pathogenesis, molecular characterization, chemotherapy and vaccine...

PATHOGENESIS, MOLECULAR CHARACTERIZATION,

CHEMOTHERAPY AND VACCINE DEVELOPMENT FOR

MYCOPLASMOSIS IN SMALL RUMINANTS

BY

MUHAMMAD KAMAL SHAH

A dissertation submitted to The University of Agriculture, Peshawar in partial

fulfillment of the requirement for the degree of

DOCTOR OF PHILOSOPHY IN PATHOLOGY

(ANIMAL HEALTH)

DEPARTMENT OF ANIMAL HEALTH FACULTY OF ANIMAL HUSBANDRY AND VETERINARY SCIENCES

THE UNIVERSITY OF AGRICULTURE, PESHAWAR

KHYBER PAKHTUNKHWA-PAKISTAN

MARCH, 2017




BY

MUHAMMAD KAMAL SHAH

A dissertation submitted to The University of Agriculture, Peshawar in partial

fulfillment of the requirement for the degree of

DOCTOR OF PHILOSOPHY IN PATHOLOGY

(ANIMAL HEALTH)

Approved by:

_________________________ Supervisor

Prof. Dr. Umar Sadique

_________________________ Member (Major)

Dr. Zahoor ul Hassan

Assistant Professor

_________________________ Member (Minor)

Dr. Aqib Iqbal

Associate Professor

_________________________ Chairman and Convener Board of Study

Prof. Dr. Umar Sadique

_________________________ Dean, Faculty of Animal Husbandry and

Prof. Dr. Nazir Ahmad Veterinary Sciences

_________________________ Director Advanced Studies and Research

Prof. Dr. Muhammad Jamal Khan

DEPARTMENT OF ANIMAL HEALTH FACULTY OF ANIMAL HUSBANDRY AND VETERINARY SCIENCES

THE UNIVERSITY OF AGRICULTURE, PESHAWAR

KHYBER PAKHTUNKHWA-PAKISTAN

MARCH, 2017

DEDICATION To

The Holy Prophet MUHAMMAD (PBUH)

And my adorable parents, siblings

Sweet and sincere wife

Little princesses Malaika, Laiba and Maria Kamal

For their eternal love

Muhammad Kamal Shah

PUBLICATION OF RESEARCH FROM THE PRESENT STUDY

Research paper published/accepted

1-Muhammad Kamal Shah*, Umer Saddique, Shakoor Ahmad, Aqib Iqbal, Abid

Ali,Waseem Shahzad, Sayyar Khan Khan and Hanif ur Rehman (2017). Molecular

characterization of local isolates of Mycoplasmas capricolum sub specie

capripneumoniae in goats (Capra hircus) of Khyber Pakhtunkhwa, Pakistan. Pakistan

Vet. Journal. 37(1): 90-94. (IF=0.822) Status published

2-Muhammad Kamal Shah1,

*Umer Saddique1, Shakoor Ahmad

1, Zahoor ul

Hassan1,Murad Ali Khan

1, Farhan Anwar

1 (2017). Molecular identification and

comparative anti-mycoplasmal activity of three indigenous medicinal plants against

Mycoplasma Putrefaciens isolated from sheep. Accepted in Journal of Animal & Plant

Sciences (JAPS), Pakistan. Paper ID. VS-17-002. (IF=0.422) Status accepted

3-Muhammad Kamal Shah, Umer Sadique, Shakoor Ahmad, Sadeeq ur Rehman,

Yousaf Hayat and Tariq Ali (2017). Prevalence and antimicrobial susceptibility profiles

of Mycoplasma mycoides subsp. capri field isolates from sheep and goats in Pakistan.

Small Ruminant Research.Elsevier, Paper ID. Rumin-D-17-8550. (IF=1.08) Status

published.

Conference proceeding/ Abstract

1-M. K. Shah*, U. Sadique, S. Ahmad, S. Qureshi, S. Khan and S. A. Shah (2017).

Prevalence of Mycoplasma mycoides subsp. capri in different climatic zones of Khyber

Pakhtunkhwa, Pakistan. Poster presentation in 3rd

International conference on

Agriculture, Food and Animal Science, Januray10-12, 2017. Sindh Agriculture

University Tandojam, Sindh, Pakistan. Page. 220

2-M. Kamal Shah, Umer Sadique, Zahoor ul Hassan, Shakoor Ahmad (2016). Comparative efficacy of commercially available antimicrobials against local isolates of

Mycoplasma mycoides subspecie capri. 21st Congress of the International Organization for

Mycoplasmology being held at the Queensland University of Technology (QUT) in

Brisbane, Australia from the 3rd – 7th July 2016. Accepted and win travel award.

3-Muhammad Kamal Shah*, Umer Saddique, Shakoor Ahmad, Zahoor ul Hassan,

Hamayun Khan, Hanif Ur Rehman, Shakir Ullah and Hayat Ullah (2015). Antibacterial

Activity of Leaf Extracts of Azadirachta indica (Neem) against Mycoplasma

putrefaciens. Oral presentation in International Workshop on Dairy Science Park. Nov.

16-18, Peshawar, Pakistan. Page. 68

4-Muhammad Kamal Shah*, Umer Saddique, Shakoor Ahmad, Abdul Jabbar Tanwir,

Hamayun Khan, Hanif Ur Rehman, Shakir Ullah and Hayat Ullah (2015). Anti-

mycoplasmal activity of Calotropis procera against local isolates of Mycoplasma

mycoides subsp. Capri in Khyber PakhtunKhwa, Pakistan. Oral presentation in Int.

Workshop on Dairy Science Park. Nov. 16-18, Peshawar, Pakistan. Page. 69

5-Muhammad Kamal Shah*, Umer Saddique, Muhammad Subhan Qureshi, Shakoor

Ahmad, Zahoor Ul Hassan, Hamayun Khan, Said Sajjad Ali Shah (2015). Comparative

study of DNA extraction protocols for Mycoplasma species. Int. Workshop on Dairy

Science Park. Nov. 16-18, Peshawar, Pakistan. Page. 70

TABLE OF CONTENTS

CHAPTER NO. TITLE PAGE NO.

LIST OF TABLES .......................................................................................... i

LIST OF FIGURES ......................................................................................... iii

LIST OF PLATES ........................................................................................... v

LIST OF ABBREVIATIONS ......................................................................... viii

ACKNOWLEDGMENT ................................................................................. ix

GENERAL ABSTRACT ................................................................................ xi

I. INTRODUCTION ......................................................................................... 1

II. REVIEW OF LITERATURE ....................................................................... 8

2.1 Respiratory complications in small ruminants .................................... 8

2.2 Mycoplasmosis in livestock ................................................................. 8

2.3 Contagious Caprine Pleuropneumonia (CCPP). .................................. 9

2.4 History of CCPP ................................................................................... 9

2.5 Susceptible hosts for Mycoplasma infection... ..................................... 10

2.6 Classification of Mycoplasma .............................................................. 11

2.7 Morphology .......................................................................................... 11

2.8 Characteristics of Mycoplasma ............................................................ 12

2.9 Growth requirement and culturing of Mycoplasma ............................. 12

2.10 Ecology ................................................................................................. 13

2.11 Pathogenic Mycoplasma species .......................................................... 14

2.12 Pathogenesis of Mycoplasma manifestation......................................... 15

2.13 Clinical complication of mycoplasmosis ............................................. 20

2.14 Pathological changes ............................................................................ 23

2.15 Gross pathology .................................................................................... 23

2.16 Histopathology ..................................................................................... 25

2.17 Diagnosis of Mycoplasma. ................................................................... 26

2.18 Isolation of Mycoplasma ...................................................................... 27

2.19 Culture and cultivation ......................................................................... 28

2.19.1 Special media requirements for Mycoplasma growth. ............. 28

2.19.2 Identification of Mycoplasma ................................................... 28

2.19.3 Biochemical tests ...................................................................... 28

2.19.4 Serological tests ........................................................................ 30

2.19.4.1 Growth inhibition test ................................................. 30

2.19.4.2 Latex agglutination test .............................................. 31

2.19.4.3 Enzyme linked immunosorbent assay (ELISA) ......... 31

2.19.4.4 PCR for identification of Mycoplasma ...................... 32

2.19.4.5 DNA sequencing ........................................................ 33

2.19.4.6 Phylogenetic analysis and DNA homology ............... 33

2.20 Chemotherapy ...................................................................................... 34

2.20.1 Antimicrobial agents ................................................................ 35

2.20.2 Antimicrobial agents used for the treatment of caprine

mycoplasmosis… ..................................................................... 35

2.20.3 Classification of antimicrobial agents ...................................... 36

2.20.3.1 Amino glycosides ....................................................... 36

2.20.3.2 Fluoroquinolone ........................................................ 37

2.20.3.3 Macrolides .................................................................. 37

2.20.3.4 Tetracycline ............................................................... 37

2.20.4 Resistance of Mycoplasma to antimicrobial agents ................. 38

2.20.5 Medicinal plants ....................................................................... 40

2.21 Vaccination and control of Mycoplasma infections ............................. 43

2.22 Detection of antibodies by serological tests ......................................... 46

2.23 Importance of Mycoplasmosis in Pakistan ........................................... 46

2.24 Study area, Khyber Pakhtunkhwa ....................................................... 48

2.25 Sheep and Goats in Khyber Pakhtunkhwa Pakistan ........................... 48

III. STUDY-I ........................................................................................................ 50

ISOLATION AND MOLECULAR IDENTIFICATION OF PATHOGENIC

MYCOPLASMA SPECIES FROM NATURALLY INFECTED SMALL

RUMINANT OF KHYBER PAKHTUNKHWA .................................................... 50

Abstract ........................................................................................................................ 51

3.1 Introduction ......................................................................................... 52

3.2 Materials and Methods ........................................................................ 54

3.2.1 Sampling ................................................................................... 54

3.2.2 Culturing of pathogenic Mycoplasma species .......................... 55

3.2.2.1 processing of samples .................................................. 55

3.2.2.2 Sterilization of glass wares .......................................... 55

3.2.2.3 Modified Hayflick medium for Mycoplasma growth .. 56

3.2.2.3a Part A (Autoclavable Part) ........................................ 56

3.2.2.3b Part B (Membrane-filtered Part) ............................... 56

3.2.2.3c Media storage ............................................................ 57

3.3 Isolation and identification .................................................................. 57

3.3.1 Morphological identification ................................................... 57

3.4 Identification and confirmation of isolates .......................................... 58

3.4.1 Biochemical tests ..................................................................... 58

3.4.2 Molecular confirmation and characterization........................... 58

3.4.2.1 DNA extraction ........................................................... 58

3.4.2.2 Quantification of extracted DNA ............................... 59

3.4.2.3 Polymerase chain reaction .......................................... 59

3.4.2.4 PCR conditions ............................................................ 60

3.4.2.5 Gel electrophoresis ...................................................... 61

3.5 Homology and phylogenetic analysis ................................................. 61

3.6 Statistical analysis ............................................................................... 61

3.7 Results ................................................................................................. 62

3.7.1 Isolation of Mycoplasma ......................................................... 62

3.7.2 Biochemical tests ...................................................................... 72

3.7.3 Molecular identification and characterization of local

isolates ...................................................................................... 76

3.7.4 Homology and phylogenetic analysis ...................................... 84

3.8 Discussion ........................................................................................... 87

3.9 Conclusion ............................................................................................ 99

3.10 Recommendation ................................................................................ 100

IV. STUDY-II ....................................................................................................... 101

STUDY ON PATHOGENESIS OF CCPP IN NATURALLY INFECTED

SMALL RUMINANTS OF KHYBER PAKHTUNKHWA ................................... 101

Abstract ........................................................................................................................ 102

4.1 Introduction ......................................................................................... 103

4.2 Materials and methods ......................................................................... 105

4.2.1 Clinico-pathological picture of ruminant mycoplasmosis........ 105

4.2.2 Necropsy .................................................................................. 105

4.2.3 Gross lesions and scoring ......................................................... 105

4.2.4 Histopathology ......................................................................... 106

4.2.4.1 Procedure for histopathology ...................................... 106

4.2.4.2 Staining ........................................................................ 107

4.2.4.3 Slide reading ................................................................ 109

4.3 Microscopic lesions scoring ................................................................ 109

4.4 Statistical analysis ................................................................................ 109

4.5 Results ................................................................................................. 110

4.5.1 Clinical finding ........................................................................ 110

4.5.2 Gross pathology ....................................................................... 112

4.5.3 Histopathology ........................................................................ 117

4.5.3.1 Trachea ....................................................................... 117

4.5.3.2 Lungs .......................................................................... 118

4.5.3.3 Intestine ...................................................................... 119

4.5.3.4 Kidney ........................................................................ 120

4.5.3.5 Spleen .......................................................................... 122

4.5.3.6 Liver ............................................................................. 123

4.5.3.7 Brain ............................................................................. 125

4.5.4 Gross lesions scoring ................................................................ 126

4.5.5 Microscopic lesions scoring ..................................................... 127

4.6 Discussion ............................................................................................ 129

4.7 Conclusion ............................................................................................ 138

4.8 Recommendation ................................................................................. 138

V. STUDY-III ...................................................................................................... 139

CHEMOTHERAPEUTIC TRIAL OF COMMONLY USED

ANTIMICROBIAL AGENTS AND INDIGENOUS MEDICINAL PLANTS

FOR THE TREATMENT OF CCPP ....................................................................... 139

Abstract ........................................................................................................................ 140

5.1 Introduction .......................................................................................... 141


5.2.1 Antimicrobial agent used in-vitro trial ..................................... 144

5.2.2 Collection and identification of medicinal plants..................... 144

5.2.3 Preparation of methanolic extract............................................. 144

5.2.4 Test organisms used in-vitro trial ............................................. 145

5.2.5 Preparation of Mycoplasma culture .......................................... 145

5.2.6 Determination of antibiogram assay ........................................ 145

5.2.6.1 Disc diffusion assay for antimicrobial agents ............. 145

5.2.6.2 Determination of minimum inhibitory concentration

(MIC) for antimicrobial agents and plants extract ....... 146

5.2.6.3 Agar well diffusion assay ............................................. 146

5.3 Statistical analysis ................................................................................ 147

5.4 Results .................................................................................................. 148

5.4.1 Comparative efficacy of antimicrobial agents against local

isolates ...................................................................................... 148

5.4.2 MIC of antimicrobial agents using broth micro dilution

method ...................................................................................... 150

5.4.3 Comparative efficacy of medicinal plants against local

isolates ..................................................................................... 154

5.4.4 MIC of medicinal plants extracts using broth micro dilution

method ..................................................................................... 157

5.5 Discussion ............................................................................................ 161

5.6 Conclusion ............................................................................................ 167

5.7 Recommendation ................................................................................. 168

VI. STUDY-IV ...................................................................................................... 169

TRIAL OF INDIGENOUS VACCINE DEVELOPMENT AGAINST THE

LOCAL ISOLATES OF MYCOPLASMA MYCOIDES SUB SP. CAPRI

(Mmc) ........................................................................................................................... 169

Abstract ........................................................................................................................ 170

6.1 Introduction .......................................................................................... 171


6.2.1 Preparation of Mycoplasma vaccine ....................................... 173

6.2.2 Culture preparation .................................................................. 173

6.2.3 Inactivation of Mmc antigen ..................................................... 173

6.2.4 Protein estimation of cultured cell ........................................... 174

6.2.5 Quality control saponized vaccine .......................................... 174

6.2.5.1 Sterility testing of vaccine ............................................ 174

6.2.5.2 Fluid thioglycollate medium ........................................ 174

6.2.5.3 TSB soybean-casein digest medium............................. 175

6.2.5.4 Mannitol salt agar (MSA) media .................................. 175

6.2.5.5 Sabourad dextrose agar media ...................................... 175

6.2.6 Safety of vaccine ...................................................................... 176

6.2.7 Vaccinal trial in experimental animal ...................................... 176

6.2.7.1 Sheep grouping and vaccine inoculation ...................... 176

6.2.7.2 Goats grouping and vaccine inoculation ...................... 177

6.2.7.3 Examination of vaccinated animals and blood

sampling ....................................................................... 177

6.2.7.4 Preparation of Mycoplasma antigen ............................. 178

6.2.7.5 Sensitization of sheep erythrocytes (RBC) .................. 178

6.2.7.6 Indirect haemagglutination (IHA) test ......................... 178

6.2.8 Data analysis ............................................................................ 178

6.3 Results .................................................................................................. 179

6.3.1 Viable counts and protein concentration of inactivated

stock culture ............................................................................ 179

6.3.2 Sterility testing ......................................................................... 179

6.3.3 Safety of whole cell saponized vaccine .................................... 179

6.3.4 Estimation of antibodies titer through IHA ............................. 179

6.4 Discussion ............................................................................................ 185

6.5 Conclusion ............................................................................................ 189

6.6 Recommendation ................................................................................. 190

VII. SUMMARY .................................................................................................... 191

LITERATURE CITED ................................................................................. 195

ANNEXURES ................................................................................................. 234

i

LIST OF TABLES

TABLE NO. TITLE PAGE NO.

