6±0.22). The uterine horn in the first groups of one
month post-infection animals showed mild inflammatory cells (1.4±0.00), moderate
congestion (1.9±0.00) and mild degeneration and necrosis (1.3±0.00). In the second
month post-infection group, the uterine horn exhibited mild degeneration and necrosis
(1.4±0.19), moderate congestion (2.3±0.13) and mild inflammatory cells (1.4±0.21).
For the third month post-infection, the uterine horn showed mild degeneration and
necrosis (1.3±0.21), moderate inflammatory cells (1.9±0.04) and moderate congestion
(2.2±0.06). The uterus of the first month post-infection group showed mild
inflammatory cells (1.2±0.00), mild congestion (0.8±0.45) and mild degeneration and
necrosis (1.4±0.45). For the second month post-infection group, the uterus showed mild
inflammatory cells (1.0±0.04), mild congestion (1.1±0.13) and mild degeneration and
necrosis (0.5±0.22). For the third month post-infection group, the uterus exhibited
moderate congestion (1.5±0.22), moderate inflammatory cells (1.6±0.22) and moderate
degeneration and necrosis (1.5±0.22). For cervix, the first month post infection group
showed presence of mild inflammatory cells with mean score of 1.2±0.45, mild
congestion with mean score of 1.4±0.33 and mild degeneration and necrosis with mean
score of 0.5±0.45. For the second month post-infection group, cervix showed cellular
changes of mild inflammatory cells with mean score of 1.0±0.00, mild congestion with
mean score of 1.1±0.13, and mild presence of degeneration and necrosis with mean
score of 0.8±0.19. For the third month post-infection group, cervix exhibited cellular
changes of mild congestion with mean score of 1.1±0.07, moderate inflammatory cells
with mean score of 1.5±0.22 and moderate degeneration and necrosis with mean score
of 1.5±0.22. In conclusion, PCR and histopathology of the reproductive tract of Katjang
does proved that C. pseudotuberculosis can successfully ascended the reproductive
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tract and penetrated the physical barrier of the genital tract causing severe lesions as
observed in this study. The presence of C. pseudotuberculosis causes the IL-1β, IL-6
and estradiol to be increased while progesterone hormone to be decreased gradually.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia
sebagai memenuhi keperluan untuk ijazah Sarjana Sains
FISIOLOGI PEMBIAKAN PENYAKIT BISUL NODUS LIMFA PADA
KAMBING BETINA KATJANG MELALUI
SUNTIKAN INTRADERMAL
Corynebacterium pseudotuberculosis
Oleh
NUR AMIRAH ABDUL LATIF
Disember 2015
Pengerusi : Faez Firdaus Jesse Abdullah, PhD
Fakulti : Perubatan Veterinar
Corynebacterium pseudotuberculosis merupakan agen penyebab kepada penyakit
zoonotik Bisul Nodus Limfa (CLA). CLA merupakan penyakit kronik dan separa
klinikal yang kebanyakkannya menjangkiti bebiri dan kambing di seluruh dunia. CLA
mengakibatkan kerugian ekonomi yang tinggi terhadap negara-negara yang endemik
dengan CLA dimana penyakit ini akan menyebabkan masalah kepada pembiakan,
pengurangan dalam penghasilan susu dan daging, kurang pertumbuhan bulu dan kes-
kes kematian yang jarang berlaku pada kambing, bebiri dan ruminan-ruminan kecil
yang lain. Oleh yang demikian, kajian ini bertujuan untuk mengenalpasti kehadiran C.
pseudotuberculosis di dalam organ pembiakkan dan nodus limfa iliac kambing betina
yang tidak bunting berkaitan perubahan selular, hormon dan sitokine yang disebabkan
oleh bakteria tersebut dalam bentuk kronik melalui suntikan intradermal. Ini merupakan
kajian pertama dalam mengkaji jangkitan kronik pada organ-organ pembiakkan dan
nodus limfa yang berkaitan dalam kambing betina yang tidak bunting. Dalam kajian
ini, lapan belas ekor kambing betina sihat yang tidak bunting berumur 2 tahun telah
dibahagikan kepada dua kumpulan. Kumpulan pertama dan kumpulan kedua terdiri
daripada sembilan ekor kambing betina dan kumpulan kedua telah dibahagikan kepada
tiga kumpulan kecil. Kumpulan pertama telah disuntik dengan 1 ml 107 cfu C.
pseudotuberculosis hidup melalui suntikan intradermal manakala kumpulan kedua telah
diberi 1 ml PBS (pH 7) melalui suntikan intradermal. Kumpulan pertama telah
dibahagikan kepada tiga kumpulan kecil dimana kumpulan kecil yang pertama (B1)
telah dijangkiti selama 30 hari, kumpulan kecil yang kedua (B2) telah dijangkiti selama
60 hari dan kumpulan kecil yang ketiga (B3) telah dijangkiti selama 90 hari. Kumpulan
yang kedua telah dibahagikan kepada tiga kumpulan kecil (C1, C2, C3) dimana setiap
kumpulan dijaga selama 30, 60 dan 90 hari selepas dijangkiti. Selepas diberi jangkitan,
kambing-kambing tersebut dilihat untuk tanda-tanda klinikal dan pengambilan darah
dilakukan pada masa yang ditentukan selama tiga bulan berturut-turut untuk analisa
yang seterusnya. Tiga ekor kambing daripada kumpulan terjangkit telah disembelih
setiap bulan dan pembedahan susulan telah dijalankan dan organ-organ pembiakkan
dan nodus limfa yang berkaitan telah dikumpulkan untuk histopatologi. Uji kaji
kehadiran dan identifikasi C. pseudotuberculosis dan analisa PCR telah dijalankan.
