NUR AMIRAH ABDUL LATIFpsasir.upm.edu.my/id/eprint/65892/1/FPV 2015 20 IR.pdfzoonotik Bisul Nodus...

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


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


By


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


necrosis (1.4±0.45). For the second month post-infection group, the uterus showed mild


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


Oleh


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



(Member)

Abd Wahid Haron, PhD

Professor



(Member)

Zunita Zakaria, PhD

Associate Professor



(Member)

BUJANG BIN KIM HUAT, PhD Professor and Dean

School of Graduate Studies


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



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



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).



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



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


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




degeneration and necrosis, and (black arrow) mild inflammatory

cells at the muscularis of vagina (H & E, 200x).

41




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


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




degeneration and necrosis, and (red arrow) mild congestion at the

myometrium (H & E, 200x).

43



after three months post-infection: (blue arrow) moderate


myometrium (H & E, 200x).

43

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4.5.13 Photomicrograph of a section of a uterus of a non-pregnant


after one month post-infection: (blue arrow) mild degeneration and

necrosis of the myometrium wall (H & E, 200x).

44



after two months post-infection: (blue arrow) moderate tissue


endothelium gland (H & E, 200x).

44



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


after one month post-infection: (blue arrow) mild degeneration and

necrosis at the muscularis of cervix (H & E, 200x).

45



after two months post-infection: (blue arrow) severe degeneration

and necrosis, and (red arrow) mild congestion in cervix of uterus

(H & E, 200x).

46




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) The table below provides important descriptive statistics for this analysis.

Descriptive Statis

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