2.1 Sensitivity of pathogenic Mycoplasma species of small ruminants to

various biochemical assays. ............................................................................. 30

3.1 List of different PCR primer sequence, annealing temperature and

expected amplicon size ..................................................................................... 60

3.2 Result of Mycoplasma growth on culture media isolated from small

ruminant suffering from respiratory syndrome suspected for (CCPP) in

three different climatic zones. .......................................................................... 63

3.3 Comperative isolation of Mycoplasma from sheep and goats suffering

from respiratory syndrome suspected for CCPP. ............................................. 63

3.4 Distribution of positive isolates on culture media collected from sheep and

goats across different climatic zones. ............................................................... 64

3.5 Gender based isolation of Mycoplasma from sheep and goat suspected for

CCPP ................................................................................................................ 64

3.6 Age wise distribution of Mycoplasma isolated from sheep and goats on

modified Hayflick media. ............................................................................... 65

3.7 Result of positive isolates identify through biochemical assays. ..................... 73

3.8 PCR based confirmed isolates of Mycoplasma across the species in

different climatic zones... ................................................................................. 76

3.9 Molecular identification and prevalence of pathogenic Mycoplasma

species from animals suspected for CCPP. ...................................................... 79

3.10 PCR result for confirmation of Mycoplasma Mycoides cluster and

proportional difference using Z- test analysis in different climatic zones. ..... 79

3.11 PCR result for confirmation of Mycoplasma Mycoides subsp. capri and

proportional difference using Z- test analysis in different climatic zones. ...... 80

3.12 PCR result for confirmation of Mycoplasma capricolum subsp.

capripnuemoniae and proportional difference using Z- test analysis across

different climatic zones .................................................................................... 80

3.13 PCR result for confirmation of Mycoplasma putrefaciens and proportional

difference using Z- test analysis across different climatic zones ..................... 81

3.14 Confirmation of Mycoplasma species by PCR from different clinical

sample of animals in different climatic zones ................................................. 81

ii

3.15 PCR result from different source of samples and proportional difference

using Z-test analysis across three climatic zones ............................................. 82

4.1 Occurence of clinical signs in (% age) in naturally infected small

ruminants suffering from respiratory syndrome. .............................................. 111

4.2 Occurence of gross lesions in (% age) in different body tissue in naturally

infected animals ................................................................................................ 113

4.3 Occurence of gross lesions (% age) in different body tissue in naturally

infected sheep and goat. .................................................................................. 116

4.4 Scoring of gross lesions in naturally infected sheep and goats suspected

for CCPP across different climatic zone. ......................................................... 127

4.5 Scoring of microscopic lesions in naturally infected sheep and goats

suspected for CCPP across different climatic zone. ......................................... 128

5.1 Antimicrobial activity of commercially available agents against local

isolates of Mycoplasma mycoides subsp. capri using agar disc diffusion

assay. ................................................................................................................ 149


isolates of Mycoplasma capricolum subsp. capripneumoniae using agar

disc diffusion assay. ......................................................................................... 149


isolates of Mycoplasma putrefaciens using agar disc diffusion assay. ............ 149

5.4 Anti-mycoplasmal activity of methanolic extract of Azadirachta indica,

Calotropis procera & Artemisia herba-alba using agar well diffusion

assay against Mycoplasma mycoides subsp. capri. .......................................... 155



assay against Mycoplasma capricolum subsp. capripneumoniae. ................... 156



assay against Mycoplasma putrefaciens. .......................................................... 157

iii

LIST OF FIGURES

FIGURE. NO TITLE PAGE NO.

3.1 Map of Khyber Pakhtunkhwa represent different districts of sample

collection ......................................................................................................... 55

3.2 Overall molecular prevalence (% age) of different pathogenic Mycoplasma

species in small ruminant. ................................................................................ 82

3.3 Overall molecular prevalence (% age) of pathogenic Mycoplasma species

in different climatic zones. ............................................................................... 83

3.4 Comparative specie based prevalence of pathogenic Mycoplasma species

in small ruminants ............................................................................................ 83

3.5 PCR confirmed Mycoplasma isolates recovered from different source of

clinical samples ............................................................................................... 84

3.6 Phylogenetic relationship of the Mycoplasma capricolum sub specie

capripneumoniae sequence obtained (Swat, Pakistan) .................................... 85

3.7 Phylogenetic relationship of the Mycoplasma mycoides subsp. capri

sequence ........................................................................................................... 85

3.8 Phylogenetic relationship of the Mycoplasma putrefaciens sequence ............. 86

4.1 Comparative distribution of gross lesions in various organs of sheep and

goats died due to CCPP .................................................................................... 117

5.1 Average MICs value of different antimicrobial agents against the local

isolates of Mmc. ................................................................................................ 153


isolates of Mccp. ............................................................................................... 153


isolates of Mycoplasma putrefaciens ............................................................... 154

5.4 Anti-mycoplasmal activity of methanolic extract of A. indica, C. procera

and A.herba-alba against the local isolates of Mycoplasma putrefaciens ...... 158


and A.herba-alba against the local isolates of Mccp ....................................... 159


and A.herba-alba against the local isolates of Mycoplasma capri .................. 160

iv

6.1 Mean GMT value of whole cell saponised Mmc vaccine antibodies titer in

sheep. ................................................................................................................ 180

6.2 Mean GMT value of lyophilized Mmc vaccine VRI, Lahore antibodies

titer in sheep. .................................................................................................... 180

6.3 Comparative GMT value of whole cell saponized vaccine and lyophilized

Mmc vaccine of VRI for sheep. ........................................................................ 181

6.4 Mean GMT value of whole cell saponized Mmc vaccine antibodies titer in

goats. ................................................................................................................ 182

6.5 Mean GMT value of antibodies titer of lyophilized Mmc vaccine VRI

Lahore in goats ................................................................................................ 183

v

LIST OF PLATES

PLATE NO. TITLE PAGE NO.

3.1 Mycoplasma positive culture in modified Hayflick broth showing turbidity

at day 5th

post incubation. ................................................................................. 66

3.2 The culture show turbidity for Mycoplasma growth of nasal discharge

taken from sheep ............................................................................................. 66

3.3 Mycoplasma putrefaciens gross colonies after 3rd

day post inoculation on

modified Hayflick agar isolated from nasal swab of sheep ............................. 67

3.4 Small tiny (0.2-.3mm) Mycoplasma capricolum subsp. capripneumoniae

(Mccp) visible gross colonies at day 5th

post inoculation on modified Hay

lick agar medium .............................................................................................. 67

3.5 Gross colonies of Mycoplasma mycoides subsp. capri after 3 days post

incubation isolated from lungs tissue of goat in Southern zone ....................... 68

3.6 Mycoplasma mycoides subsp. capri colonies with nipple like appearance

on day 3rd

post incubation on modified Hayflick agar at 10X. ......................... 68

3.7 Mycoplasma capricolum sub specie capripneumoniae colony with typical

fried egg appearance on day 7th

psot incubation in modified Hayflick ...........

3.8 Mycoplasma cluster colonies with nipple like appearance on day 4th

post

incubation on modified Hayflick agar at 4X …………… ............................... 69

3.9 Typical Mycoplasma capricolum subsp. capripneumoniae colonies

showing nipple like appearance with pleomorphism on modified Hayflick

agar ................................................................................................................... 69

3.10 Mycoplasma putrefaciens colonies having pleomorphism on day 2nd

post

incubation in modified Hayflick agar. .............................................................. 70

3.11 Mycoplasma mycoides subsp. capri colony with typical nipple like

appearance on day 3rd

post incubation on modified Hayflick agar at 10X ...... 70

3.12 Modified Hayflick broth showing turbidity for pure growth of

Mycoplasma ..................................................................................................... 71

3.13 Tube in center showed turbidity for pure Mycoplasma growth after 3rd

passage collected from pleural fluid ................................................................. 71

3.14 Result of Glucose fermentation test with yellow color represent positive

for Mycoplasma mycoides subsp. capri while red color in center is

negative control. ............................................................................................... 72

vi

3.15 Mycoplasma culture positive for casein hydrolysis test, showing growth

along with line of culture. ................................................................................ 73

3.16 Mycoplasma culture showing digestion of serum along the line of growth

of culture. ......................................................................................................... 74

3.17 Arginine hydrolysis test (aerobic) the tube on both Side showed positive

result in center control tube. ............................................................................. 74

3.18 Arginine hydrolysis test (anaerobic) tubes showing positive for

Mycoplasma culture and negative control in center. ....................................... 75

3.19 Tetrazolium reduction test, control tube (uninoculated) in the center with

Tetrazolium anaerobic positive in the right side and Tetrazolium aerobic

positive tube in the left. .................................................................................... 75

3.20 PCR result of Mycoplasma myocoides cluster and Mccp with an amplicon

size of 548 and 316 bp in samples collected from goat ................................... 77

3.21 PCR gel product of Mycoplasma mycoides subsp. capri with an amplicon

size of 194bp, isolated from lungs tissue of goat ............................................. 77

3.22 PCR product of Mycoplasma putrefaciens with an amplicon size of 540 bp .. 78

4.1 Important clinical sign in small ruminants suffering from respiratory

syndrome suspected for CCPP ......................................................................... 112

4.2 Gross lesion in various organs of animals at postmortem examination

suffering from CCPP. ....................................................................................... 114

4.3 Gross lesion in various organs of animals at postmortem examination

suffering respiratory syndrome suscepted for CCPP. ...................................... 115

4.4 Tracheal section of goat showing sloughing of epihelial layer ........................ 118

4.5 Lungs of goat suffering from respiratory syndrome showing sloughing of

ciliated epithelium in bronchioles(long arrow) . .............................................. 119

4.6 Lungs of goat suffering from respiratory sundrome showing emphysema

(long arrows) and rupture of aveoli (arrow head) ............................................ 119

4.7 Intestine of goat showing sloughing of villi and leukocytic infiltration

H&E stain, 400X .............................................................................................. 120

4.8 Histo-micrograph of kidney of the goats infected with Mycoplasma .............. 121

4.9 Histo-micrograph of kidney of the sheep infected with Mycoplasma. ............ 121

4.10 Spleen of sheep suffering from respiratory syndrome showing congestion

and extensive leukocytic infiltration. ............................................................... 122

vii

4.11 Spleen of goat suffering from respiratory syndrome showing mild

leukocytic infiltration. ...................................................................................... 122

4.12 Liver of the goat infected with CCPP .............................................................. 123

4.13 Liver of the sheep infected with mycoplasmosis. ............................................ 124

4.14 Liver of the sheep infected with mycoplasmosis showing swollen

hepatocytes (arrow). ......................................................................................... 124

4.15 Brain of goat suffering from respiratory syndrome showing mild

congestion (long arrow) and few inflammatory cells ....................................... 125

4.16 Brain of sheep suffering from respiratory syndrome showing normal

histological structure. ....................................................................................... 125

5.1 MIC of different antimicrobial agents using broth micro dilution method

against Mycoplasma mycoides subsp. capri in PPLO broth. .......................... 150


against Mycoplasma capricolum subsp. capripneumoniae in PPLO broth. ... 151


against Mycoplasma putrefaciens in PPLO broth. ........................................... 151

5.4 Indigenous medicinal plants 1= Calotropis procera, 2= Artemisia herba-

alba, 3= Azadirachta indica ............................................................................. 152

6.1 Whole cell indigenous saponized and lyophilized Mmc vaccine, VRI

Lahore ............................................................................................................... 184

6.2 Vaccine inoculation in Kari sheep at experiemnetal Livestock

farm………………………………………………………………………… .. 184

viii

LIST OF ABBREVIATIONS/ACRONYMS

B wt. Body weight

BSA Bovine serum albumin

c- ELISA Competitive enzyme-linked immunosorbent assay

Cfu Colony forming unit

CBPP Contagious bovine pleuropneumonia

CCPP Contagious Caprine Pleuropneumonia

DIC Disseminated intravascular coagulation

df Degree of freedom

FAO Food and Agriculture organization

FATA Federally Administered Tribal Area

GMT Geometric mean titer

GI Growth Inhibition

HCL Hydrochloric acid

IHA Indirect haemagglutination

KP Khyber Pakhtunkhwa

LC Large Colony

H & E Hematoxylin and Eosin

M Mycoplasma

MBG7 Mycoplasma Bovine Group 7

Mm Mycoplasma Mycoides

MIC Minimum inhibitory concentration

Mmc Mycoplasma mycoides subsp. capri

Mmm LC Mycoplasma mycoides subsp. mycoides Large Colony

Mmm SC Mycoplasma mycoides subsp. mycoides Small Colony

Mmc Mycoplasma mycoides subsp. capri

Mcc Mycoplasma capricolum subsp. capricolum

Mccp Mycoplasma capricolum subsp. capripneumoniae

Mp Mycoplasma putrefaciens

NAOH Sodium Hydroxide

OIE Office International Des Epizootics

OD Optical density

PBS Phosphate buffer saline

PCR Polymerase Chain Reaction

PPLO Pleuropneumonia Likes Organism

RT-PCR Real Time Polymerase Chain Reaction

TBE Tri buffer acetate

VRI Veterinary Research Institute

ix

ACKNOWLEDGMENT

All praises be to ALLAH Almighty WHO sent the final messenger

MUHAMMAD (S.A.W) for the eternal guidance of mankind, WHO taught with the

pen and WHO Taught man what he knew not.

Special appreciation goes to my supervisor and Chairman, Prof. Dr. Umar

Sadique for his supervision and constant support. His invaluable help of constructive

comments and suggestions throughout the experimental and thesis work have

contributed to the success of this research. I would like to thank my co-supervisor Dr.

Zahoor Ul Hassan and Dr. Aqib Iqbal for his support and understanding throughout

the process. The enthusiasm of both these teachers as pathologists and molecular

biologist for this study made a strong impression on me.

I would like to thank the rest of my supervisory committee: Prof. Dr. Nazir

Ahmad, Dean FAH&VS and Prof. Dr. Muhammad Subhan Qureshi, Ex-Dean for

their encouragement, insightful comments and hard questions. My gratitude goes to

Prof. Dr. Sarzamin Khan, Prof. Dr. Abdur Rahman, Chairman Dept. of Livestock

Management, for their valuable sugessetion and correction in thesis write up. Special

thanks to Dr. Syed Muhammad Suhail, Dr. Shakoor Ahmad Qureshi, Dr. Farhan

Anwar, Dr. Sadeeq Ur Rahman, Dr. Hamayun Khan, Dr. Murad Ali Khan Dr.

Waseem Shahzad, Dr. Muhammad Mushtaq, Dr. Faisal Anwar for their technical

support during my studies. I am also very thankful to Dr. Yousaf Hayat and

Muhammad Iftekhar who kindly assisted me in the statistical analysis of this huge

data. My sincere thanks also go to my fellow lab mates at the Department of Animal

Health Dr. Farida Tahir, Dr. Said Sajjad Ali Shah, Dr. Azmat, Dr. Mushtaq

Ahmed, Dr. Hayatullah Khan, Dr. Faisal Ahmad, Dr. Asfand Yar Khan, Dr.

Subhan, Dr. Tasbeeh Ullan and Dr. Nazir for the stimulating discussions, for the

tireless work and their nice company during lab experiemts since last four years.

I feel pleasure to express my cordial thanks to Por. Dr. Sohail Ahmad, Dr.

Sayyar Khan and Dr. Abid Ali, IBGE, the University of Agriculture Peshawar for

their sincere guidance, kind help and support during my molecular research activities

and optimization of experiement in their labs. They also really helped me in the

molecular identification and interpretation of my results.

x

I extend my heartiest thanks to Dr. Iqbal Khattak, Director VRI, Peshawar,

Dr. Rehmat Jan PRO, VRI Peshawar and Dr. Hanif ur Rehman, RO for their

guidance and technical support to conduct valuable research work regarding vaccine

preparation and trails in VRI Kohat and Peshawar. I extend my special thanks to Dr.

Shakib Ullah Khan, Principal, Dr. Shakir Ullah, Dr. Muhammad Tariq, Dr. M.

Shoaib, Dr. Madiha Hassan, Nasurllah Khan, Haji Muhammad Nazir, Azaz

Rasool and Adil Rizwan Gomal College of Veterinary Sciences, Gomal University D.

I. Khan for their encourgment and moral support during the whole study.

I extended my special thanks to my laboratory staff Lawad Khan Khattak

(Lab supervisor), Ilyas U Din (Lab superintendent), Wali Ullah (Office clerck),

Muhammad Saeed Khan, Nehad Khan (Lab Assiatant), Muhammad Ijaz (Lab

attendent) and Muhammad Ilyas Khan (Lab assiatant) to facilitate me in my research

activities.

I am highly thankful to Pakistan Science Foundation (PSF) for funding the

project PSF/NSLP/KP-AU (219) and enabling this tudy possible.

Last but not least, my deepest gratitude goes to my family, my beloved parents,

brothers Muhammad Jamal and Muhammad Rehman for supporting me spiritually

and financially throughout my life, also to my all brothers and sisters and my sweet

wife for their endless love, prayers and encouragement during my entire study. I

offered my sincere love to my little princesses Malaika, Laiba and Maria Kamal for

their pataince and innocent love and pray to complete this study. Thank you all very

much.

Muhammad Kamal Shah

xi




Muhammad Kamal Shah and Umer Sadique

Department of Animal Health

Faculty of Animal Husbandry and Veterinary Sciences

The University of Agriculture, Peshawar-Pakistan

March, 2017

GENERAL ABSTRACT

The project was designed to investigate the pathogenesis, molecular characterization,

chemotherapy and vaccine development for Mycoplasmosis in small ruminants. The

project was completed in four different studies as described below:

In study-1, the isolation and molecular characterization of Mycoplasma spp. was

carried out in small ruminants suffering from respiratory syndrome in natural infection

suspected for Contagious Caprine Pleuropneumonia (CCPP). A total of 1980 samples

were collected from different sources comprising of nasal discharge (n=1500), tracheal

swab (n=300), lungs tissue (n=147) and pleural fluids (n=33) from animals exhibiting

respiratory signs suspected for CCPP. A detail history about specie, age, sex of animals

was also documented on preformed questionnaire. Out of total, 737 (37.22%) were

positive for Mycoplasma growth, showing mass turbidity, whirling movement in

culture broth and typical fried egg colonies on agar media. The results revealed that

significantly (P˂0.001) higher isolation rate (% age) of Mycoplasma isolates was noted

in northern zone (43%) followed by southern (34.6%). Similarly, significantly

(P˂0.01) higher ocuurenace was observed in goats (58.8%) as compared to sheep

(41.2%). On PCR analysis, a total of 553 (27.92%) isolates were confirmed as

Mycoplasma with species distribution of 13.53%, 5.5% and 7.97% Mycoplasma

mycoides subsp. capri, Mycoplasma capricolum subsp. capripneumoniae and

Mycoplasma putrefaciens, respectively. It was revealed that highest isolates were

recovered from pleural fluids (63.6%) followed by lungs tissue (58.5%), nasal

discharge (25.5%) and least from tracheal swab (21%). The phylogenetic study of all

the three species were also documented having distinct nucleotide sequence as

compared with the available isolates at National Center for Biotechnology Information

(NCBI). This revealed the local isolates of Mycoplasma capricolum subsp.

capripneumoniae (Mccp) different from the strains of USA and France but having close

homology with the strain of neighboring countries i.e. China and India.