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Kajian berjaya menunjukkan kehadiran C. pseudotuberculosis di dalam organ
pembiakkan dan nodus limfa iliac kambing betina yang di uji kaji selepas dua bulan
daripada satu ekor kambing betina yang dijangkiti manakala dua ekor kambing betina
yang lain hanya menunjukkan data positif di dalam ovari. Pada bulan kedua selepas
jangkitan, seperti yang di jangka, kesemua kambing betina yang dijangkiti
menunjukkan positif C. pseudotuberculosis di dalam nodus limfa iliac dan kesemua
organ-organ pembiakkan dalam kambing betina yang dijangkiti dengan C.
pseudotuberculosis. Kepekatan IL-1β dan IL-6 pula menunjukkan peningkatan selepas
tiga bulan jangkitan. Manakala, tindak balas hormon dalam kambing betina yang di
jangkiti menunjukkan penurunan dalam kepekatan progesteron dan kenaikkan hormon
estradiol sepanjang tiga bulan jangkitan. Perubahan kepekatan hormon seterusnya
disokong dengan perubahan pada tisu organ pembiakkan kambing betina yang
berpenyakit. Histopatologi untuk kumpulan yang dijangkiti menunjukkan kehadiran
sel-sel radang, kesesakkan sel darah, degenerasi dan nekrosis pada skala minima
sehingga teruk dalam kesemua organ-organ pembiakkan dan nodus limfa iliac untuk
kesemua kumpulan yang dijangkiti. Nodus limfa iliac menunjukkan sel-sel radang yang
minima (1.6±0.00), kesesakan sel darah yang minima (1.0±0.45), degenerasi dan
nekrosis yang sederhana (1.5±0.00) untuk satu bulan jangkitan. Bagi kumpulan yang
menerima jangkitan selama dua bulan, nodus limfa iliac menunjukkan kehadiran sel-sel
radang skala sederhana (1.6±0.21), kesesakan sel darah yang minima (1.2±0.18) dan
degenerasi dan nekrosis yang sederhana (1.5±0.22). Bagi kumpulan yang menerima
jangkitan selama tiga bulan, nodus limfa iliac menunjukkan kehadiran sel-sel radang
skala sederhana (1.6±0.22), kesesakan sel darah yang sederhana (1.5±0.11), degenerasi
dan nekrosis skala teruk (2.6±0.22). Kumpulan yang menerima jangkitan selama
sebulan menunjukkan peresapan minima sel-sel radang (1.4±0.00), kesesakan sel darah
yang minima (1.4±0.55), degenerasi dan nekrosis yang minima (1.4±0.55) pada ovari.
Dalam kumpulan yang menerima jangkitan selama dua bulan, ovari menunjukkan sel-
sel radang yang minima (1.4±0.18), kesesakan sel darah yang minima (1.4±0.26) dan
degenerasi dan nekrosis yang minima (1.4±0.18). Kumpulan yang dijangkiti selama
tiga bulan menunjukkan kehadiran degenerasi dan nekrosis skala sederhana (2.4±0.15),
bilangan sel-sel radang skala sederhana (2.3±0.17) dan kesesakan sel darah yang
minima (1.4±0.19). Dalam kumpulan pertama yang dijangkiti selama sebulan, faraj
menunjukkan sel-sel radang yang minima (0.6±0.45), kesesakan sel darah skala
sederhana (1.5±0.55) dan degenerasi dan nekrosis yang teruk (2.6±0.45). Bagi
kumpulan yang dijangkiti selama dua bulan menunjukkan faraj dengan kesesakan sel
darah yang sederhana (1.5±0.21), degenerasi dan nekrosis yang teruk (0.9±0.08) dan
sel-sel radang yang minima (1.5±0.22). Kumpulan yang dijangkiti selama tiga bulan,
faraj menunjukkan keradangan sel yang sederhana (2.5±0.22), kesesakan sel darah
yang teruk (2.6±0.22), dan degenerasi dan nekrosis yang teruk (2.6±0.22). Tanduk
rahim haiwan dalam kumpulan pertama selepas sebulan jangkitan menunjukkan sel-sel
radang (1.4±0.00), kesesakan sel darah yang sederhana (1.9±0.00) dan degenerasi dan
nekrosis yang minima (1.3±0.00). Dalam kumpulan yang dijangkiti selama dua bulan
menunjukkan degenerasi dan nekrosis yang minima (1.4±0.19), kesesakan sel darah
yang sederhana (2.3±0.13) dan sel-sel radang yang minima (1.4±0.21). Untuk
kumpulan yang dijangkiti selama tiga bulan, tanduk rahim menunjukkan degenerasi
dan nekrosis yang minima (1.3±0.21), sel-sel radang yang sederhana (1.9±0.04) dan
kesesakan sel darah yang sederhana (2.2±0.06). Rahim kumpulan yang dijangkiti
selama sebulan menunjukkan sel-sel radang yang minima (1.2±0.00), kesesakan sel
darah yang minima (0.8±0.45) dan degenerasi dan nekrosis yang minima (1.4±0.45).