In study-2, a total of 1800 diseased and 180 necropsied animals were surveyed for

recording of the clinico-pathological picture of contagious caprine pleuropneumonia

(CCPP) in naturally infected sheep and goats. Out of total examined animals,

pneumonia was recorded in 61.55% followed by pyrexia (58.2%), cough (56.83%)

watery nasal discharge (52.22%) and lacrimation (40.77%). The most frequent lesions

were recorded in the lungs 53.88%, followed by trachea 37.7% and pleural effusion

18.33%. On histopathological examination majority of lung sections showed

emphysema, atelectasis, thickning of alveolar wall and extensive leukocytic infiltration.

Some section also showed chronic inflammatory changes consisted of aggregation of

xii

macrophages, fibroblast and plasma cells. The multi-systemic involvement is the

common feature of the present findings. The internal organs including liver, spleen,

kidneys and intestine revealed congestion, hemorrhages and leukocytic infiltration. Few

brain sections showed mild congestion and few inflammatory cells; however most of

the brains were showing normal histological details. The gross and microscopic lesion

scoring revealed that maximum lesions were observed in respiratory tissue. The overall

lesion scoring indicated more severe nature of disease in goat as compared to sheep.

In study-3, chemotherapeutic trials were conducted to investigate the effects of

commonly used antimicrobial agents and three indigenous medicinal plants against the

local isolates of Mycoplasma recovered from small ruminants. Five different

commercially available antimicrobial agents like tylosin, oxytetracycline, enrofloxacin,

gentamycin and ceftofer sodium and three medicinal plants including Calotropis

procera, Azadirachta indica and Artemisia herba-alba were tested invitro as broth

micro dilution and disc diffusion assay. The results of disc diffusion assay revealed that

maximum zone of inhibition 19.0±0.71 mm was produced by enrofloxacin, followed by

gentamycin 11.0±0.45 mm and tylosin 6.8±0.37 mm against Mmc. The isolates showed

resistance against oxytetracycline and ceftofer sodium. The results of broth micro

dilution revealed that enrofloxacin exhibited strong antibacterial activity with minimum

inhibitory concentrations (MICs) value of 0.001, 0.001 and 0.01 mg/mL against

Mycoplasma mycoides subsp. capri (Mmc), Mycoplasma capricolum subsp.

capripneumoniae (Mccp) and Mycoplasma putrefaciens (Mp), respectively.

Gentamycin was moderately effective against all isolates of Mycoplasma. Tylosin,

oxytetracycline and ceftofer sodium exhibited high MICs value against the tested

isolates. Among the tested methanolic plant extracts A. herba-alba showed maximum

zone of inhibition 16.3±0.33, 14.0±0.44 and 15.4±0.12 mm at 30 mg against Mmc,

Mccp and Mp, respectively. It was concluded that enrofloxacin is the most potent agent

for the treatment of caprine mycoplasmosis. Among the tested medicinal plants A.

herba-alba was showing high anti-mycoplasmal activity against the local tested isolates

of Mycoplasma.

The study-4 was aimed to prepare a saponized vaccine from the local isolates of

Mycoplasma mycoides subsp. capri (Mmc). The PCR confirmed local isolates of Mmc

having 0.2 mg/mL protein content was inactivated with saponin at the dose rate of 3.0

mg/mL. The indigenous saponized vaccine and commercially available lyophilized

Mmc vaccine were inoculated in experimental animals consisted of sheep and goats for

evaluation and comparison of its immunogenic potential. In sheep the maximum

antibodies titer was achieved with geometric mean titer (GMT) values of 147.1 and 128

for saponized and lyophilized vaccine on day 35 post vaccination. The antibodies titer

with highest GMT values of 224 and 192 was recorded on day 28 post vaccination in a

challenged group vaccinated with saponized and lyophilized vaccine, respectively. No

abnormal signs were observed in all experimental animals throughout the experimental

trial. This study confirmed that the vaccine prepared from local field strain of Mmc

confer better protection as compared with the commercially available vaccine.

Key words: Mycoplasma, goat, nasal discharge, lungs, PCR, lesions, antimicrobial

agents, disc diffusion, medicinal plants, saponin, vaccine.

1

I. GENERAL INTRODUCTION

The livestock industry is an emerging long market chain that provided an

employment to at least 1.3 billion people globally. In the developing world about 600

million small farmers are directly supported by this sector (Thornton, 2006). Livestock

products fulfill the nutritive requirements of the human population by contributing 33%

protein and 17% kilocalories globally (Rosegrant et al., 2009). In Pakistan, Agriculture

plays a vital role in the national economy and livestock contributes major share to meet

the demand of meat, milk, fat, hide and provides a source of ready cash for the poor

farmers to fulfill their daily requirements. Livestock is considered a more secure source

of income for the handless poor and small farmers by providing employment to the

mass community and plays a role in poverty alleviation. It contributed approximately

58.3 % in agriculture and shared 11.4 % to the national gross domestic production

(GDP) (Economic survey, 2016-17). Majority of the rural population is involved in the

livestock sector and about 08 million families are engaged in raising livestock and

derivning more then 35% income from that sector (Economic survey, 2016-17). Sheep

and goat rearing brings incredible importance in rural economy for nonagricultural or

low earning mass community of the country. In subcontinent goat is recognized as poor

man’s cow (Shahzad et al., 2013). Pakistan is the 3rd

largest goats and 12th

sheep

producing country of the world by sharing approximately 102 million small ruminant

population that yielded up to 930 Matric tons (MT) of milk and 701 MT of mutton

(Economic survey, 2016-17).

Small ruminant population is under continuous stress and faces various

challenges in the form of harsh environmental conditions, scarcity of feed stuff, poor

husbandry practices and fatal diseases. Amongst different infectious diseases, the

mycoplasmosis is a major threat to small ruminant causing high morbidity and

mortality (Regassa et al., 2010). Mycoplasmosis is multi systemic disease referred to

the infection collectively caused by various Mycoplasma mycoides cluster and non-

cluster pathogenic Mycoplasma species. Ruminant mycoplasmosis is prevalent

throughout the world particularly in the developing countries of south East Asia,

Middle East and Africa, which is inflicting heavy economic losses to the small

ruminant industries (Tigga et al., 2014; Ongor et al., 2011; Srivastava et al., 2010).

This important disease is also widely prevalent in Pakistan with history of several

2

outbreaks that caused huge economic losses in the northern and southern regions of the

country (Banaras et al., 2016; Shahzad et al., 2013; Sadique et al., 2012; Rahman et al.,

2006; Hayat et al., 1990).

Among the caprine mycoplasmosis the contagious caprine cleuropneumonia

(CCPP) is caused by six different pathogenic Mycoplasma species including

Mycoplasma capricolum sub specie capripneumoniae (Mccp), Mycoplasma mycoides

subsp. mycoides small colony (MmmSC), Mycoplasma mycoides subsp. capri (Mmc),

Mycoplasma mycoides subsp. mycoides Large Colony (MmmLC), Mycoplasma

capricolum subsp. capricolum (Mcc) and Mycoplasma bovine group 7 (MBG7). All

these species have interrelated group called as Mycoplasma mycoides (Mm) cluster that

is further divided into two sub clusters (Manso-Silvan et al., 2007; Cottew et al., 1987).

The non-cluster species like Mycoplasma agalactiae, Mycoplasma ovipneumoniae and

Mycoplasma putrefaciens have been isolated from sheep and goats in many countries of

the world (Banaras et al., 2016; Ongor et al., 2011; Noah et al., 2011; Awan et al.,

2009; DaMassa et al., 1992).

Among the ruminant mycoplasmosis CCPP is highly significant respiratory

disease caused by Mccp. This disease mainly confined to the thoracic cavity

characterized by fibrinous-pleuropneumonia, pyrexia and high mortality (OIE, 2014;

Gelagay et al., 2007; Nicholas, 2002). It is wide spread disease in the world posing a

serious threat to the small ruminant population and incuded in list B diseases by world

organisation for animal helath (OIE, 2013). CCPP is widespread in Pakistan and

causing high morbidity and mortality in goat population in southern and northern

regions (Shahzad et al., 2016; Sadique et al., 2012; Awan et al., 2009; Rahman et al.,

2003; Hayat, 1990). The disease causes huge economic losses directly in the form of

high mortality, decrease in milk and meat production, poor carcass and indirectly the

diagnosis, treatment, control cost and trade embargo (OIE, 2014).

Mycoplasma is unique prokaryote of the class Mollicutes, smallest bacteria (100

to 250 nm) that lack rigid cell wall, highly pleomorphic and mostly pathogenic in

nature (Razin, 2000). It invade both phagocytic and non-phagocytic cell and utilizes

several immunomodulatory mechanisms to evade the host immune system. These

characteristics of Mycoplasma affect surveillance and control mechanisms such as

serologic testing and vaccination. Their genomic size is varying from 196 to 1350 bp.

3

Being delicate organism survival rate is limited in the environment and is parasitic in

nature exhibiting strict host and tissue tropism. They are widely distributed in nature as

parasites of mammals, birds, reptiles, fish, arthropods, and plants (Carlton et al., 2010).

Many of these organisms cause diseases of livestock that heavily impact on production

parameters such as weight gain, milk yield and weight loss. Mycoplasma is surrounded

by cell membrane that contains important protein called as lypoglycan. In small

ruminants lypoglycan stimulate acute inflammation in pulmonary tissue that leads to

excessive purulent exudate and pleural effusion. In goats it can causes acute septicemia

with pulmonary capillary thrombosis (Rosendal, 1993).

Pathogenic Mycoplasma species also causes different systemic and

inflammatory conditions in sheep, goats and some wild ruminants. Among other

clinical manifestation, respiratory signs are very important and commonly observed in

many outbreaks. In mycoplasmosis the main clinical findings are painful respiration,

pneumonia, persistent cough, nasal discharge, lacrimation and keratoconjunctivitis

(Mondal et al., 2004), other inflammatory complications such as arthritis, mastitis,

hepatitis, peritonitis, cervical abscesses and in rear cases of meningitis (Abtin et al.,

2013; Schumacher et al., 2011; Sharif and Muhammad 2009; Jubb et al., 1985). In

mycoplasmosis the typical multi-systemic manifestation is called MAKePS (mastitis,

arthritis, keratitis, pneumonia and septicemia) syndrome (Egwua et al., 2001;

Thiaucourt et al., 1996; Bolske et al., 1988). The affected animals show extreme

depression, anorexia, suspended rumination and high rise of temperature upto 106 °F or

41°C (Thiaucourt and Bolske, 1996; McMartin et al., 1980). Other signs include

lameness, diarrhea, stiff neck and lie down on ground with lateral recumbancy in

advance stage of disease (Sadique et al., 2012; OIE, 2008). In fully susceptible flocks

the morbidity is usually reached up to 100% and mortality recoded 70% (Mondal et al.,

2004; Madanat et al., 2001). In some animals nervous signs are also noticed, like

animal reluctant to move, stiffed neck and circling movement (Shahzad et al., 2013;

Schumacher et al., 2011).

Pathological lesions play vital role in the diagnosis of a disease and directed the

clinician for intervention and selecting proper therapy for early recovery. Some

pathognomonic lesions are tissue specific and helpful for the diagnosis of a particular

disease and pave away to design strategies by the researcher and physician for accurate

4

treatment and eradication of the disease. The different pathogenic species of

Mycoplasma has the ability to produced lesions in different tissues, organs and system

of the body provided key for its diagnosis and therapeutic intervention (Riaz et al.,

2012; Laura et al., 2006; Mondal et al., 2004; Leach et al., 1993). The severity of

lesions in Mycoplasma infection depends on many factors like age, breed, sex, immune

status of animal, environmental factors and the pathogenicity and virulencey of

Mycoplasma species (Yousuf et al., 2012).

Mycoplasma also produced significant pathological changes in different tissues

and organs of the host body. Gross lesions observed are inflamed and consolidated

lungs, with marble appearance, lungs hepatization fibrinous pleuropneumonia and

accumulation of straw color fluid in pleural cavity. Unilateral or bilateral pneumonia

are commonly present (Sadique et al., 2012; Thiaucourt et al., 1996). Bronchial and

mediastinal lymph nodes are swollen and edematous. Plural adhesion with wall of chest

cavity and whitish pleura is a common feature. In some cases pericardial sac is filled

with serosanguinous fluid. Liver and kidneys get enlarged with hemorrhages and multi

focal necrotic foci. Congestions and hemorrhages of varying degree is also seen in

mucosal surface of trachea and intestine (Riaz et al., 2012; Sadique et al., 2012;

Gelagay et al., 2007; Mondal et al., 2004). The development of microscopic

pathological changes in different visceral tissues dependent on the specie of

Mycoplasma which caused the CCPP infection (Laura et al., 2006).

Microscopic lesions are characterized by fibrinopurulent pleuropneumonia with

thickening of interlobular septa (Abbas et al., 2013; Laura et al., 2006; DaMassa et al.,

1992). Hemorrhages are present in tracheal section with sloughing of lining epithelium

and leukocytic infiltration including lymphocytes, plasma cells and macrophages. In

lungs emphysema and atelectasis is frequently present accompanied by hemorrhages,

necrosis and the most characteristics finding is micro thrombosis in the lumina of small

vessels and fibrin deposition in alveoli (Riaz et al., 2012; Sadique et al., 2012; Laura et

al., 2006; Mondal et al., 2004; Gutierrez et al., 1999). In Mmc infection the lesions

were not limited to thoracic cavity but recorded in various tissues. Serosanguinous fluid

was accumulated in pericardial and peritoneal sacs (Nicholas et al., 2008). The trachea

is congested and lumen contains exudates. Hemorrhages and leukocytic infiltration in

5

liver, kidneys and spleen are frequently recorded in Mycoplasma infection (Laura et al.,

2006; Mondal et al., 2004).

The strategies to effectively treat the Mycoplasma infection are complicated and

need proper selection of antimicrobial agent for effective elimination from the host

body. Mycoplasma species are often intrinsically resistant to many conventional

antimicrobial drugs due to lacking of cell wall. A number of antimicrobial agents that

act on nucleic acid and protein synthesis are used for the treatment throughout the

world with varying degree of success. The commonly used antibiotics are including

tylosin, oxytetracycline, gentamycin, enrofloxacin and penicillin with different degree

of success (Laura et al., 2006). The Mycoplasma has the adaptive capability to

modulate its structure and evade itself from the host immune system. This unique

feature of Mollicutes make them unaccessble to therapeutic agents and thus make the

host chronic carrier. Furthermore, the indiscriminate use of these agents in small

ruminant has been shown to be associated with increased resistance (Scott and

Menzies, 2011). The resistance patterns of pathogenic Mycoplasma to commonly used

antibiotics restricted their treatment options and control. Therefore medicinal plants

extract are being used as alternative agent to minimize resistance issue, having

minimum side effect and recomended in food animals (Ashokkuma and Ramaswamy,

2014; Chin et al., 2006). Plants are rich source of secondary metabolites such as

flavonoid, alkaloids, tannins, terpenoids and phenolic compounds that are having strong

antimicrobial properties (Nasir et al., 2015; Bakht et al., 2014).

Immunization is the possible way to effectively control and prevent infectious

diseases. The past human and animal diseases like polio, small pox, diphtheria and

rinderpest are completely eradicated due to use of efficient vaccine (Ghanem et al.,

2013). Inspite of availability of vaccine several infections are still not controlled

properly and responsible for million of deaths in human and animal population (Cruttis,

2011). The specie specific vaccine is useful tool to combate many infectious diseases of

livestock (OIE, 2013). Saponin inactived Mycoplasma vaccine has been use in different

regions of the world with variable efficacy and immunogenic potential (OIE, 2014;

Nicholas and Churchward, 2012). In several studies, whole cell culture formalized and

saponized vaccine were successfully used for eradication of different diseases of

livestock (Ahmad et al., 2013; Nicholas et al., 2009; Jaffri et al., 2006). In Pakistan

6

only one specie specific vaccine i.e. Mmc is available and carried out in different areas

of the country (Shahzad et al., 2012). Inspite of vaccination, the disease outbreak is

frequently reported from every corner of the country and poses a serious threat to the

small ruminant population. The failure of vaccine has justified that in practice one

specie specific vaccine could not correctly mitigate the disease effectively. The reason

for this failure might be due to prevalence of several pathogenic Mycoplasma species

and the difference in the antigenic structure of vaccinal strain from the local strain.