Bagi kumpulan yang dijangkiti selama dua bulan, rahim menunjukkan sel-sel radang
yang minima (1.0±0.04), kesesakan sel darah yang minima (1.1±0.13) dan degenerasi
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dan nekrosis yang minima (0.5±0.22). Bagi kumpulan yang menerima jangkitan
selama tiga bulan, rahim menunjukkan kesesakan sel darah skala sederhana (1.5±0.22),
sel-sel radang yang sederhana (1.6±0.22) dan degenerasi dan nekrosis yang sederhana
(1.5±0.22). Untuk pangkal rahim, kumpulan yang dijangkiti selama sebulan
menunjukkan kehadiran sel-sel radang yang minima dengan nilai min 1.2±0.45,
kesesakan sel darah yang minima dengan nilai min 1.4±0.33 dan degenerasi dan
nekrosis yang minima dengan nilai min 0.5±0.45. Bagi kumpulan yang dijangkiti
selama dua bulan, pangkal rahim menunjukkan perubahan selular seperti sel-sel radang
yang minima dengan nilai min 1.0±0.00, kesesakan sel darah yang minima dengan nilai
min 1.1±0.13, dan kehadiran degenerasi dan nekrosis yang minima dengan nilai min
0.8±0.19. Bagi kumpulan yang dijangkiti selama tiga bulan, pangkal rahim
menunjukkan perubahan selular seperti kesesakan sel darah yang minima dengan nilai
min 1.1±0.07, sel-sel radang yang sederhana dengan nilai min 1.5±0.22 dan degenerasi
dan nekrosis yang sederhana dengan nilai min 1.5±0.22. Secara kesimpulannya, PCR
dan histopatologi saluran pembiakan Katjang betina membuktikan bahawa C.
pseudotuberculosis telah berjaya memasuki saluran pembiakan dan menembusi
halangan fizikal yang terdapat dalam organ pembiakan dan telah menyebabkan luka
yang teruk seperti yang diperhatikan dalam kajian ini. Kehadiran C. pseudotuberculosis
menyebabkan peningkatan dalam kepekatan IL-1β, IL-6 dan estradiol sementara
kepekatan hormon progesteron berkurangan secara beransur-ansur.
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ACKNOWLEDGEMENTS
Praise be to Allah S.W.T Most Gracious, Most Beneficient
My deepest appreciation to my supervisor Dr. Faez Firdaus Jesse bin Abdullah
for his concern, continuous advice and encouragement throughout the master’s project.
My gratitude also to my co-supervisors and his team, Prof. Dr. Mohd Zamri bin Saad,
Prof. Dr. Abd Wahid bin Haron, Assoc. Prof. Dr. Zunita binti Zakaria, Mr. Yap Kee
Heng and Mr. Jefri bin Norsidin for being patience and gave valuable guidance based
on their many years of experienced on CLA.
I would like to record my sincere thanks to all staff in the Department of
Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia
(UPM) for assist me and turn this research into valuable experiences.
My utmost appreciation to my beloved family and friends for their
understanding, patience and support throughout the project.
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been
accepted as fulfillment of the requirement for the degree of Master of Science. The
members of the Supervisory Committee were as follows:
Faez Firdaus Jesse Abdullah, PhD
Senior Lecturer
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Chairman)
Mohd Zamri Saad, PhD
Professor
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Member)
Abd Wahid Haron, PhD
Professor
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Member)
Zunita Zakaria, PhD
Associate Professor
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Member)
BUJANG BIN KIM HUAT, PhD Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
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Declaration by graduate student
I hereby confirm that:
This thesis is my original work; Quotations, illustrations and citations have been duly referenced; This thesis has not been submitted previously or concurrently for any other
degree at any other institutions;
Intellectual property from the thesis and copyright of thesis are fully-owned by Universiti Putra Malaysia, as according to the Universiti Putra Malaysia
(Research) Rules 2012;
Written permission must be obtained from supervisor and the office of Deputy Vice-Chancellor (Research and Innovation) before thesis is published (in the
form of written, printed or in electronic form) including books, journals,
modules, proceedings, popular writings, seminar papers, manuscripts, posters,
reports, lecture notes, learning modules or any other materials as stated in the
Universiti Putra Malaysia (Research) Rules 2012;
There is no plagiarism or data falsification / fabrication in the thesis, and scholarly integrity is upheld as according to the Universiti Putra Malaysia
(Graduate Studies) Rules 2003 (Revision 2012-2013) and the Universiti Putra
Malaysia (Research) Rules 2012. The thesis has undergone plagiarism detection
software.