For effective treatment and control of a disease it is utmost important to

diagnose and confirm its causative agent. Different conventional and non-conventional

techniques are used for the diagnosis of CCPP in small ruminants with varying degree

of success. The isolation and culturing of Mycoplasma is difficult task because of its

unique nature, growth requirements, need special media and expertise. The successful

isolation is usually failed in field practices due to extensive use of antibiotics. The

serological and biochemical assays cannot confirm the exact specie of Mycoplasma

because of sharing its antigenic epitopes by several mycoides cluster species (Manso-

Silvan et al., 2009; Cottew et al., 1987). The advanced molecular techniques are

effective tools for accurate diagnosis and confirmation of the exact causative specie. In

the era of advanced molecular technology a vast array of primers are available and

successfully used for the diagnosis of different diseases including the CCPP in small

ruminant (Banaras et al., 2016; Manso- Silvan et al., 2009; McAuliffe et al., 2005;

Dominique et al., 2004). The PCR with specie specific primer amplified 16S- rRNA

gene of Mycoplasma that allowed the identification of genus and specie (Kumar et al.,

2011; Manso-Silvan et al., 2007; Hotzel et al., 1996).

In Pakistan, for the first time, Mmc in goats is reported that was identified

through biochemical tests (Khan et al., 1989). Later on several biochemical and

serological tests were used for preliminary identification of several Mycoplasma

species (Rahman et al., 2003; Hayat et al., 1990). The introduction of molecular

diagnostic techniques makes possible the accurate identification of causative pathogen.

For the 1st time Mccp was confirmed in Baluchistan (Awan et al., 2010), and Mmc in

Khyber Pakhtunkhwa (Sadique et al., 2012). Later on mycoides cluster and non-cluster

species like M. putrefaciens was confirmed in sheep and goats in Baluchistan (Banaras

et al., 2016; Hira et al., 2015; Awan et al., 2012). The DNA sequencing and

7

phylogenetic analysis of 16S rRNA gene is useful tool to establish relationship between

different Mycoplasma species. The gene sequencing also provides the evolutionary

history of the organisms and to define mutational changes in the genetic makeup of

pathogen (Manso-Silvan et al., 2007; Thiaucourt et al., 2000; Pettersson et al., 1994). A

little work has been conducted on molecular identification and characterization of the

pathogenic Mycoplasma species. It is therefore important to investigate and charaterize

that how many pathogenic species of Mycoplasma are causing the diseases in sheep and

goats in Pakistan. Therefore the present study was designed to use advance molecular

techniques to find out the causative species prevailing in this region of the country.

General Objectives:

This study has been designed with the following objectives:

1. To study the prevalence, molecular characterization and spatial distribution of

the local isolates of Mycoplasma responsible for CCPP in the study area.

2. To investigate the pathogenesis of CCPP in small ruminant of Khyber

Pakhtunkhwa.

3. Chemotherapeutic trials of commonly used antimicrobial agents and indigenous

medicinal plants against the local isolates of Mycoplasma.

4. Trial to develop indigenous vaccine from the local isolates for the control of

Mycoplasmosis.

8

II. REVIEW OF LITERATURE

2.1 Respiratory complications in small ruminants

Pneumonia is one of the most important threats to the livestock population

throughout the world. Different pathogens including bacteria, fungi, viruses and

parasites are responsible for several respiratory complications in small ruminants

besides poor managemental practices. Among the bacterial diseases, mycoplasmosis is

causing huge economic losses in the small ruminants throughout the world especially in

the under developed countries (Regassa et al., 2010). Several Mycoplasma species are

prevalent in the different regions of the world with different pathogenic potential. The

most important pathogenic Mycoplasma infections consisted of avian mycoplasmosis,

bovine mycoplasmosis and caprine mycoplasmosis. Caprine mycoplasmosis is

prevalent throughout the world particularly in the developing countries of south East

Asia and Africa and inflicting heavy economic losses to the small ruminant production

(Tigga et al., 2014; Ongor et al., 2011; Srivastava et al., 2010). Both pathogenic and

non-pathogenic species of Mycoplasma are normally present in the respiratory tract of

the small ruminant with no serious complications (Razin et al., 1998). However the

different stress conditions predispose the animal to succumb to infection by the

frequent multiplication of those opportunistic micro-organisms (Browning et al., 2007).

2.2 Mycoplasmosis in livestock

Pakistan is an agro-climatic country having estimated livestock population (191

millions) and among which contribution of small ruminant is 102 million (Economic

Survey, 2016-17). In small ruminants, respiratory complication is the major health

problem caused by pathogenic Mycoplasma species that are present in the normal flora

of the respiratory tract. Some pathogenic Mycoplasma species causes important disease

in cattle and small ruminants called as mycoplasmosis (Blood et al., 2007). These

diseases CCPP, contagious agalactiae (CA) in small ruminant and contagious bovine

pleuropneumonia (CBPP) in cattle. These diseases are contagious in nature with high

morbidity and mortality of any age and sex. The mycoplasmosis is highly contagious

disease and transmission is mainly occuring through aerosol and contaminated feed,

water and milk (Thiaucourt and Bolske, 1996). Mmc cluster and non-cluster species like

9

M. putrefaciens, M. agalactiae and M. ovipneumoniae are manily involved in the

infection

2.3 Contagious Caprine Pleuropneumonia (CCPP)

Contagious caprine pleuropneumonia (CCPP) is highly fetal respiratory disease

of small ruminants. CCPP is caused by six different pathogenic Mycoplasma species

called “mycoides cluster”. According to many researchers the principal causative agent

of the disease is important mycoides cluster member is the Mccp. This pathogen is

mainly targated the host respiratory system and the manifestation of the disease is

restricted to the thoracic cavity (OIE, 2014). The primary host of the pathogen is goat,

but also reported in sheep and wild ruminants with high mortalities (Arif et al., 2007).

2.4 History of CCPP

Mycoplasma is mainly responsible for the respiratory syndrome in livestock

population. It causes pleuropneumonia in cattle and small ruminants throughout the

world. CCPP is the major threat to the goat farming industry in the developing

countries (Lorenzon et al., 2002). The disease is pandemic in Asia, Africa, Eastern

Europe and the Middle East (Nicholas and Churchward, 2012; Manso-Silvan et al.,

2011; Kopcha, 2005). CCPP is a highly fatal disease which was for the 1st time

reported in 1873 in Algeria (McMartin et al., 1980), latter on disease was spread by

shipment of Angora goat in the cape colony of South Africa in 1881 (Hutcheon, 1889;

Hutcheon, 1881). It is documented that CCPP is prevalent in more than 40 countries but

Mccp has been only isolated in 20 countries (Nicholas et al., 2003). However, now it

has been confirmed that Mccp is prevalent in many country of the world including

Turkey (Ozdamiret al., 2005), China (Chu et al., 2011), Tajikistan (FAO, 2012),

Pakistan (Shahzad et al., 2016; Peyraud et al., 2014).

In the last decade two members of mycoides cluster MmLC and Mmc were

considered the causative agents of disease due to the production of pleuropneumonia in

small ruminants. Mycoplasma F38 associated with respiratory disease was considered

the cause of respiratory syndrome in small ruminants (MacOwan and Minnette, 1976).

Six pathogenic Mycoplasma species called as mycoides cluster are responsible for the

disease in small ruminants. Several scientists confirmed that CCPP is caused by Mccp

10

bio type F38, which was for the time first isolated in Kenya. The specie was later on

reported in many countries of Africa and Asia (OIE, 2014; Awan et at., 2010;

Thiaucourt et al., 2008; Manso-Silvan et al., 2007; Rurangirwa et al., 1987a; Mac

Owan and Minette, 1976).

In Pakistan, the disease was reflected to be caused by Mmc till the molecular

confirmation as Mccp in Baluchistan by Awan et al. (2010). Recently a collaborative

study was conducted in different countries of Asia and Africa for the sero prevalence of

CCPP using c-ELISA kit formatted at CIRAD, France. The seroprevalence of CCPP

caused by Mccp was estimated 2.7% and 44.2% in Gilgit and Diamer districts of

Norther Pakistan, 14.6% in Afar regions of Ethiopia, 10.1% in the Shuro-Obod District

of Tajikistan and 15% in Mauritius (Peyraud et al., 2014). Similarly using same c-

ELISA kit the seroprevalence of CCPP caused by Mccp was reported 8.52% in

different areas of Punjab, Pakistan (Shahzad et al., 2016).

2.5 Susceptible hosts for Mycoplasma infection

The genus Mycoplasma is causing infectious diseases in bovine, ovine, caprine,

camel and wild ruminants. Among the susceptible host, goat (Capra hircus) is the

primary and most common animal susceptible to the Mycoplasma infection in natural

outbreak (Madanat et al., 2001). Other susceptible species are sheep (Ovis aries) and

wild ruminants including wild goats (Capra aegagrus), Nubian Ibex (Capra ibex

nubiana), Gerenuk (Litocranius walleri), Lasristan mouflon (Ovis orientalis

lasristanica), Sand gazelles (Gazella subgutturosa marica), Tibetan antelope

(Pantholops hodgsonii) and Arabian oryx (Oryx leucoryx) (Giangaspero et al., 2010).

The pathogenic member of Mm cluster and non- cluster species like M. agalactiae and

M. putrefaciens are mainly responsible for small ruminant mycoplasmosis with multi-

systemic involvement (OIE, 2014; Nicholas et al., 2008; Arif et al., 2007). In large

ruminants, cattle (Bostaurus) and Camels (Camelus dromedarius) also infected by M.

bovis and MmLC respectively (Shoieb and Sayed-Ahmed, 2016), M. hyosynoviae and

M. hyorhinis causes swine mycoplasmosis (Thacker, 2006). The disease also reported

in largest ruminants like giraffe (Giraffa Camelopardalis reticulata) and elephants

(Loxodonta africana and Elephas maximus (Elizabeth et al., 2003; Clark et al., 1994).

11

2.6 Classification of Mycoplasma

All the Mycoplasmas belong to class Mollicutes that are distinct from all other

bacteria due to lacking of cell wall and minute genome size ranging from 600 to 2200

Kbp. Mollicutes consisted of more than 120 species and eight genera namely

Mycoplasma, Ureapalsma, Acholeplasma, Spiroplasma, Asteroplasma, Anaeroplasma,

Mesoplasma and Entamoplasma (Tully et al., 1993). The genus Mycoplasma consisted

of important pathogenic species and sub specie responsible for animal and human

diseases. Among the different species, Mm clusters are considered to be the main

pathogens causing disease in small ruminant. The mycoides cluster consisted of six

different pathogenic species and sub species comprises of Mcc, Mccp, MmmSC and

large colony (LC) types, Mmc and bovine group7 (Manso-Silvan et al., 2007). Many

members of Mycoplasma species share genomic and antigenic structure that often

causes immunological cross reaction (Cottew et al., 1987).

2.7 Morphology

Mycoplasma are the smallest prokaryotic cell that was described more than 100

years ago. They are broadly distributed in nature and inhabiting human, animals, plants

and insects (Rottem and Naoh, 1998). The organism is characterized as the smallest

self-replicating bacteria. Highly pleomorphic having pear shaped, helical filaments,

flask shaped cells of various lengths, but some species also have a cytoskeleton and the

single coccoid cell has a diameter of about 0.3 nm (Razin et al., 1998). The spherical,

pear shaped, filamentous and branched Mycoplasma cells are usually 0.3-0.8 µm in

diameter. They have length from few micrometers to almost 150µm. Some pathogenic

species are capable of forming biofilms in-vitro and in-vivo, which increase their

resistance to heat, desiccation, co mplement mediated lysis, antibiotics and body

immune system. The biofilm formation by Mycoplasma species leads to immune

surveillance, thus the body defense system failed to encounter the infection (McAuliffe

et al., 2006). They have length from few micrometers to almost 150 µm.

12

2.8 Characteristics of Mycoplasma

Family and genera of Mycoplasma primarily distinguished by means of some

properties like smallest genomic size, pleomorphic shape, cholesterol requirement,

NADH oxidase location, urease reaction, habitat and the effect of oxygen and

temperature requirements. Mycoplasma are the smallest (100 to 250nm), self-

replicating microorganism devoid of rigid cell wall due to lacks genes for cell wall

synthesis (Razin et al., 1998). Mycoplasma has very small genome approximately 0.58-

2.20 Mb, having minimal G-C contents which is required for the growth and replication

of bacteria. Fraser et al. (1995) reported M. genitalium with minimum size of genome

(580 kb). The morphology of Mycoplasma species depend on several factors like

specific growth rate, osmotic pressure, temperature, pH and medium with specific

nutrients (Henderson and Miles, 1990). Most of the Mycoplasma species required 5%

CO2 for its growth during culturing. The colonies with the diameter of 10-600 µm

usually grow within two to twelve days at 37 oC, most species showed growth within 3-

5days and forming large colonies of 2-3 mm size. These can be best seen as

transparent, flat, typical fried egg and nipple like appearance with the help of dissecting

microscope or stereomicroscope (Al-Momani et al., 2006; Freundt, 1974). All the

Mycoplasmas produced fried egg colonies except M. ovipneumoniae which yields

centerless colonies (Jones and Gilmour, 1983). Mycoplasma reproduce by binary

fission like other bacteria but the cytoplasmic division is slow than the genomic

replication and lead to development of multinucleated filaments (Razin et al., 1998).

Mycoplasma species are usually host specific and having tissue tropism due to limited

biosynthetic potentials (Rottem and Yogev, 2000).

2.9 Growth Requirement and Culturing of Mycoplasma

Most of the Mycoplasma species are established as facultative whereas few are

reported as obligate anaerobes in nature. Sterol (cholesterol) and fatty acids are

essential component for the growth of Mycoplasma species. It lacks many genes

including those responsible for the production of all 20 amino acids and other important

biosynthetic genes (Razin et al., 1998). Due to its fastidious nature, it is very difficult to

grow Mycoplasma on ordinary laboratory media. Therefore, some special media are

used for the growth and isolation of different pathogenic Mycoplasma species.

Mycoplasma can grow on media which enriched with some special components like

13

10% horse or pig serum, sodium pyruvate and glucose (Nicholas, 2002; Thiaucourt et

al., 1992). Pleuro pneumonia like organism (PPLO) broth and modified Hay media are

commonly used for the isolation and culturing of various Mycoplasma species by many

researchers (Kabir and Bari, 2015; Sadique et al., 2012; Noah et al., 2011; Ongor et al.,

2011). During culturing, bacterial and fungal contamination is one of the most common

problems. Therefore, thallium acetate, fluconazole and penicillin are commonly used in

the preparation of media. Some special and modified media are also used for selective

isolation of several Mycoplasma species. The ager non selective media ICCA

(Mycoplasma Experience Ltd. product) which allow the development of Mccp as red

colonies at seven days post incubation. Mccp has been successfully grown and isolated

on modified Hayflick medium by several researchers (Manso-Silvan et al., 2011;

Balikci et al., 2008). Most of the Mycoplasma species by providing enriched media, 05

% CO2, humidity and temperature of 37 °C, can produce colonies of 1-3 mm within 3-5

days post incubation. The important member of mycoides cluster Mccp can grow

slowly as compared to all other Mycoplasma species and normally take 5 to 12 days

(Thiaucourt et al., 1996). However the pure culture of Mycoplasma after 3-5 passage

can grow fastly in 36-96 hours (OIE, 2014).

2.10 Ecology

Mycoplasma is basically host specific and may cause disease in wide range of

hosts, for example Mcc, Mccp and Mmc have been isolated from pneumonic lungs,

pleural fluids, nasal discharge and pericardial fluids of the sheep and goats (Shahzad et

al., 2013; Sadique et al., 2012; Awan et al., 2010). Many species of Mycoplasma now

have been isolated not only from livestock but also from aquatic animals, man and also

from insects and plants (Razin, 1992). A number of species have been found to cause

serious disease in small ruminants, some are associated with other pathogens and

potentially involved in multiple inflammatory conditions (Stalheim, 1984).

14

2.11 Pathogenic Mycoplasma Species

Most of the animal species are commonly infected by the Mycoplasma species.

Among the domestic animals, small ruminants are known to have a serious and

economically important disease, the CCPP. The Mm cluster is a group of Mycoplasmas

that are famous pathogens of small ruminant and cattle (Cottew et al., 1987). These

organisms are well documented to create the most well-known taxonomic problems

within the genus Mycoplasma (DaMassa et al., 1992). They are consisting of six

closely related Mycoplasmas species that share genetic characters caused by MmmLC.

Among these atypical pneumonia and agalactiae caused by M. ovipneumoniae and M.

agalactiae in sheep are very common. Mcc was 1st time described from goat with

polyarthritis. This specie also causes peracute or acute manifestation when introduced

experimentally. M. conjunctivae cause ovine and caprine conjunctivitis and mostly

isolated from eyes and nasopharynx (Fernandez-Aguilar et al., 2013; Motha et al.,

2003). Goats on the other hand have very important Mycoplasmal diseases caused by

Mmc targeting multiple tissues including lungs, pleura, kidneys, eyes, and joints.

Pneumonia, pleuropneumonia, pleuritis, hydrothorax, keratoconjunctivitis, mastitis, and

arthritis are the most commonly noted pathological manifestations. At present era it is

proved and well documented that contagious caprine pleuropneumonia (CCPP) caused

by Mccp is mainly confined to the thoracic cavity and still the most important disease

in goats in many parts of the world including Pakistan (Samiullah, 2013). Different

pathogenic species of Mycoplasma in small ruminants has been isolated in Asia,

Europe, Middle East and Africa and are listed as;

i. Mycoplasma capricolum sub specie capricolum

(Awan et al., 2009; Giadinis et al., 2008)

ii. Mycoplasma mycoides sub specie mycoides large-colony

(Antunes et al., 2007; Gutierrez et al., 1999)

iii. Mycoplasma mycoides sub specie mycoides small-colony

(Manso-Silvan et al., 2007)

iv. Mycoplasma mycoides sub specie capri

(Sadique et al., 2012; Laura et al., 2006)

v. Mycoplasma capricolum sub specie capripneumoniae

(Peyraud et al., 2014; Thiaucourt et al., 2008)

15

vi. Mycoplasma agalactiae (Al-Momani et al., 2011)

vii. Mycoplasma putrefaciens (Banaras et al., 2016)

viii. Mycoplasma ovipneumoniae (Ongor et al., 2011; Nicholas et al., 2009)

ix. Mycoplasma arginini (Abbas et al., 2013)

x. Mycoplasma conjunctivae (Fernandez-Aguilar et al., 2013; Motha et al., 2003)

xi. Mycoplasma gallinarum (Taylor et al., 1994)

xii. Mycoplasma bovis (Nicholas et al., 2004; Flitman-Tene et al., 1997)

2.12 Pathogenesis of Mycoplasma manifestation

Interactions between the pathogen, host and the environment determine the

outcome of infections. Host is equipped with numerous mechanism of protection from

the lethal insult of antigen, while the pathogens have the capabilities to adopt various

strategies to evade itself from the immune mechanism of the host (Carlton et al., 2010).