Signature:
Name and Matric No: Nur Amirah binti Abdul Latif GS40404
Date:
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Declaration by Members of Supervisory Committee
This is to confirm that:
The research conducted and the writing of this thesis was under our supervision; Supervision responsibilities as stated in the Universiti Putra Malaysia (Graduate
Studies) Rules 2003 (Revision 2012-2013) are adhered to.
Signature:
Name of
Chairman of
Supervisory Committee: Dr. Faez Firdaus Jesse bin Abdullah
Signature:
Name of
Member of
Supervisory Committee: Prof. Dr. Mohd Zamri bin Saad
Signature:
Name of
Member of
Supervisory Committee: Prof. Dr. Abd Wahid bin Haron
Signature:
Name of
Member of
Supervisory Committee: Assoc. Prof. Dr. Zunita binti Zakaria
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TABLE OF CONTENTS
Page
ABSTRACT i
ABSTRAK iv
ACKNOWLEDGEMENTS vii
APPROVAL viii
DECLARATION x
LIST OF TABLES xiv
LIST OF FIGURES xv
LIST OF ABBREVIATIONS xviii
CHAPTER
1 INTRODUCTION
1.1 Background of study 1
1.2 Objectives 3
1.3 Hypotheses of this study 3
2 LITERATURE REVIEW
2.1 Caseous lymphadenitis (CLA) 4
2.1.1 General 4
2.1.2 Economic significance of CLA 5
2.1.3 Global prevalence of CLA 6
2.1.4 Route of infection 7
2.1.5 Transmissions 7
2.1.6 Clinical signs 8
2.2 Corynebacterium pseudotuberculosis 8
2.2.1 History 8
2.2.2 Characteristics of Corynebacterium
pseudotuberculosis 9
2.2.3 Corynebacterium pseudotuberculosis susceptibility
to antimicrobial agents
10
2.2.4 Pathogenesis of Corynebacterium
pseudotuberculosis
10
2.2.5 Virulence factors 12
2.2.6 Combating CLA 12
2.3 Hormonal responses (progesterone and estrogen) 13
2.4 Cytokines analysis (IL-1β and IL-6) 14
2.5 Histopathological changes 16
3 MATERIALS AND METHODS
3.1 Animals and management 18
3.1.1 Ethical consideration 18
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3.1.2 Estrus synchronization 18
3.1.3 Corynebacterium pseudotuberculosis inocula 19
3.1.4 Samples collection and culture 19
3.2 Polymerase Chain Reaction 21
3.2.1 Agarose gel preparation 21
3.3 Hormonal assay 21
3.3.1 Progesterone assay 22
3.3.2 Estradiol assay 22
3.4 Cytokines assay 22
3.4.1 Cytokines analysis 23
3.5 Histological preparation 23
3.5.1 Lesion scoring 24
3.6 Statistical analysis 26
4 RESULTS
4.1 Molecular detection of Corynebacterium
pseudotuberculosis by PCR
27
4.2 Hormonal profiles alteration in non-pregnant Katjang does
inoculated with Corynebacterium pseudotuberculosis
following intradermal route of infection in chronic form
29
4.3 Cytokines effects in non-pregnant Katjang does infected
with Corynebacterium pseudotuberculosis following
intradermal route of infection in chronic state
31
4.4 Correlation between hormone and cytokine concentration
in non-pregnant Katjang does following experimental
chronic infection with Corynebacterium
pseudotuberculosis by intradermal route
33
4.5 Cellular changes in reproductive organ and associated
lymph nodes of Katjang does infected with
Corynebacterium pseudotuberculosis following chronic
infection via intradermal route
35
5 DISCUSSION 47
6 SUMMARY, GENERAL CONCLUSION AND
RECOMMENDATION FOR FUTURE RESEARCH
54
REFERENCES 55
APPENDICES 68
BIODATA OF STUDENT 97
LIST OF PUBLICATIONS 98
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LIST OF TABLES
Table Page
3.5.1 Histopathological lesion scoring
25
4.1.1 Bacteriology of organ tissue Group 1 (intradermal) taken from non-
pregnant Katjang does following intradermal route of infection.
28
4.1.2 Bacteriology of organ tissue taken from Group 2 (control) non-
pregnant Katjang does following intradermal route of PBS
administration.
28
4.4.1 Pearson correlation between pro-inflammatory cytokine and
reproductive hormones in does infected intradermally with C.
pseudotuberculosis.
34
4.5.1 Table showing pathological lesions scoring in reproductive organs
and iliac lymph nodes of non-pregnant Katjang does following
chronic intradermal infection with C. pseudotuberculosis.
37
A3.1 Polymerase chain reaction (PCR) technique for bacteria
identification.
71
A4.1 The amount needed in each labeled tube for progesterone assay.