For successful infection and manifestation of disease, various factors play significant

role including the entry of pathogen into the host, reaching to the predilection site,

adherence, invading the target tissue, tissue tropism, multiplication and dissemination

(Blood et al., 2007). During this process, the invading pathogen uses its lethal

weaponry system to cause tissue damages and get nutrients from the host for

multiplication and modulate itself to evade host immune system to make the host

carrier for transmitting the infection (Poumarat et al., 1996).

In the respiratory tract, several physical and biochemical defense mechanisms

exist to protect the animal against foreign microbial colonization and infection

(Howard, 1984). The protective mechanism includes intact epithelium, mucociliary

apparatus, surfactant, surfactant proteins and alveolar macrophages (Fales-Williams et

al., 2002). The activity of the mucociliary movement repels the microbial adherence

and proliferation with respiratory mucosa. The mucinous and serous secretions of the

air way are enriched with some factors that inhibit activity of invading pathogens.

These include lysozymes, lactoferrin, phospholipase A2, surfactant proteins,

peroxidases, secretory leukoprotease inhibitor, bactericidal permeability-inducing

factor, cathelicidin, defensins, serprocidins and anionic peptides (Ganz and Weiss,

1997).

16

Primary viral or bacterial infections may suppress the innate immune response

of the host (Brogden et al., 1998). Soon after infection, the upper and lower respiratory

tissue like bronchi, bronchioles and alveoli contains dense neutrophils infiltration,

fibrin, seroproteinaceous fluid and blood. The exudate is associated with extensive

parenchymal necrosis caused by bacterial toxins such as lipopolysaccharide,

leucotoxin, and polysaccharide accompanied by inflammatory factors released by

leukocytes of acute inflammation. Neutrophil constituents that potentially contribute to

the tissue damage include enzymes, cytokines, oxidative radicals and chemokines

(Ackermann and Brogden, 2000). The most characteristic lesions are the hepatization

and consolidation of lungs. They also cause unilateral or bilateral pleuropneumonia

with tickining of the interlobular septa accompanied by serofibrinous fluids in the

thoracic and abdominal cavities. Hepatitis and multifocal splenitis is also recorded

(Abbas et al., 2013; DaMassa et al., 1992; Jones, 1989).

Mycoplasma is normal inhabitant of respiratory and urogenital tract epithelial

lining but rarely invade tissue (Razin, 1999). Some special protein called binding

protein like VIhA in Mycoplasma gallisepticum and galactan, P26 in M. mycoides sub

sp. mycoides small colony play role in adherence to tissue surfaces. Some other

variable surface protein (Vsps) play role in adhesion of PG45 on continuous line of

embryonic bovine lungs cell line (Sachse et al., 2000). The adherence followed by

multiplication and colonization spread infection locally in respiratory and urogenital

tract. The infection leads to contamination of the body surface secretions and also

penetrates epithelial barriers and spread hematogenously. In acute stage of disease

caused by some pathogenic species like Mmc resulted in septicemia, pyrexia and high

mortality in goat kids (Thiaucourt et al., 2000; Sadique et al., 2012). In chronic cases,

Mycoplasma localization occurs in serosal cavities or joints lead to polyserositis,

arthritis and polyarthritis (Elizabeth et al., 2003).

Biofilm formation is the important characteristic of Mycoplasma infections in

which bacteria attached to a substratum, or each other, mostly bounded by an

extracellular polysaccharide material (Donlan and Costerton, 2002). It makes the

pathogen to remain in the host tissue inspite of immune response (McAuliffe et al.,

2006). The formation of biofilm masks the Mycoplasma that minimizes the efficacy of

therapy and gets resistance against antimicrobial chemotherapeutic agents. It is reported

17

that there is significant difference in the capability of many Mycoplasmas to form

biofilm. Among the pathogenic species, M. agalactiae, M. putrefaciens, M. bovis, M.

ovipneumoniae, Mcc, Mmc, MmmLC having the ability of biofilm formation

(McAuliffe et al., 2006). Some Mycoplasma species having notable capability to

change surface antigenic protein which help in evading host immune response

(Bradbury, 2005). These factors justify the chronic nature and difficulty in eradication

of Mycoplasma from infected tissue and cell culture (Razin, 1999).

The mechanisms of the disease development of CCPP are exactly unknown, but

it is well known that most of Mycoplasma species adopted complex strategies to inter

into the host (Sachse et al., 1996). It showed tissue tropism than establish the infection

at cellular level in the predilection site followed by pathological alteration with multiple

clinical complications. Many factors may influence Mycoplasma-associated arthritis

and other inflammatory disease expression, including maternal or herd immunity, strain

virulence, management practices or biosecurity, breed and environment (Thacker,

2006). The process of systemic dissemination remains unknown, but having affinity for

serosal surfaces and mammary tissue that may leads to acute inflammation of the serosa

of body cavities and synovial membrane. However, it is currently unidentified that how

these factors may contribute in the pathogenesis (Darzi et al., 1998).

In experimental study, it was observed that Mycoplasma adheres to the

polymorph nucleated cells and completely altered the phagocytic activity of these cells.

It has been documented that Mm cluster can invaded both the phagocytic and non-

phagocytic cell (Thomas et al., 1991). It has been reported that Mmc produce peroxide

free radicals in tracheal tissue of the experimental animal that is an important factor for

the pathogenesis of CCPP (Howard, 1984; Cherry and Taylor, 1970). The modulating

capabilities of the mycoides cluster suppress the immune response of the host that

ultimately leads to persistence of infection for longer duration and causes chronic

inflammation (Browning et al., 2007). The M. bovis has the ability to stimulate the

apoptosis process in the lymphocytes with help of some unknown factors and proteins

(Vanden-Bush and Rosenbusch, 2004). The other important characteristic is anti-

phagocytic capabilities make them able to survive in host immune attack (Thomas et

al., 1991). Mycoplasma has also the ability to cross the respiratory epithelium and make

enter into the tissue intracellular spaces which enables them to persist for longer period

18

of time. This strategy of the Mycoplasma makes them capable to evade the host

immune system and ultimately leads to chronic infections (Rodriguez et al., 1996;

Howard, 1984). Mm cluster can also enter into the cardiovascular system causes

hemorrhagic pericarditis and hydropericardium in small ruminant (Sadique et al.,

2012). Some pathogenic member of Mycoplasma cluster like Mmc has a capsular toxin

called as galactan which causes thrombosis in micro vessels lead to disseminated

intravascular coagulopathy and toxemia (Gutierrez et al., 1999).

After establishing the infections, the microorganism get multiplied and entered

into the blood stream lead to septicemia resulting in acute inflammation and developed

lesions in distant organs of the body with a poor prognosis (Rosendal, 1993; Bolske et

al., 1989). Several pathogenic species of Mm cluster like MmLC and Mmc and non-

cluster group like M. putrefaciens and M. agalactiae causes multiple systemic

complications with different degree of severity. The important disease syndrome

developed during Mycoplasma infection comprises of mastitis, keratoconjunctivitis,

arthritis, serosanguinous fluids in pericardial and peritoneal sacs, and sometime

meningitis in small ruminants (Abtin et al., 2013; Schumacher et al., 2011). In

abdominal cavity, the spleen becomes enlarged with necrotic foci. Hepatic

abnormalities are noted in the form of enlargement of liver, focal hemorrhages and

congestive necrosis (Mondal et al., 2004). The inflamed and congested intestinal

mucosa shows desquamation and sloughing of villi. There is also enlargement of

mediastinal lymph node (Sadique et al., 2012). In such systemic manifestation

pathological lesions are recorded in multiple organs that also can helpful for successfull

isolation of Mycoplasma from various tissue of the body (Laura et al., 2006; Mondal et

al., 2004; Darzi et al., 1998).

One of the most important member of mycoides cluster is Mccp that was

consider to be the principal cause of CCPP in small ruminants (OIE, 2014). This

member of the cluster has tissue tropism to the thoracic cavity only and its lesions are

confined to the respiratory tissue and actively invade the peumocytes II cells of the

alveoli (Johnson et al., 2000). The invasion of phagocytic cells by Mccp leads to

immunosuppression and provide an opportunity for its rapid multiplication and

damages to the alveoli and adjacent tissue. The degeneration of alveoli and surrounding

tissues lead to emphysema, atelectasis, micro vascular thrombi, thickened inter alveolar

19

septa and leukocytic infiltration (Gutierrez et al., 1999). As the disease progress the

gross pathological changes become evident in the form of unilateral or bilateral sero-

fibrinous pleuropneumonia with severe pleural effusion and hepatization (OIE, 2014;

Mondal et al., 2004). In some acute cases straw colored fluids are evident in the pleural

cavity with fibrin flocculations (Rurangirwa and McGuire 2012). In per acutecases,

minimal clinical signs are noted with high mortality within 1-3 days (OIE, 2014;

Samiullah, 2013). It is reported that Mccp when get attached to the acinar epithelium of

the host respiratory tissue it inflicted acute inflammatory response in the host

respiratory tissues (Darzi et al., 1998). In chronic form of infection the fibroblastic

growth factor are get activated with extensive fibrosis causing pleural adhesion resulted

to reduced lungs capacity that ultimately leads respiratory distress (Mondal et al.,

2004).

Free radical production is the key factor in the pathogenesis of many species of

Mycoplasma infections. One of the most common important free radical is the

hydrogen peroxide that produced during Mycoplasma infection causes tissue damages

and inflicted acute inflammation. The strain of Mccp has the ability to produced large

amount of hydrogen peroxide during the oxidation of NADH by lysed cells

(Houshaymi et al., 2002). This free radical altered the membrane channel of the host

cell that leads to cellular degeneration followed cell necrosis. In a study, it was reported

that during Mycoplasma cluster infection there is extensive loss of K+ channels in the

ciliated tracheal epithelium resulting ballooning degeneration and desquamation (Izutsu

et al., 1996). The free radicals like hydrogen peroxide and super oxide are the toxic

metabolites of Mycoplasma infection causes tissue damages and provoke the release of

inflammatory mediators comprises of tumor necrosis factor (TNF) alpha, interleukin-6

(IL) and nitric oxide. The release of theses mediators disturbed the hemodynamics, the

thermoregulatory system and causes systemic manifestation (Razin et al., 1998). In

another study it was reported that Mcc infection produces oxygen free radicals and

stimulate the mechanism of chemotaxis by draining the macrophages to the

inflammatory site that lead to production of very potent oxidant the per-oxynitrite

(Darzi, et al., 1998; Avron and Gallily, 1995).

These free radicals also target the lipid part of cell membrane and inflicted cell

membrane injury that ultimately resulted to cell lysis. The oxygen free radical also

20

causes damages by inhabiting the activity of catalase, by promoting the huge

production of hydrogen peroxide (Almagor et al., 1984). The other member of the

cluster the Mmc has the potential to produce hydroxyl (OH-) and superoxide radicals.

Some other species of Mycoplasma called the fermentative species including Mccp, M.

putrefaciens (Mp), Mycoplasma bovine serogroup 7 and many others are reported for

the production of free radical like hydrogen peroxide during the oxidation of glucose

and glycerol (Miles et al., 1991).

2.13 Clinical complications of mycoplasmosis

Clinical signs and symptoms exhibited by the diseased animal reflected the

tissues damages caused by invading microorganisms in a particular organs or system.

The severity of the signs and symptoms shown by the animals, are also presenting the

pathogenicity, virulencey of the pathogens, the extent of damages and losses of normal

physiology. Some other factors also contribute in disease progression including

maternal or herd immunity, strain virulence, biosecurity, management practices and

environment (Thacker, 2006). The signs and symptoms of a disease give an insight to

the clinician to decide the intervention procedure and prognosis. Clinical signs and

symptoms are providing the basic diagnostic approach about many diseases. A number

of Mycoplasma species are associated with livestock and causes different diseases by

involving different body system (Adler et al., 1980). Among the livestock, the small

ruminants are predisposed to many infectious agents particularly the Mycoplasma.

Some important species of Mycoplasma are responsible for small ruminant infections.

The most common Mycoplasma infectious disease is CCPP inflicted high morbidity

and mortality in the small ruminants throughout the world particularly in Africa and

Asia (Sadique et al., 2012; Regassa et al., 2010; OIE, 2008; Bergonier et al., 1997).

The virulent species of the Mm cluster is comprising of six different members,

which are mainly responsible for disease in small ruminant called CCPP (Laura et al.,

2006). Some other non-cluster pathogenic species like M. putrefaciens and M.

agalactiae also reported in mixed type of infections by involving different systems

(Banaras et al., 2016; Hira et al., 2015). A typical clinical signs produced by

Mycoplasma cluster are pyrexia (41-43 °C), high morbidity and mortality in infected

animals accompanied by dysponea, purulent nasal and ocular discharge, painful cough,

21

abducted fore limb, diarrhoea, anorexia and occasionally abortion (OIE, 2009;

Nicholas, 2002).

In some cases, upper respiratory tract is also asscociated with excessive

lacrimation, keratoconjunctivitis accompanied by corneal opacity (Mondal et al., 2004).

The infection also causes severe digestive problem, diarrhea and anorexia in small kids

(Laura et al., 2006). In Mmc and MmLC infections the disease is septicemic in nature

and the various sign reflect multiple organs involvement. Several pathogenic species of

Mm cluster like MmLC and Mmc and non-cluster group like M. putrefaciens and M.

agalactiae causes septicemia and multiple systemic complications. This multi-systemic

manifestation is called MAKePS (mastitis, arthritis, keratitis, pneumonia and

septicemia) syndromes (Egwua et al., 2001; Thiaucourt and Bolske, 1996). Some

Mycoplasma species also causes different systemic and inflammatory condition like,

cervical abscesses, hepatitis, peritonitis, spleenitis and in rare cases meningitis

(Schumacher et al., 2011; Madanat et al., 2001; Jubb et al., 1985). The incubation

period of CCPP normally take 3-15 days. The disease is fatal in per-acute cases; goat

may die within one to three days with minimal clinical signs (OIE, 2008). In chronic

cases the infection persisted for longer period of time from weeks to months. In such

case the animals become carrier for the rest of the life and transmit the disease to

healthy animals during favourable environment. The stress and hard climatic conditions

provides an opportunity for the recurrence and spreading of disease (Yousuf et al.,

2012; Regassa et al., 2010).

The non-cluster pathogenic species like M. agalactiae and M. bovis are

responsible for ruminant and bovine disease. Both the organism phenotypically and

genotypically closely related and share sizable number of related proteins and common

epitopes. They have mammary, articular and ocular tissue tropism with additional

possibilities of respiratory disease (Al- Momani et al., 2011; Flitman-Tene et al., 1997).

The M. agalactiae causes a typical disease called contagious agalactiae of sheep and

goats. Both sexes of sheep and goats are susceptible but female are more frequently

infected. Many authors reported that some other pathogenic species like M.

putrefaciens, Mcc, MmLC can also produce a typical “syndrome” with similar clinical

picture including mastitis leading to agalactiae (Bergonier, 1997). Interestingly, the

contagious agalactiae in different geographical areas also depends on the causative

22

agent. In the United States, Mmc is the most prevalent caprine Mycoplasma, although

M. agalactiae has been recently isolated. In Spanish dairy sheep farms M. agalactiae is

the most prevalent specie (Verbisck-Bucker et al., 2008). In various areas of the

northern Jordan, Mmc and M. agalactiae are the two main cause of contagious

agalactiae in small ruminant population (Al-Momani et al., 2011). The disease is

generally mild, acute or chronic in nature having incubation period of 1 to 5 weeks in

sheep and goats. The main signs are generalized sickness, fever, anorectics, udder

painful to touch, mastitis, sudden decrease in milk production, altering milk quality and

agalactiae (Fox et al., 2005). In some infected animal, severe keratoconjunctivitis may

also be developed. Several infections may also lead to pneumonia and occasionally

abortion. In chronic cases the organism settled in the joints and leads to polyarthritis

(DaMassa et al., 1992; Azevedo et al., 2006; Nicolas, 2008). Loria et al. (2007)

reported contagious agalactiae in sheep caused by M. agalactiae that has been isolated

from the brain of sheep with lesion of non-purulent encephalitis.

However, it has been documented that the classical form of CCPP is caused by

Mccp that mainly restricted to the thoracic cavity (OIE, 2014). The disease chiefly

infected the small ruminant particularly the goat and characterized by high fever (41-

43°C), acute fibrinous pneumonia, high morbidity followed by mortality in susceptible

herds. After 2-4 days, post pyrexia the other signs are developed including accelerated

and painful respiration accompanied by grunt, productive and violent coughing. In the

terminal stage of disease, the animals is unable to move, neck become stiff, abducted

leg, continuous salivation from the mouth and lie down on lateral recumbancy followed

by death (OIE, 2014; Gelagay et al., 2007). The M. bovis is responsible for the disease

in bovine called contagious bovine pleuropneumonia. It also causes mastitis in dairy

animals in many parts of the world with significant economic losses (Sulyok et al.,

2014; Francoz et al., 2005). This pathogenic specie was isolated for the first time in

United States in 1961 from the milk of cow having mastitis. It is normal inhabitant of

the upper and lower respiratory tract of healthy animals and cause a disease in

immunocompromised animals under favourable environmental conditions. M. bovis is

the second most pathogenic Mycoplasma throughout the world and inflicted significant

economic losses to the dairy industry. The signs and symptoms of the disease is not

specific which depend on many factors including age, sex, breed, immune status of

animal and other environmental stresses (Sherif et al., 2012; Regassa et al., 2010). It

23

was reported that in 5 days old calf the signs were fever, depression, loss of appetite,

dysponea, cough, hyperventilation and nasal discharge (Stipkovits et al., 2001). In adult

animals, it causes mastitis, bronchopneumonia, arthritis accompanied by varying degree

of morbidity and mortalities (Khan et al., 2013; Maunsell et al., 2011; Gerchman et al.