74
A4.2 The amount needed in each labeled tube for estradiol assay. 76
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LIST OF FIGURES
Figure Page
3.1.4 Flow chart of experimental design.
20
4.2.1 Comparative progesterone analysis (pg/ml) in non-pregnant does
in control group (G2) and experimentally inoculated with C.
pseudotuberculosis (G1) following intradermal route of infection
in chronic form. Each bar allocates the standard error mean
(mean± SE).
30
4.2.2 Comparative estrogen analysis (ng/ml) in non-pregnant does from
control group (G2) and experimentally inoculated with C.
pseudotuberculosis (G1) following intradermal route of infection
in chronic form. Each bar allocates the standard error mean
(mean± SE).
30
4.3.1 Inflammatory cytokines (IL-1β) concentrations in non-pregnant
does experimentally infected with C. pseudotuberculosis via
intradermal route and the control group. Each bar allocates the
standard error mean (mean± SE).
32
4.3.2 Inflammatory cytokines (IL-6) concentrations in non-pregnant
does experimentally infected with C. pseudotuberculosis via
intradermal route and the control group. Each bar allocates the
standard error mean (mean± SE).
32
4.5.1 Photomicrograph of a section of an iliac lymph node of a non-
pregnant Katjang does inoculated intradermally with C.
pseudotuberculosis after one month post-infection: (blue arrow)
mild degeneration and necrosis of the cortex (H & E, 400x).
38
4.5.2 Photomicrograph of a section of an iliac lymph node of a non-
pregnant Katjang does inoculated intradermally with C.
pseudotuberculosis after two months post-infection: (blue arrow)
mild necrosis and degeneration and (black arrow) mild
inflammatory cells of the cortex (H & E, 400x).
38
4.5.3 Photomicrograph of a section of an iliac lymph node of a non-
pregnant Katjang does inoculated intradermally with C.
pseudotuberculosis after three months post-infection: (blue arrow)
severe degeneration and necrosis, (black arrow) moderate
inflammatory cells and (red arrow) mild congestion of the cortex
(H & E, 200x).
39
4.5.4 Photomicrograph of a section of an ovary of a non-pregnant
Katjang does inoculated intradermally with C. pseudotuberculosis
after one month post-infection: (blue arrow) moderate
degeneration and necrosis, (black arrow) mild inflammatory cells,
39
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and (red arrow) mild congestion of the stromal cell (H & E, 200x).
4.5.5 Photomicrograph of a section of an ovary of a non-pregnant
Katjang does inoculated intradermally with C. pseudotuberculosis
after two months post-infection: (blue arrow) moderate
degeneration and necrosis, and (black arrow) mild inflammatory
cells of the stromal cell (H & E, 200x).
40
4.5.6 Photomicrograph of a section of an ovary of a non-pregnant
Katjang does inoculated intradermally with C. pseudotuberculosis
after three months post-infection: (blue arrow) severe degeneration
and necrosis, (black arrow) mild inflammatory cells, and (red
arrow) moderate congestion seen in medulla of ovary (H & E,
200x).
40
4.5.7 Photomicrograph of a section of a vagina of a non-pregnant
Katjang does inoculated intradermally with C. pseudotuberculosis
within one month post-infection: (blue arrow) mild degeneration
and necrosis, and (black arrow) mild inflammatory cells of the
major vestibular gland (H & E, 200x).
41
4.5.8 Photomicrograph of a section of a vagina of a non-pregnant
Katjang does inoculated intradermally with C. pseudotuberculosis
after two months post-infection: (blue arrow) moderate
degeneration and necrosis, and (black arrow) mild inflammatory
cells at the muscularis of vagina (H & E, 200x).
41
4.5.9 Photomicrograph of a section of a vagina of a non-pregnant
Katjang does inoculated intradermally with C. pseudotuberculosis
after three months post-infection: (blue arrow) severe degeneration
and necrosis, and (black arrow) mild inflammatory cells at the
muscularis of vagina (H & E, 200x).
42
4.5.10 Photomicrograph of a section of uterine horn of a non-pregnant
Katjang does inoculated intradermally with C. pseudotuberculosis
after one month post-infection: (blue arrow) moderate
degeneration and necrosis, (black arrow) mild inflammatory cells,
and (red arrow) mild congestion of the myometrium (H & E,
200x).
42
4.5.11 Photomicrograph of a section of uterine horn of a non-pregnant
Katjang does inoculated intradermally with C. pseudotuberculosis
after two months post-infection: (blue arrow) moderate
degeneration and necrosis, and (red arrow) mild congestion at the
myometrium (H & E, 200x).
43
4.5.12 Photomicrograph of a section of uterine horn of a non-pregnant
Katjang does inoculated intradermally with C. pseudotuberculosis
after three months post-infection: (blue arrow) moderate
degeneration and necrosis, and (red arrow) mild congestion at the
myometrium (H & E, 200x).
43
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4.5.13 Photomicrograph of a section of a uterus of a non-pregnant
Katjang does inoculated intradermally with C. pseudotuberculosis
after one month post-infection: (blue arrow) mild degeneration and
necrosis of the myometrium wall (H & E, 200x).
44
4.5.14 Photomicrograph of a section of a uterus of a non-pregnant
Katjang does inoculated intradermally with C. pseudotuberculosis
after two months post-infection: (blue arrow) moderate tissue
degeneration and necrosis, and (red arrow) mild congestion at the
endothelium gland (H & E, 200x).
44
4.5.15 Photomicrograph of a section of a uterus of a non-pregnant
Katjang does inoculated intradermally with C. pseudotuberculosis
after three months post-infection: (blue arrow) moderate tissue
degeneration and necrosis, (black arrow) moderate congestion, and
(red arrow) mild congestion of the myometrium (H & E, 200x).
45
4.5.16 Photomicrograph of a section of a cervix of a non-pregnant
Katjang does inoculated intradermally with C. pseudotuberculosis
after one month post-infection: (blue arrow) mild degeneration and
necrosis at the muscularis of cervix (H & E, 200x).
45
4.5.17 Photomicrograph of a section of a cervix of a non-pregnant
Katjang does inoculated intradermally with C. pseudotuberculosis
after two months post-infection: (blue arrow) severe degeneration
and necrosis, and (red arrow) mild congestion in cervix of uterus
(H & E, 200x).
46
4.5.18 Photomicrograph of a section of a cervix of a non-pregnant
Katjang does inoculated intradermally with C. pseudotuberculosis
after three months post-infection: (blue arrow) severe degeneration
and necrosis, and (red arrow) mild congestion in cervix of uterus
(H & E, 200x).
46
A9.1 Flow chart of reproductive pathophysiology. 96
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LIST OF ABBREVIATIONS
% Percentage
CLA Caseous lymphadenitis
Cfu Colony forming unit
C. pseudotuberculosis Corynebacterium pseudotuberculosis
DPX Distyrene plasticizer xylene
E3 Estrogen
P4 Progesterone
Fig. Figure
IL-1β Interleukin-1β
IL-6 Interleukin-6
N Number of animal(s)
PBS Phosphate buffer saline
PCR Polymerase chain reaction
RIA Radioimmunoassay
ELISA Enzyme-linked immunosorbent assay
UPM Universiti Putra Malaysia
GnRH Gonadotropin releasing hormone
FSH Follicle stimulating hormone
LH Luteinizing hormone
ml Milliliter
SEM Standard error of mean
SPSS Statistical Package for Social Sciences
TPU Taman Pertanian Universiti
DNA Deoxyribonucleic acid
MgCl2 Magnesium chloride
TAE Tris-acetate-EDTA
bp Base pair
UV Ultra violet
RT Room temperature
QC Quality control
W Watt
PLD Phospholipase D
Std. Standard
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CHAPTER 1
INTRODUCTION
1.1 Background of study
Caseous lymphadenitis (CLA) has been a prevalent disease among farmed small
ruminants in the majority of goat-rearing countries since 1888 (Jesse et al., 2013a). As
it is a chronic disease, introduction of control measures in many countries has been
slow or absent, resulting in a steady increase in the incidence of the disease. This, in
turn, has impacted upon small ruminant livestock producers with respect to significant
economic losses, not to mention issues of animal welfare, through (in extreme cases)
chronic ill-thrift, carcass condemnation at abattoirs and reduced wool yields (Baird &
Fontaine, 2007).
CLA is generally multicontinental and it has been reported to be widely prevalent in
Europe, Africa, Australia, South and North America, and the Middle East (Robins,
1991; Paton et al., 2005). CLA, in many of these countries, had been generally
considered as a devastating disease which is characterized by economic losses and
other problems associated with animal (Cesari, 1930). In Malaysia, CLA was first
detected in imported sheep during meat inspection at the Johor Bahru abattoir in 1960
(Department of Veterinary Services, 2015). A decade later, another case of CLA was
reported when the causative organism was successfully isolated from a goat at the
Veterinary Research Institute, Ipoh (VRI) and one year later, the bacterium was
isolated from an ovine case (Stoops et al., 1984; Komala et al., 2008).
Corynebacterium pseudotuberculosis is a Gram-positive actinomycete which is the
cause of chronic CLA in farmed small ruminants (Khuder et al., 2012). In addition, C.
pseudotuberculosis infection in humans has been reported on several occasions, thus,
CLA is a zoonotic disease (House et al., 1986). The significance of the disease is
evidenced by the scientific research and political debate which has been ongoing in
relation to this topic for over a century.
Infection of small ruminants with C. pseudotuberculosis usually results in the
formation of pyogranulomatous lesions which present in two different forms (Baird &
Fontaine, 2007). The external form, also known as cutaneous or superficial, is
characterized by the development of abscesses within the superficial lymph nodes or
within the subcutaneous tissue resulting in the loss of overlying hair and often leads to
rupture of abscess. The second form of CLA is the visceral form which is characterized
by the formation of lesions within the animal that cannot be observed externally
(Dorella et al., 2006; Baird & Fontaine, 2007). The site of these lesions is commonly
the internal lymph nodes (primarily the mediastinal lymph nodes) or lungs, although
other tissues may also be affected which primarily includes the liver, kidneys or the
mammary gland and less frequently the heart, brain, spinal cord, testes, uterus and
joints (Dorella et al., 2006; Baird & Fontaine, 2007).