2009; Gagea et al., 2006; Fox et al., 2005). In bovine, it can be isolated from different

clinical specimen like milk, synovial fluid. As bovine milk is often used to feed young

goat kids for supplementation, this practice providing an opportunity for the pathogen

to seed in mouth, oropharynx, lower trachea and lungs and this practice leads to

infection (DaMassa et al., 1992).

2.14 Pathological Changes

Pathological lesions play vital role in the diagnosis of a disease and directed the

clinicians for intervention and help them in selection of proper therapy for early

recovery. It will also decide the fate and future consequences of disease. Some

pathognomonic lesions are tissue specific and helpful for the diagnosis of a particular

disease and paved away to design strategies by the researchers and physicians for

accurate treatment and eradication of a disease (Sadique et al., 2012). The different

pathogenic species of Mycoplasma has the ability to produced lesions in different

tissues, organs and system of the body provided key for its diagnosis and therapeutic

intervention (Riaz et al., 2012; Laura et al., 2006; Mondal et al., 2004; Leach et al.,

1993).

2.15 Gross Pathology

The severity of lesions in Mycoplasma infection depends on many factors like

age, breed, sex, immune status of animal, environmental factors and the pathogenicity

and virulencey of Mycoplasma species (Yousuf et al., 2012; Mekuria and Asmare,

2010). The Mycoplasma cluster causes CCPP in small ruminant with different lesions

in the form of inflamed and consolidated lungs having marble appearance, fibrinous

pleuropneumonia and accumulation of straw color fluid in pleural cavity (Sadique et

al., 2012; Thiaucourt et al., 1996). The unilateral or bilateral pneumonia are the

common feature of CCPP with frequent involvement of enlarged mediastinal lymph

node. In many infections different pathogenic species like MmLC, Mmc, M.

putrefaciens and M. agalactiae are responsible for mixed type of infection along with

24

septicemia. In such septicemic case, kidneys become congested with necrotic foci

having pus in the pelvis. The liver get enlarged pale in color with multi focal

hemorrhagic and necrotic areas. The mucosal surface of intestine becomes thick and

hemorrhagic with enlarged mesenteric lymph node. In some cases, there is involvement

of heart by presenting hemorrhagic pericarditis and accumulation of sero-fibrinous fluid

in the pericardial sac (Sadique et al., 2012; Nicholas et al., 2008; Laura et al., 2006;

Gutierrez et al., 1999). In another study lesions were noted on lungs surface as

yellowish foci and fibrin deposition. The pleura were observed thickened along with

fibrin deposition and adhesions to the chest wall. In some animals lesions may

restricted to one lung and the entire lob become solidified (Kabir and Bari, 2015).

In experimental study goat kids were infected with Mmc and the lesions were

observed in all visceral organs including splenomegaly and hemorrhages on the

capsular surface of spleen (Gutierrez et al., 1999). In another study, it was revealed that

the Mycoplasma cluster developed lesions in different visceral organs of varying degree

in trachea, lungs, liver, kidneys, spleen and intestine (Riaz et al., 2012; Sadique et al.,

2012; Gelagay et al., 2007; Mondal et al.,2004). Kabir and Bari, (2015) recorded

lesions in Black Bangal goats in Bangladesh. In most of the necropsied goats trachea

showed hemorrhages and catarrhal exudation. Several small yellowish foci and fibrin

layer on the surface of the lungs. The pleura becomes thickened, fibrin deposit and

there were adhesions to the chest wall. Some lungs showing yellowish pea sized

nodules accompanied by marked congestion around the nodules. In most animals, the

lesions seen unilaterally and affected the entire lobe. In some animals both lungs

showed multiple area of hepatization (Kabir and Bari, 2015).

The other most common specie of Mycoplasma is M. agalactiae causing

mastitis in sheep and goats (Al-Momani et al., 2011). The route of entry is common

wounds of the mammary tissue or through the descending routes by contamination of

the teat canal with infected soil and mud (Fox et al., 2005). The M. agalactiae infection

is mostly restricted to the udder and causing acute inflammation of the mammary tissue

that become hard and swollen. Milk shows yellowish or bluish colored fluids with salty

test. In later stage of infection the milk production is reduced and contained purulent

exudates and followed by cessation of milk (Egwua et al., 2001). The udder becomes

hard in consistency due to extensive fibrosis that ultimately leads to loss of infected

25

quarter. This specie has also a tissue tropism for other organs including eyes and joint

tissue. The M. agalactiae alone or in combination with other Mm cluster produced a

syndrome called “MAKePS” and the lesions are produced in various organs. When

pathogen localize in the ocular tissue, congestion seen in the conjunctiva followed by

keratitis, keratoconjunctivitis and vascularisation of the cornea which leads to loss of

eye vision (Kwantes and Harby, 1995). In some cases, the organism is localized in the

joints causing arthritis that commonly seen in knee and hock joints. Joints become

swollen and synovial fluids are infiltrated with multinucleated cell followed by fibrosis

and ankylosis (De la Fe et al., 2009; Kwantes and Harby, 1995; Real et al., 1994).

The lesion of Mccp is mainly confined to the thoracic cavity either unilateral or

bilateral sero-fibrinous pleuropneumonia with severe pleural effusion and hepatization

(OIE, 2014; Mondal et al., 2004). In early progression of the disease pea-sized grey

yellowish nodules are seen in the lungs followed by marked congestion. The lesions

mainly located unilateral and affect the entire lobe. Bronchial and mediastinal lymph

nodes are swollen and edematous. Plural adhesion with wall of chest cavity and whitish

pleura is common feature (Sadique et al., 2012). In some cases pericardial sac is filled

with serosanguinous fluid, pleural cavity contains excess straw colored fluids with

fibrin flocculation. Extensive pleuritis is a common feature and observed in most of the

outbreak with various stage of hepatization and marked dilatation of interlobular septa

(Rurangirwa and McGuire, 2012; OIE, 2008).

2.16 Histopathology

The development of microscopic pathological changes in different visceral

tissues depends on the specie of Mycoplasma that causes the CCPP infection. Some

infections are acute showing early vascular and cellular changes and cell hypertrophies

in tissue while other chronic in nature and produced lesions in the form of fibrosis,

granuloma and metaplasia. The pathological changes depend upon specie of

Mycoplasma, tissue involvement, age and sex of animal, health status and immune

response towards the foreign pathogen (Regassa et al., 2010). The environmental

factors like stress, hot or cold climatic condition, rainy season and husbandry practices

also contribute in the occurrence of moderate to severe lesions (Yousuf et al., 2012;

Knowles et al., 1995).

26

In CCPP, general microscopic lesions in lungs are characterized by

fibrinopurulent pleuropneumonia with thickening of interlobular septa (Laura et al.,

2006; DaMassa et al., 1992; Jones, 1989). Hemorrhages are present in tracheal section

with sloughing of lining of epithelium and leukocytic infiltration. In some infections

trachea showed erosion of the superficial layer and hemorrhages in sub mucosa and

muscular layer. Intestinal mucosa showed, hemorrhages, sloughing of villi with

degenerative changes along with infiltrateration of mononuclear cells including

lymphocytes, plasma cells and macrophages (Laura et al., 2006; Mondal et al., 2004).

In lungs emphysema, atelectasis is frequently present accompanied by hemorrhages,

necrosis and the most characteristics feature is micro thrombosis in the lumina of small

vessels (Nicholas et al., 2008). There is sloughing of alveoli and deposition of fibrin in

alveolar spaces is commonly observed. The adjacent alveoli due to eruption combine to

each other to form bullae (Sadique et al., 2012). Thrombosis of blood capillaries,

congestion and hemorrhages along with perivascular cuffing of leukocytes were also

observed in some tissue. The affected lung lobe shows prominent interlobular edema,

peribronchial and perbronchiolar lymphoid hyperplasia with mononuclear cell

infiltration (Gutierrez et al., 1999). In chronic cases focal abscesses surrounded by a

fibrous core infiltrated with chronic inflammatory cells.

Hemorrhages and leukocytic infiltration in liver, kidneys and spleen are

frequently seen. Mmc also causes acute multifocal purulent splenitis showing

microabscsses in splenic parenchyma (Sadique et al., 2012; Laura et al., 2006;

Gutierrez et al., 1999). The urinary tubules showed distension and cast were observed

in section of the kidneys. The tubular epithelial cells were showing degenerative

changes followed by necrosis. The mediastinal lymph nodes showed hyperplasia,

congestion, necrosis and numerous leukocytic infiltrations. Liver showed congestion,

swelling of hepatocytes and necrosis around the central veins. The necrotic focal area

and polymorph nucleated cells are scattered (Gelagay et al., 2007; Wesonga et al.,

2004).

2.17 Diagnosis of Mycoplasma

In field or in an outbreak, the history, clinical finding and postmortem lesions

are helpful for initial diagnosis of disease. Some lesions like fibrinous

pleuropneumonia, marked hepatization and pleural adhesion are helpful in field

27

diagnosis of CCPP. The desirable tissue and swab may also be taken for further

biochemical and molecular identification. The diagnosis of CCPP in natural outbreaks

is some time also become very difficult due to mixed infections caused by other

pathogens which produced same clinical picture like Mycoplasma infection. The

respiratory tract infections in small ruminant like pasteurellosis and pest des petits

ruminants (PPR) exhibit similar clinico-pathological symptoms like mycoplasmosis. So

the best way is the isolation followed by molecular identification of the causative agent.

A lot of work has been carried out for the diagnosis and identification of

Mycoplasma species by using different conventional and non-conventional techniques

with various degree of success. The conventional methods of identification are usually

failed to address the issue properly because of its shortcoming. In the present era,

different molecular techniques are frequently used for the confirmation and

identification of different pathogens. A definitive diagnosis can be made by detecting

Mycoplasma species from different clinical samples like nasal and tracheal discharge,

milk, conjunctival and ear swab, synovial fluids, lungs tissue, pleural and regional

lymph fluids (Amores et al., 2010). In several ceases, pathogenic species like Mccp is

directly detected from lung tissues, pleural fluid or regional lymph nodes. Samples may

also be taken from active involved tissue due to heavy pathogens load (Lorenzon et al.,

2008).

2.18 Isolation of Mycoplasma

Mycoplasma is one of the most fastidious pathogen which needs special care

and requirements for growth. It is not easy to grow Mycoplasma on routine and

ordinary laboratory media used for other bacteria but it need special media for

successful growth and isolation. Different media are used for the isolation of

Mycoplasma like pleuro pneumonia like organism (PPLO) broth media, modified

Hayflick media, B4 media, Friis medium, SP4 medium, modified Newing tryptic agar

broth medium (Kibor and Waiyaki, 1984). These media used for the Mycoplasma

isolation containing some special ingredients like glucose, sodium pyruvate, serum

(horse or swine) rich protein base (heart infusion), yeast extract. Bacterial and fungal

contamination is the common problem during isolation. Therefore antifungal agents

like thallium acetate or fluconazole and antibiotics like penicillin, amphotericin B is

added in the media to encounter the growth of unwanted pathogens (Thiaucourt and

28

Bolske, 1996; Thiaucourt et al., 1992). The modified Hayflick medium was

considerably used by many scientists/researchers for the isolation and identification of

different species of Mycoplasma (OIE, 2008; Gelagay et al., 2007; Mondal et al., 2004;

Wesonga et al., 2004; Woubit et al., 2004; Rodriguez et al., 1996).

2.19 Culture and Cultivation

2.19.1 Special media requirements for Mycoplasma growth

After inoculation the culture are incubated at 37 °C, providing 5% carbon

dioxide and humid atmosphere. Broth must be examined daily for evidence of growth,

which are changes in the color of media, appearance of turbidity and floccular material.

Plate culture should be examined after 3-5 days under sterio microscope for the

appearance of typical fried egg shape or nipple like colonies (Mondal et al., 2004;

Wesonga et al., 2004). Cloning and purification is performed by repeated transfer of

single colony representing each morphological type. In early passage of Mycoplasma

cultivation the colonies produced are bizarre type often small, center less and irregular

shape but with the passage the isolate demonstrate typical fried egg shape colony (OIE,

2008). This procedure for inoculation and obtaining the pure culture of Mycoplasma

were adopted by many researchers (Gelagay et al., 2007; Laura et al., 2006; Mondal et

al., 2004; Wesonga et al., 2004).

2.19.2 Identification of Mycoplasma

Various conventional and molecular tests are used for identification and

confirmation of different species of Mycoplasma. But these tests have some limitations

due to the fact that some time it gives false positive and negative results due to cross

reactivity among different species and with other bacterial contamination. Some of the

common tests used for Mycoplasma identification are enlisted below.

2.19.3 Biochemical tests

Different biochemical tests are widely used based on nutritional and specific

enzymatic activities for the initial identification of pathogenic Mycoplasma species in

clinical and experimental cases (Noah et al., 2011). Some tests distinguish Mycoplasma

from the other genera, while some used for the identification of cluster or other

29

pathogenic species of the genus Mycoplasma. These tests are not absolutely

confirmatory but routinely used for the identification of several Mycoplasma species. It

is early useful tool both for preliminary screening as well as providing supportive data

for serological and molecular PCR based analysis (OIE, 2008; Eshetu et al., 2007;

Gelagay et al., 2007; Woubit et al., 2004; Mondal et al., 2004). The most commonly

conducted tests are serum digestion, glucose fermentation, phosphates activity,

digitonin sensitivity, arginine hydrolysis, tetrazolium chloride reduction, and film and

spot formation (Nicholas et al., 2009).

Mycoplasmas are distinguished from Acholeplasma by Digitonin sensitivity and

serum digestion differentiated members of the Mm cluster from all other ruminant

Mycoplasmas (FAO, 2012). Phosphates production separates Mcc from other members

of the Mycoides cluster, while metabolic differences (such as maltose positive reaction

for Mccp) allow differentiation between Mcc and Mccp (Bradbury, 1983). Mccp is also

positive for glucose fermentation, phosphates activities reduction of tetrazolium

chloride while negative for arginine hydrolysis (Nicholas et al., 2008; Gelagay et al.,

2007; Adehan et al., 2006).

The interspecies variation in some biochemical reactions is often notable,

rendering their application valueless (Rice et al., 2000; Jones, 1989). The lacking of

arginine catabolism by Mccp may help to differentiate it from Mcc (Noah et al., 2011),

but in some strains of Mcc arginine catabolism is reported to be lacking or very difficult

to detect (Rurangirwa, 1996; Leach et al., 1993; Jones, 1992). The MmLC, Mccp and

Mmc are reacting positively to casein hydrolysis, glucose fermentation, serum

digestion. Similarly, M. agalactia reacts positively to phosphatase activity, digitonin

sensitivity and formation of spot and film. The M. putrefaciens are positive for glucose

fermentation, serum digestion and tetrazolium reduction test (Nicholas et al., 2009).

The sensitivity of important pathogenic Mycoplasma is summarized in Table 2.1.

30

Table 2.1 Sensitivity of pathogenic Mycoplasma species of small ruminant to

various biochemical assay.

Mycoplasma

specie

Glucose

fermentation

Arginine

hydrolysis

Film

and

spot

Casein

digestion

Phosphatase

activity

Tetrazolium

reduction

aerobic

Tetrazolium

reduction

aerobic

Mm LC + - - + - + +

Mmc + - - + - + +

Mccp + - - + - Varies varies

Mcc + + - + + + +

M. agalactiae - - + - + + +

M. arginini - + - - - - +

M.

Conjunctivae + - - - - - +

M.

ovipneumoniae + - - - - varies +

M. putrefaciens + - + - + varies +

MmLC=Mycoplasma mycoides sub sp. Large colony Source (Nicholas et al., 2009)

Mmc=Mycoplasma mycoides subsp. capri

Mcc=Mycoplasma capricolum subsp. capricolum

Mccp=Mycoplasma capricolum subsp. capripneumoniae

2.19.4 Serological Tests

Serological tests are nowadays not widely used for the identification of

causative agent of mycoplasmosis as a routine laboratory diagnosis. However, in

endemic outbreak of CCPP with mycoides cluster can produce a background of positive

titers to this organism in a significant proportion among healthy animals (Jones and

Wood, 1988). Complement fixation test (CFT) and indirect haemagglutination (IHA)

are widely used to evaluate the antibodies titer of Mccp in goats (DaMassa et al., 1992).

The CFT is more specific that is used for the detection of CCPP as compared to IHA

(Thiaucourt and Bolske, 1996; MacOwan and Minnette, 1976). Similarly many species

of Mm cluster and non-cluster species shared antigenic structure thus showing false

positive results on serological and biochemical assay that render the proper diagnosis of

exact specie (Thiaucourt et al., 1994).

2.19.4.1 Growth Inhibition Test

Growth inhibition (GI) test are used for the possible identification of mycoides

cluster (Dighero et al., 1970). Hyper immune serum is raised in animals and used in

different technique such as metabolic inhibition, indirect immunofluorescent test (IFT)

and growth inhibition (Lauerman, 1994). The GI works by stopping the growth of

31

microbes on agar media using a disc having specific antibody that detect the antigen

(Dighero et al., 1970). Recently Mcc and Mp were isolated from nasal and lungs tissue

and identified by GI and biochemical test in Balochistan, Pakistan (Awan et al., 2009).

The GI and direct immunofluorescence (IF) tests were used for the identification of

local isolates of Mmc (Singh et al., 2004). The test was commonly used for the

preliminary identification of several pathogenic Mycoplasma species by several

researchers (OIE, 2008; Wesonga et al., 2004; Poveda and Nicholas, 1998; Rodriguez

et al., 1996; Thiaucourt and Bolske, 1996).

2.19.4.2 Latex agglutination test

This test detects antibodies in serum of CCPP infected animals, it is sensitive

than CFT and can be easily used in field conditions requiring blood as well as

serum/plasma with a quick result (Cho et al., 1976). The latex agglutination test has

been successfully used as pen side test by many diagnostic laboratories of the world

(OIE, 2008). In this test latex beads are coated with polyclonal IgG raised in

experimental animal against the polysaccharide antigen of Mycoplasma. The test is also

used to detect antigen in sheep and goat serum. In the past this test was usually used in

Kenya for the investigation of CCPP outbreak. It can also be performed easily at the

pen side by using a single drop of blood (Rurangirwa et al., 1987a).

2.19.4.3 Enzyme linked immunosorbent assay (ELISA)

The ELISA tests are also useful tool for the diagnosis of CCPP in several

outbreaks. Several researchers used this technique for initial screening of animals to

check status of herd health (Sachse et al., 1996). Similarly, a competitive ELISA with

modification was developed which is specific and sensitive for the diagnosis of CCPP

(Thiaucourt et al., 1994). Several ELISA including commercially available kits have

been described for the serological identification. A study was conducted using c-ELISA

that indicates 35.29% seroprevalence in goat population (Wesonga et al., 2004). ELISA

test is also extensively used as a diagnostic tool for screening of CCPP and CBPP

(Nicolet and Martel, 2007). In other investigation the seroprevalence survey indicated

33.67% CCPP in district Nagpur, India (Ingle et al., 2008). An international

collaborative study was conducted through monoclonal antibody based c-ELISA

technique Kit (IDEXX-Montpellier SAS & CIRAD). The seroprevalence of CCPP

32

caused by Mccp was 14.5% in Afar region of Ethiopia, 2.7% and 44.2% in Gilgit and

Diamer Districts of Norther Pakistan, 6-90% in Kenya and 10.1% in the Shuro-Obod

District of Tajikistan (Peyraud et al., 2014). Using the same c-Elisa kit the

seroprevalence of Mccp was 8.52% in different District of Punjab, Pakistan (Shahzad et

al., 2016).

2.19.4.4 PCR for identification of Mycoplasma

The relative difficulty in isolation by culture and confirmation by biochemical

tests is long and time consuming process with false positive results. The

scientists/researchers had been made various attempts to introduce more reliable and

sensitive technique to identify the actual causative agent (Hotzel et al., 1996). Some

researchers developed oligonucleotide probes for targeted 16-sRNA gene (Mattsson et

al., 1994). Latter on several scientists developed a PCR based analysis for

differentiating between M. mycoides subsp. SC and M. mycoides subsp. LC by the

cleavage of amplified DNA template with the help of restriction enzyme (Bashiruddin

et al., 1994; Taylor et al., 1992). Bascunana et al. (1994) developed a specific set of

primer of 16s-rRNA using amplified template DNA for detection of Mccp.

All other diagnostic assay was replaced by PCR for confirmation, identification

and characterization of CCPP because of its high sensitivity and accuracy. In the recent

era different set of primers being developed for the specie specific CCPP diagnosis

(Woubit et al., 2004). Use of PCR for the confirmation of Mm cluster member and

Mccp is very useful for the exact species identification. By using the specie specific

primer the amplicon size of 316 bp was obtained for Mccp (Woubit et al., 2004). In

another study Laura et al. (2006) implemented PCR scheme of Hotzel et al. (1996) with

slight modification by applying two sets of primers. The first set of primers was cluster

specific and second specie specific that successfully identified the species of Mm

cluster. PCR is also used for the rapid and specific detection of M. agalactiae directly

from the ear swab (Amores et al., 2010). Some scientists made modification in PCR

technique like multiplex PCR for the diagnosis of contagious agalactiae of sheep and

goat from ear swab (Greco et al., 2001). Similarly the milk samples were also screen by

PCR for the detection of M. agalactia (Lorusso et al., 2007). The advancement in

molecular detection was the introduction of real time PCR for the confirmation and

quantification of pathogenic Mycoplasma species by using syber green probe which

33

detect target specie both in clinical samples and culture (Lorenzon et al., 2008). In the

present era, several researchers have been used PCR for successful confirmation of

several pathogenic Mycoplasma species like Mycoides cluster, Mmc, Mccp, Mcc, M.

ovipneumoniae, M. putrefaciens and M. bovis (Banaras et al., 2016; Hira et al., 2015;

Sadique et al., 2012; Ongor et al., 2011; Peyraud et al., 2003).

2.19.4.5 DNA Sequencing

DNA sequencing is an important tool for the confirmation and characterization

of any organisim. Once the unique clones is identified, their nucleotide sequence would

be determine and examine for interspecies heterogenecity. Then the phylogenetic tree

could be constructed by using all available sequence of specific pathogen in NCBI gene

data bank (Daniel et al., 2011). The sequence information is useful to establish

relationship between different Mycoplasma species. The gene sequencing also

facilitated the researchers about variation and evolution in the Mycoplasma genetic

makeup. Manipulation of fusA gene is a rapid tool for identification and phylogenetic

positioning by PCR and sequencing (Manso-Silvan et al., 2007; Pettersson et al.,

1994).

2.19.4.6 Phylogenetic analysis and DNA homology

Phylogeny is the classification for characterization of the organism that based

on sequencing of 16S-rRNA gene that provides the evolutionary history of the

organisms. Sequencing is accurate tool to identify the pathogen and confirm his

relationship near or far within the genus and specie or sub-specie. The phylogenetic tree

was helpful and make possible to characterize the same specie of pathogen and their

genetic variation and mutational changes for comparison with starins of different

countries (Pettersson et al., 1996). Some researcher has also reported the phylogenetic

relationship on the basis of beta subunit of F1 F0-type ATPase 23S-rDNA molecule

and elongation factor (EF-Tu, EF-G) (Razin, 2000).

The phylogenetic analysis of Mycoplasma through 16S-rRNA gene revealed

that they are derived from the Gram positive bacteria by the process of degenerative

evolutionary changes accompanied by loss of several biosynthetic capabilities

(Weisburg et al., 1989). DNA homology study of the different isolates were carried out

34

that revealed 80% genetic similarities in between Mcc and strain F-38 biotype while

40% for Mycoplasma mycoides species (Thiaucourt et al., 2000). Similarly, the DNA

hybridization study revealed homology of 70-90% in between Mcc and Mccp and for

the different strains within each group of Mccp and Mcc organisms (Christiansen and

Erno, 1982).

2.20 Chemotherapy

Bacterial pneumonia is a common and often life-threatening respiratory

problem in small ruminants. Among the bacterial infections the mycoplasmosis is a

major cause of respiratory pneumonia inflicting high mortality and reduce animal

production. The accurate and early diagnosis of the disease is vital for devising

strategies to use proper chemotherapeutic agents to efficiently encounter the infection

and reduce the economic losses (Gautier-Bouchardon et al., 2002). The class of

Mycoplasma is consisted a variety of species and sub-species having different response

to variuos chemotherapeutic agents. The effective treatment of mycoplasmosis is

depending upon timely response and selection of accurate antimicrobial agent with

proper dose and duration. By lacking proper diagnosis of the exact specie of

Mycoplasma usually leads to therapeutic failure (Nicholas and Ayling, 2003). For

effective treatment and eradication of mycoplasmosis, the detail understanding of the

mechanism of antibiotics distribution in the host and its mode of action against the

pathogen is prerequisite.

Mycoplasma having the ability to change its surface protein by the mechanism

of genetic modulation that make difficulties in disease diagnosis and treatment, thus

create difficulty in management and controlling of this lethal disease (Behrens et

al.,1994). The duration of disease varies according to environmental conditions, health

and immune status of the animal (OIE, 2009). The infected animal may survive as long

for month or even recover by providing good management and treatment (Thiaucourt et

al., 2008). The formation of biofilm is also important characteristic of the pathogenic

Mycoplasma in which the bacteria attached to a substratum, or each other, mostly

bounded by an extracellular polysaccharide material (Donlan and Costerton, 2002).

The formation of biofilm masks the Mycoplasma that minimizes the efficacy of therapy

and gets resistance against chemotherapeutic agents. Therefore, many antibiotics not

mitigated the infection properly and persisted for long time. It is often necessary in the

35

control of Mycoplasmal infections to complement barrier measures by the use

antimicrobial therapy. This will reduce economic losses and lateral and vertical

transmission (Gautier-Bouchardon et al., 2002).

2.20.1 Antimicrobial Agents

Antimicrobial are an agents or compounds that inhibit or eliminate the growth

of microorganisms, such as bacteria, fungi and protozoans. They are widely used in

humans, plants and animals to treat different ailments. It prevents infections from

growth and distribution in the host body. It also provides a favorable environment for

the host to use its potential up to the optimum level to survive and increase its

productivity (Hirsh, 2000).

2.20.2 Antimicrobial agents used for the treatment of caprine mycoplasmosis

The Mycoplasma is wallless bacteria and having ability to invade both

phagocytic and non-phagocytic cell of the host. The survival ability and its growth in

the host cell are different from other bacteria. The treatment strategies for effectively

elimination and control of Mycoplasma infection are quite complicated from the host

body because of proper selection of antimicrobial agents. Lack of cell wall in

Mycoplasma narrow the range of chemotherapy in human and animals infections. The

formation of biofilm by several pathogenic species also makes difficulties in the

treatment. Due to its unique characteristic the common antimicrobial agents are usually

fail to treat the Mycoplasmas infection in animals. However, some agents that act on

protein and nucleic acid synthesis are commonly used for the treatment of ruminant

mycoplasmosis globally with varying degree of success (Clothier et al., 2012). The

Mycoplasma has the capability of structural modulation that evades itself from the host

immune mechanism and access of therapeutic agents thus leads to survive in the host

for longer period of time. Being peculiar morphology, the Mycoplasma species are not

affected by agents that interfere with the synthesis of folic acid or that targets the cell

wall such as the ß-lactams and fosfomycin (Puglisi et al., 2000). This characteristic

narrows the range of available antimicrobials to treat the Mycoplasma infections. The

effective antimicrobials against Mycoplasma are tetracyclines, macrolides (tylosin,

erythromycin, tiamulin and clindamycin), aminoglycosides, chloramphenicol and

fluoroquinolones (Bebear et al., 1998). The antibiotics having fewer efficacies against

36

Mycoplasma in-vitro are likely to perform similarly in-vivo. However it is not essential

that the agents possessing strong activities in-vitro will show the same performance in

the natural infection of field condition (Ayling et al., 2000).

2.20.3 Classification of antimicrobial agents

Broadly, antimicrobial agents can be classified as bacteriostatic and

bacteriocidal depending on its mechanism of action. Bacteriostatic such as

sulphonamides and tetracycline inhibit the growth of organisms and mainly depend

upon the host immune system to kill and remove the bacteria. Bacteriocidal drugs such

as penicillin and streptomycin have a rapid lethal action and kill bacteria directly. In

routine practice, various antimicrobial agents are used having bacteriocidal and

bacteriostatic efficacy of varying degree (Yao and Mollering, 2007). Bacteria need

different nutritional requirements and fulfill it from the host cell. In the environment

where these nutritional requirements are lacking, survival for bacteria becomes

difficult. Most of the bacteria are well known for their nutritional requirements and

scientist can easily plan to arrest bacteria multiplication in-vivo. These strategies are

successful for the treatment of many bacterial infection including Mycoplasma in

human as well as animals.

The other important classification of these agents based on their modes of action

that target the specific pathway of microbial growth. This different pathway includes

interference with cell wall synthesis, i.e penicillin, vancomycin, cephalosporin,

fosfomycin and Beta-lactamase inhibitors. The ploymyxin act to inhibit the synthesis of

cytoplasmic membrane. The Fluoroquinolones group act on nucleic acid to inhibit the

synthesis of DNA and RNA. Similarly the other groups that inhibit protein synthesis

like tetracycline, aminoglycosides and linezolid, the mycolic acid synthesis inhibitors

like Isoniazid and those cause inhibition of the metabolic pathway for folic acid

synthesis e.g. sulphonamides and trimethoprim (Yao and Mollering, 2007).

2.20.3.1 Aminoglycosides

Aminoglycosides are the class of antibiotics with bactericidal properties against

most of the bacterial pathogens. This class of antibiotics has the potential to penetrate

bacterial cell than bind with 30-S ribosomal subunit that alters the protein synthesis

37

(Patricia et al., 2007). The antibiotics include kanamycin, streptomycin, gentamicin,

dihydrostrep-tomycin, neomycin, tobramycin and amikacin. Some Mycoplasma like M.

pneumoniae is susceptible to streptomycin (Taylor-Robinson and Bebear, 1997).

2.20.3.2 Fluoroquinolones

Presently there are several brands of fluoroquinolones that are frequently used

in veterinary medicine. These preparations included enrofloxacin (sheep, goats, cattle,

dogs, cats and poultry), danofloxacin (cattle), sarafloxacin (poultry), difloxacin,

marbofloxacin (dogs) and orbifloxacin (dogs and cats) (Walker, 2000). The mechanism

of action of this group of antibiotics targeted the bacterial cell by the inhibition of the

enzymes DNA gyrase or topoisomerase IV that alter the supercoiling of bacterial

chromosomal material and deprived it for essential protein synthesis. These agents are

fast in action as bactericidal and ensure quick clinical recovery against several

Mycoplasmas infections in different species of animals (Reinhardt et al., 2002;

Sanchez-Pescador et al., 1988).

2.20.3.3 Macrolides

These agents are bacteriostatic in nature and alter RNA function by inhibiting

protein synthesis. They bind to the 23-S RNA in the 50-S ribosome subunit of the

targeted pathogen and block the translocation reaction of polypeptide chain elongation.

Normally these agents act as bacteriostatic but in some tissue like lungs also having

bactericidal effects at high concentration. Tylosin is consider a drug of choice in

respiratory infections and widely used for many Mycoplasma infections of animal

origin. The erythromycin and its derivatives are gaining importance in treating the

respiratory tract diseases in human but it has limited uses in veterinary practices and

less effective in animal Mycoplasma infections (Yao and Mollering, 2007).

2.20.3.4 Tetracycline

Tetracycline is one of the old antibiotics used for the treatment of many

bacterial infections including mycoplasmosis. The commonly used agents of this group

are oxytetracycline, doxycycline and chlortetracycline for the treatment of animal and

human diseases. These agents target the 30-S ribosomal subunits, preventing the

38

attachment of aminoacyl-t RNA to the ribosomal acceptor of A-site in the RNA-

ribosome complex and inhibit the protein synthesis (Chopra and Roberts, 2001).

Tetracycline is to be considering the most effective drug that encounters many bacterial

infections with less toxic effects. However its prolong use in the calf and children

impart tooth discoloration. Doxycycline, due to its lipophilic nature having better

penetration capabilities in the cell, is considered to be more effective than

oxytetracycline (Ayling et al., 2000).

A number of antimicrobial agents have been used for the therapy of caprine

mycoplasmosis. The streptomycin treated goat recovered on third day of treatment

suffering from natural and experimental CCPP infection (Rurangirwa and McGuire,

2012). In other study it was reported that morbidity and mortality of CCPP among the

herd was stopped by treating with long acting oxytetracycline (Giadinis et al., 2008). It

was also reported that danofloxacin was more effective agent for the treatment of CCPP

infected goats (Ozdemir et al., 2006). In an In-vitro study, the enrofloxacin against M.

agalactiae was found the most effective antibacterial agent with MIC 0.125 to

0.500µg/mL and MIC50 of 0.203 µg/mL followed by tylosin with MIC50 0.292 µg/mL

(Loria et al., 2003). It has been reported that a single injection of long acting

tetracycline along with local application was effective in keratoconjunctivitis caused by

M. conjunctivae. Similarly, floronphinicol and spiramycin were effective in-vitro

against MmLC (Kidanemariam et al., 2005). In comparative antimicrobial therapeutic

study against M. agalactiae the enrofloxacin was found most potent followed by

lincomycin, tylosin and tetracycline among the tested agents (Loria et al., 2003). It is

also revealed in an experiment that enrofloxacin and its metabolite ciprofloxacin has

effective against some species of mycoides cluster like MmLC and Mcc (Antunes et al.,

2007).

2.20.4 Resistance of Mycoplasma to Antimicrobial Agents

Microbial resistance against commonly used antibiotics is one of the emergent

issues of the 21st century which get a serious health concern throughout the world. At

the dawn of discovery of these antimicrobial agents most pathogenic organisms were

highly susceptible; however the efficacy of broad-spectrum antibiotic has been

decreased due to its indiscriminate use and acquisition of genetic mutation in the

susceptible microorganism (Gautier-Bouchardonet al., 2002; Bradbury et al., 1994). It

39

is revealed by the World Health Organization (WHO) that more than 50% of the

antimicrobials produced in the world are used in animal sector. These agents are widely

used as growth promoters, treatment of infections and prophylaxis as well (Mathew et

al., 2007). This recent trend of extensive use of antibiotics exposes the microbes for

genetic adaptations, modulation, continuous mutation that leads in the form of multi-

drug-resistant strains. Some of the multi-drugs resistant infections including, ESBL

(Extended spectrum beta-lactamase), VISA (vancomycin-intermediate S. aureus),

MRSA (methicillin resistant Staph. aureus), VRSA (vancomycin-resistant S. aureus),

VRE (Vancomycin resistant Enterococcus) and MRAB (Multi-drug resistant A.

baumannii) (Appelbaum, 2007). The major factors contributing in the emergence of

drug resistance in livestock population are self-medication, inappropriate and misuse of

antibiotics, poor quality and incomplete course of therapy (Bushra et al., 2016; Canton

et al., 2013; Mathew et al., 2007).