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Skin abrasions due to ear tagging, castration, shearing, docking and by environmental
hazards such as nails, wired fences, splintered wood and metal edges are the possible
causes of CLA transmission within a herd (Batey, 1986; Dorella et al., 2006; Baird &
Fontaine, 2007; Jesse et al., 2013a). Abscesses from the affected animal can be
surgically drained and flushed with iodine solution. However, draining the abscess will
increase the risk of transmission of the organism to other animals if they are exposed to
the pus (Dorella et al., 2006). In a case where the abscess has ruptured, usually the
infected animal will be moved to an isolation pen to minimize contamination of the
environment (Braga et al., 2006).
The insidious effects of CLA in reproduction often make the veterinarians and farmers
overlooked its direct relation to fertility reduction and this contributes to the fact that
farmers have little concern about CLA. Although many studies have mentioned that
chronic diseases such as CLA affect the reproduction of farm animals, yet these studies
did not adequately highlight the mechanisms involved in the pathophysiology of the
reproductive system of affected animals (Williamson, 2001; Arsenault et al., 2003;
Paton et al., 2003; Peterhans et al., 2004). Therefore, the pathophysiologic mechanisms
leading to infertility due to prolong C. pseudotuberculosis infection through
intradermal route is the main focus of this study. Hence, this study provides better
information on how C. pseudotuberculosis affects reproductive efficiency by
pathologic changes of reproductive organs that influence reproductive hormones
concentration, proinflammatory cytokines concentration in does following chronic
challenged with C. pseudotuberculosis via intradermal route.
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1.2 Objectives
The objectives of this study were to:
1. Ascertain the progesterone and estrogen concentrations in non-pregnant Katjang goats following chronic intradermal infection with Corynebacterium
pseudotuberculosis.
2. Determine the cytokines concentration (interleukin-1 and interleukin-6) of non-pregnant Katjang goats following chronic intradermal infection with
Corynebacterium pseudotuberculosis.
3. Investigate the reproductive pathophysiology of chronic caseous lymphadenitis in non-pregnant Katjang goats following intradermal infection
with Corynebacterium pseudotuberculosis.
1.3 Hypotheses of the study
The hypotheses of this study were as follows:
HA1= Infection of Corynebacterium pseudotuberculosis in non-pregnant Katjang
does through intradermal route in chronic form may alter the progesterone and
estrogen concentrations.
HA2= There might be changes in the cytokines concentrations associated with
Corynebacterium pseudotuberculosis infection following chronic intradermal
infection.
HA3= There will be pathologic changes of reproductive organs in non-pregnant
Katjang goats following chronic intradermal infection with Corynebacterium
pseudotuberculosis.
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APPENDICES
Appendix A1
Preparation of Brain Heart Infusion (BHI) Broth
1. Suspend 37 grams of the medium in 1 L of distilled water. Mix well while letting the medium dissolve by heating with frequent mixing.
2. Boil for one minute until complete disintegration. Dispense into appropriate containers and sterilize at 121°C for 15 minutes.
3. The prepared medium is stored at 2-8°C. The medium colour will be amber. For best results, the medium should be used on the same day or, if not, heated in a
boiling water bed to expel the dissolved oxygen and left to cool before using.
4. The dehydrated medium should be homogeneous, free−flowing and light toasted in colour. If there are any physical changes, the medium need to be discarded.
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Appendix A2
Preparation of Blood Agar
1. Suspend 40 g of Blood agar powder in 1 L of distilled water by heating in a boiling water bath or in a current of steamed.
2. The Blood agar solution was brought for autoclaved at 121°C for 15 minutes.
3. The solution was left to cool for 50°C to 45°C.
4. A 40 ml of sterile defibrinated horse blood was added and mix slowly as to avoid bubble formation.
5. Ready to be poured into a petri dish.
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Appendix A3
Preparation of Polymerase Chain Reaction (PCR)
1. All positive plates of C. pseudotuberculosis, the apparatus and the reagents involved (DNAzol, 100 µl of pipette and micropipette tips, 10 µl of pipette and
micropipette tips, 1000 µl of pipette and micropipette tips, and ddH2O) were
prepared.
2. To obtain ddH2O, distilled water need to be sterilized or autoclaved at 121°C for 15 minutes. Deionized distilled water need to be used inside an air chamber to
maintain its sterility.
3. 2 µl of DNAzol was added into each PCR tubes using 10 µl of pipette. Then, clean sterilized 10 µl pipette tip was inserted to each PCR tubes except for the
negative indicator tubes. Wait for 15 minutes and the master mix was prepared
(Table A3.1).
4. After the master mix have been mixed well, the micropipette tips were removed and 48 µl of master mix was added in each PCR tubes using 100 µl pipette.
5. Finally, the PCR tubes were arranged in the SensQ Thermocycler machine and were set with 30 cycles of amplification.
6. The PCR amplification process starts with an initial denaturizing step at 94°C for 5 minutes, denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute,
synthesis at 72°C for 2 minutes, and final synthesis at 72°C for 2 minutes.
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Table A3.1. Polymerase chain reaction (PCR) technique for bacteria
identification.
Prepare 1.5 ml of Eppendorf tube for master mix
N=1 N=10
Template (DNAzol) 2 µl 20 µl
Taq buffer 2 µl 20 µl
MgCl2 2 µl 20 µl
i-PCR Nucleotide Mix ½ µl 5.0 µl
Forward primer ½ µl 5.0 µl
Reversed primer ½ µl 5.0 µl
Taq polymerase ½ µl 5.0 µl
ddH2O (deionize distilled
water)
42 µl 420 µl
TOTAL
50 µl
500 µl
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Preparation of Gel Electrophoresis
1. 0.3 g of Agarose powder was prepared and added into 100 ml of conical flask.
2. 30 ml of 1x TBE buffer was poured into the same conical flask and was vigorously shake.
3. The conical flask was placed inside the microwave oven for 1.5 minutes at 540 watt (W). The conical flask was removed after 1.5 minutes and 1.5 µl of DNA
stain was quickly added by using pipette (10 µl).
4. The Agarose solution was poured slowly into the wells to avoid bubbles formation. The Agarose solution was left to solidify for 1 hour. Now, the Agarose
gel was ready to be loaded with samples of DNA.
5. 6x DNA loading dye and 1kb DNA ladder were prepared. All of DNA samples and reagents were inserted inside the agarose gel according to one desire. 1 µl of
DNA loading dye was added to each 7 µl of DNA samples (n=8) including with
the negative well (n=1).
6. The gel electrophoresis machine was set up for 68 minutes, 60 volt and 350 mA (milli Ampere) and the machine was filled with 1x TBE buffer until the well
submerged.
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APPENDIX A4
Radioimmunoassay (RIA) Procedure for Progesterone (P4) Assay in Female Goats
Serum
1. All chemical reagents were thaw at room temperature (18°C - 25°C) before used for 30 minutes.
2. A 100 µl of pipette, micropipette tips, and 2µl of pipette syringe were prepared for later used.
3. The progesterone hormone test tubes were organized accordingly.
4. All reagents and samples were mixed thoroughly before used.
5. A 50 µl of progesterone calibrator was added to respective tubes by using 100 µl pipette (Table A5.1).
6. All tubes were loaded with 2 µl of progesterone buffer including the “T” tubes.
7. All racks were covered with aluminium. Mixed them well at room temperature (1 hour, 350 rpm). The racks were tie perfectly using a celotape to avoid them from
falls down during shaking processes.
8. Next, all samples were brought to radioactive room to be counted in a Gamma Counter Wizard 1470.
9. The unknown samples concentrations were calculated using integrated computer software programmed and later, the data were analyze using SPSS software
version 21.
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Table A4.1. The amount needed in each labeled tube for progesterone assay.
Volume (µl) Progesterone calibrator concentration
(ng/ml)
Tube(s)
50 µl 0 Tube N and tube S0
50 µl 0.16 Tube S1
50 µl 0.53 Tube S2
50 µl 2.3 Tube S3
50 µl 11.0 Tube S4
50 µl 55.2 Tube S5
50 µl CS PPG (0.88-1.64) Tube QC
50 µl Samples Tube 1 – tube 38
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Radioimmunoassay (RIA) Procedure for Estrogen (E2) Assay in Female Goats Serum
1. All reagents provided in estradiol kits were equilibrated at room temperature (18°C - 25°C) before used for 30 minutes.
2. Apparatus such as 100 µl pipette with micropipette tips (Table A5.2), duplicate plain tubes for total counts (T) and non-specific binding (NSB) were
prepared and labeled.
3. A 50 µl of estradiol tracer was added to estradiol buffer and was thoroughly mixed. All reagents were mixed thoroughly before used including serum. A 2
µl of estradiol buffer were added to all respective tubes.
4. Covered the tube racks with aluminum foil before mixed with a shaker. Let reagents be on the shaker for 3 hours at 350 rpm (RT).
5. All samples were brought to radioactive room to be counted with the gamma counter and all data were calculated using integrated computer software. The
data were finalized again using SPSS software version 21.
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Table A4.2. The amount needed in each labeled tube for estradiol assay.
Volume (µl) Estradiol calibrator
concentration (pg/ml) Tube(s)
100 µl 0 Tube N and S0
100 µl 19 Tube S1
100 µl 52 Tube S2
100 µl 152 Tube S3
100 µl 556 Tube S4
100 µl 1718 Tube S5
100 µl 5322 Tube S6
100 µl Control (61-112) Tube QC
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APPENDIX A5
Progesterone Analysis – Repeated Measures ANOVA Using SPSS Statistics
1) The column label referring to the dependent variable “control group” that is measured at each month points.
Within-Subjects Factors
Measure: control
month Dependent Variable
1 Progestrone_Control1
2 Progestrone_Control2
3 Progestrone_Control3
2) The table below provides important descriptive statistics for this analysis.
Descriptive Statis