Antibiotic resistance can be acquired as a result of gene mutation or the

acquisition of new genetic material (Silletti and Lorian, 1986). Mycoplasma has the

ability of higher mutation rates and genetic modulation than other bacteria, this

property confer the development of antimicrobials resistance. The mechanism of

resistance in human Mycoplasma infections is some what known for fluoroquinolones

(Bebear et al., 1998), tetracyclines and macrolides (Lucier et al., 1995). But limited

data are available in the literature concerning the acquisition and mechanisms of

antimicrobial resistance against Mycoplasma of veterinary importance (Gautier-

Bouchardon et al., 2002). However, some studies were conducted for the evaluation

and sensitivity and susceptibility of pathogenic Mycoplasma species against different

antimicrobials. In many advanced countries of Europe microbial resistance were

developed by different Mycoplasma species against tylosin, oxytetracycline and

spectinomycin. Some MmmSC developed resistance against tylosin (Laura et al., 2006;

Ayling et al., 2005; Ayling et al., 2000). In some areas M. agalactiae were not sensitive

to nalidixic acid and erythromycin (Antunes et al., 2007). With the rise in the

antimicrobial resistance (AMR) to many antibiotics, there is considerable interest in the

development of other classes of antimicrobial for the control of infection. The finding

of study revealed that frequent use of antibiotics in sheep is associated with increase

resistance, highlighting the careful use of such drugs in veterinary practices (Scott and

Manzies, 2011).

40

2.20.5 Medicinal Plants

The use of plants in treating diseases is as old as civilization and traditional

medicines still provides a major share in treatments of different maladies (Alviano and

Alviano, 2009; Fabricant and Farnsworth, 2001). The developments of drug resistance

issue of antibiotics to various pathogens further signify the role of herbal medicines.

Nowadays, due to historical and cultural reasons, folk medicine is still important in

developing countries due to poverty and scarce health services. The plenty of plants on

the earth surface has been attracted human mind to investigate different medicinal

plants extracts as potential sources of new antimicrobial agents (Bonjar et al., 2004).

Therefore, medicinal plants has extensively used in Unani, Ayurveda and Homeopathic

medicine (Girish and Shankara, 2008; Kausik et al., 2002). It is estimated that only 1%

out of 0.26 million flowering plants on earth has been studied for their phyto-active

compounds as a medicinal use (Verpoorte, 2000; Cox et al., 1994).

The World Health Organization decleraed that 80% of the world’s population

depend on traditional therapies and using different plant to cure various diseases

(WHO, 1993). Medicinal plants are a good source for the discovery of new drugs and

provide base to treat the multi-drugs resistance (MDR) pathogens. It provides bio-

active ingredients in traditional folk medicine, pharmaceutical intermediates, food

supplements and lead various compounds in the manufacturing of modern drugs

(Neube et al., 2008). In recent era, multi-drug resistance in human, animals and plants

pathogens has been developed due to extensive use of synthetic drugs. Therefore, the

search for novel bioactive compounds from medicinal plants has gained immense

importance as the plant based drugs are safe, biodegradable and have fewer side effects

(Prusti et al., 2008; Srivastava et al., 2000).

Plant based medicines are simple, cheap, safe, effective having broad spectrum

activity, it also minimize the side effects of various chemotherapeutic agents and

improve general health status (Ashokkumar and Ramaswamy, 2014; Chin et al., 2006).

Plants are rich source of a variety of secondary metabolites such as flavonoid, alkaloids

tannins, terpenoids and phenolic compounds which have been shown in-vitro to have

antimicrobial properties (Nasir et al., 2015; Bakht et al., 2014).

41

Different solvents are used to isolates phyto-active compounds from medicinal

plants with various degree of success. Plant methanolic, ethanolic, acetone, chloroform

and aqueous extracts are currently used as antibacterial, antifungal, antipyretic and anti-

mycoplasmal preparations (Shetty et al., 2013; Evans, 1997). Use of herbal medicines

are continuously rising up due to their rich source of bio-active compounds, less side

effects and also no known resistance issue (Aburjai et al., 2001). Screening of

medicinal plants for animals infections especially for caprine anti mycoplasmal activity

are neglected chapter. The Phyto-chemical compound after manipulation provides new

and improved drugs for the treatment and management of these infectious diseases.

Plants are naturally available at every land on the earth thus provide cheaper and easily

available source for the development of new drugs discovery (Newman et al., 2007;

Tomoko et al., 2002). The northern regions of Pakistan are gifted with large reservoirs

of flora having high scope for herbal medicines. Plants have been used in the

preparation of medicine as antimicrobial agents since ancient times can provide a gifted

solution for drug resistant pathogens (Ismail et al., 2012). Many herbal/medicinal plants

have been used as medicine since ancient time and long been known as antibacterial,

antiviral, antiparasitic and antifungal (Shetty et al., 2013). The Calotropis procera

reported with minimum inhibitory concentration (MIC) of 80 ug/mL, while Artemisia

herba-alba with MIC 3.12 mg/mL (Al-Momani et al., 2007; Muraina et al., 2010;

Agarwal et al., 2012).

Azadirachta indica commonly known as “Neem” in subcontinent belong to the

family Meliaceae. It has been known for medicinal properties and used in Ayurvedic

treatment for more than 4000 years ago (Khatkar et al., 2013; Pankaj et al., 2011). It is

evergreen tree found in most tropical countries of the world. The genus Azadirachta is

native to India and Burma, growing in tropical and semi-tropical regions of the world.

It is well grown in South East Asia and West Africa and cultivated in many countries

including Singapore, Philippines, Pakistan, Malaysia and Australia (Hashmat et al.,

2012). Small scale successful plantation also carried out in Europe and United States

(Kumar and Navaratnam, 2013). The tree is found in hot and humid regions of the

country including Bannu and Dera Ismail Khan Districts of Khyber Pakhtunkhwa.

Similarly it is abundantly present in most parts of Punjab and Sindh. It is a fast growing

tree with average height of 15-30 meters (Bhowmik et al., 2010). It is used in folk

medicine as a principal therapeutic agent in different formulations. The leaf, seed, bark

42

and oil are well knwon for antiviral, antibacterial, antifungal and antimalarial activities

(Biswas et al., 2002). The U.S. National Academy of Science in a scientific report in

1992 declared “Neem a tree for solving global problem”. About 135 active Phyto-

chemical compounds like flavonoids, terpenoids, tannins and steroids has been isolated

from different parts of Neem (Emran et al., 2015; Biswas et al., 2002). The extract of

different parts of Neem like leaf, bark and seed oil showed wide therapeutic indications

like antimalarial, anti-inflammatory, antidiabetic, antifungal, antiparasitic,

antiprotozoal, antibacterial and antioxidant (Sultana et al., 2007; Subapriya and Nagini,

2005; Talwar et al., 1997). The leaves and seeds of Neem containing important

compounds like azadirachtins, nimbin and nimbiodol that have been used as alternative

feed supplements to control certain diseases in livestock and poultry industry. There is

no proper study on anti-mycoplasmal activity of Neem against different pathogenic

species of Mycoplasma in animals. The finding of another study revealed that

methanolic extract of Neem exhibited the antimicrobial activity at 60 mg/mL

concentration against different pathogen isolated from oral cavity. But the aqueous

extract did not produce any antibacterial and antifungal activities at high concentration

(Adyanthaya et al., 2014).

Calotropis procera commonly khwon as “milk weed” belong to family

Asclepiadaceae consisted of 280 genera and 2000 species. It is widely distributed

throughout the world and abundantly found in the tropics and sub-tropics areas of India,

Pakistan, Bangladesh and Afghanistan. In different studies the ethanoic, methanolic and

chloroform extracts exhibited good antibacterial properties (Kareem et al., 2008).

Different active compounds such as triterpinoids, cardenolide, alkaloids, resins,

calotropin, anthocyanins and proteolytic enzymes in latex, flavonoids, tannins,

saponins, mudarin, sterol, cardiac glycosides. Flowers contain terpenes, multiflorenol

and cyclisadol has been isolated (Verma et al., 2013; Al-Yahya et al., 1990). In a study,

it is reported that C. procera showed minimum inhibitory concentration (MIC) at 80

ug/mL (Arjoon et al., 2012; Al-Momani et al., 2007).

Artemisia herba-alba belongs to the family Asteraceae commonly known as

“whit wormwood” consisted of 500 species are mainly found widely in the northern

hemisphere (Bremer and Humphries, 1993). The Artemisia has different species

throughout the world and about 150 in China and Asia, 175 in Russia, 51 in Japan and

43

57 in Europe (Maria et al., 2012; Shinskin and Bobrov, 1995). Only about 30 Artemisia

species are investigated for phytochemical analysis for their medicinal uses (Maria et

al., 2012). It has been used as folk since ancient time as antidiabetic, antispasmodic,

antihypertensive and antibacterial (Zeggwagh et al., 2008; Laid et al., 2008). The plants

of Artemisia is dwarf shrub, commonly grow in Federally Administered Tribal area

(FATA) regions and northern areas of Pakistan and also in the Western border of

Pakistan including the major areas of Afghanistan. It is traditionally used for the

treatment of diabetes mellitus, liver diseases, skin infections, anthelmintic,

antispasmodic and anticancer (Willcox et al., 2009). The different parts of plant are

used for medicinal purposes like the essential oil has antibacterial, antifungal and

antigenotoxic effects (Bakkali et al., 2008; Aburjai et al., 2001). Some important

compounds like terpenin, camphor, davonone, herbalbin, flavonoides,

acetate and borneolhas been isolated from leaves, flowers, seed, root and stem (Moufid

and Eddouks, 2012). In an experimental study the A. herba-alba was found most

effective among tested plants with MIC 3.12 mg/mL against several pathogenic

Mycoplasma species (Al-Momani et al., 2007).

2.21 Vaccination and control of Mycoplasma infections

The vaccine itself does not confer any immediate protection against pathogen

but act as immunogen. Antigens stimulate the host body to produce specific antibodies

in the blood against invading pathogen. The vaccine classified as live, inactivated or

killed antigen which stimulates the body to produce specific antibodies. The foreign

substances like bacteria, viruses, their metabolites and certain other complex substances

like saponin can be recognized by the body as foreign antigens. All living organisms

encounter invading pathogens every day that have the potential to make host sick. The

host body depends on its immune system that finally produced the antibodies which

detect these pathogens and prevent them from causing an infection. The antigen at an

early entry into the host body recognized by sentinel cells an important blood cell.

These cells detect an antigen, processed and simulate a series of biochemical reactions

which finally produce antibodies. The new produced antibodies are highly specific in

their nature and function to encounter the infection. The antigen antibody complex

attracts scavenger cells that then destroy the antigens and help to prevent disease. The

antigen-antibodies complex attracts phagocytic cells and compliment system which

https://en.wikipedia.org/wiki/Camphor

https://en.wikipedia.org/wiki/Borneol

44

destroy the invading antigen and prevent disease. Mycoplasma whole cell inactivated

by saponin has immunogenic potential to produced larg amount of antibodies. The

saponine has the property to preserve the major antigenic part of immunogen and

ensure good results.

Immunization is the possibel way to effectively control and prevent infectious

diseases (Sumithra et al., 2013). A number of human and animal’s diseases like polio,

small pox, diphtheria and rinderpest become completely eradicated due to use of

efficient vaccine (Ghanem et al., 2013). Inspite of this, a number of infection

responsible for millions of death in human and animals due to unavailability of

effective vaccine (Curtiss, 2011). The specie specific vaccine is useful tool to encounter

many diseases and also recommended by many researchers (OIE, 2013). Saponin

inactived Mycoplasma vaccine also use in different regions with variable efficacy

(Nicholas and Churchward, 2012). In many studies whole cell culture formalized and

saponized vaccine are successfully used for eradication of different diseases of

livestock.

Autogenous vaccine has been used in Iran for last centuries in which a piece of

CCPP infected lungs were minced with vinegar and garlic and injected into ear that

render the host immune system (Tadjbakhsh, 1994). Similarly in the late 19th

century,

lungs extract of infected animals were introduced subcutaneously by Hutcheon

(McMartin et al., 1980). These findings clearly revealed that control is possible by

active immunization. In Europe animal vaccine has been used since 1970 but it become

intensively in practice after 1990 (Foggie et al., 1970; Tola et al., 1999). The early

vaccine prepared as prophylaxis against different species of Mycoplasma in small

ruminant included M. agatectiae, M. putrefaciens, MmmLC and Mcc (Greco et al.,

2002; De la Fe et al., 2007; Buonavoglia et al., 2010)

High prevalence of mycoplasmosis, poor response to antibiotics, development

of antibiotic resistance and concern of consumers about drugs residue in meat of the

treated animals have spurred interest in the control of CCPP through vaccination (OIE,

2004). Currently vaccination against CCPP is the single most important control

intervention to comabate the disease. A variety of vaccines containing either whole cell

of Mycoplasma or their components have been developed and tested under filed

conditions (Nicholas et al., 2009). A few of these vaccines have been found and widely

45

used in field condition. One of the hallmarks of these vaccines is a considerable

variation in duration of immunity (Shivachandra et al., 2011). Recently plain broth

formalin killed bacterins, alum precipitated and aluminum hydroxide gel adjuvanted

vaccines have been used against many bacterial infenctions in livestock (Sotoodehnia et

al., 2005).

Different chemicals are used for inactivation of Mycoplasma with various

degree of success. Formaline is extensively used as an inactiveated agent but has some

undesirable effects like local irritation, carnogenic properties and unpleasant odour in

food animals. The saponine provide an alternate with least side effects, good inactivant

and adjuvant agent. In addition, it could also highly efficiently and fastly lyse the

cholesterol/lipid rich membranes of Mycoplasma (Razin and Argaman, 1963). Saponin

is an extract from the bark of the South American tree Guillaia saponaria, has been

successfully used both as inactivant and adjuvant for Mycoplasma (Ahmad et al., 2013;

Kensil et al., 1991) and is recommended for use in food animals (Mulira et al., 1988).

Several preparation with modification has been attempted which confer solid immunity

lasting for six months to one year. Such vaccine were composed of sonicated

Mycoplasma antigens adjuvanted with incomplete Freund’s media and lyophilized F38

was inactivated with saponin and used freshly (Rurangirwa et al., 1987b; Rurangirwa et

al., 1984).

In an experimental study, saponin based inactivated M. bovis vaccine was

revealed as highly effective, safe and confers protection against virulent M. bovis

infection. Vaccines are carried out for the prevention of contagious agalactia caused by

M. agalactiae in the Middle East and Europe. However no single vaccine and method

of preparation has been globally applied (Nicholas et al., 2009). In Pendik Institute,

Istanbul Turkey, live attenuated vaccines for contagious agalactia caused by MmmLC

has been used for the last many years and was considered more effective than

inactivated vaccine (Turkaslan, 1990). The strain F38 vaccine inactivated by saponin

confers 100% protection in natural outbreak (Litamoi et al., 1989). However the

saponised vaccine has been successfully used in Kenya as a prophylaxis for the last

several years. This method need incubation of 12 hours at 4 °C for proper inactivation

of Mycoplasma cells (OIE, 2014).

46

In the present era, vaccine against Mycoplasma is available and carried out in

different area of Pakistan. In an experimental study saponin inactivated vaccine

prepared from field isolates of Mmc had been used as prophylaxis. The immunogenic

potential of this vaccine was investigated in 40 buck of Beetal breed about 9 months to

one year of age kept at Livestock Research Institute Bhadurnagar Okara, Pakistan

(Shahzad et al., 2012). In other study saponin adjuvanted inactivated M. bovis vaccine

confer protection in challenge calves (Ahmad et al., 2013; Kensil et al., 1991).

Vaccination against Mccp commercially produced in different countries of the world,

such as CCPPV (killed) and capridoll (live) and Pulmovac in Ethiopia and Turkey

respectively (Samiullah, 2013). Lyophilized Mmc vaccines are being prepared by

veterinary research institute (VRI), Lahore, Pakistan (Shahzad et al., 2012).

2.22 Detection of antibodies by serological tests

Numbers of serological test has been used for the detection of antibodies against

Mycoplasma and other bacteria. The indirect haemagglutination (IHA) test was used

successfully by many researchers for the detection of antibodies against Mycoplasma

species (Ahmad et al., 2013; Gagea et al., 2006). In an experimental study M. bovis

antibodies raised by saponized vaccine in calves was successfully evaluated by IHA

(Ahmad et al., 2013) and against Mmc antibodies raised in rabbit and goats by Rahman

et al. (2003). Similarly IHA test was conducted in an experimental study for the

detection and eveluation of antibodies produced in buffalo calves by haemorrhagic

septicemia oil adjuvant and alum precipitated vaccine (Jaffri et al., 2006). The

complement fixation test (CFT) was also used for the evaluation of antibodies in sera of

vaccinated animals and CCPP infection. They reported that CFT was more specific but

less sensitive than IHA and also need more technical expertise for performance

(Thiaucourt et al., 1996; Muthomi and Rurangirwa, 1983). The CFT was generally used

for the seroepidemiological study in different parts of the world by many researchers

(Yousuf et al., 2012; Gelagay et al., 2007).

2.23 Importance of Mycoplasmosis in Pakistan

Pakistan is one of the good geographical habitats for small ruminants,

comprising 30 and 72 million sheep and goat population respectively with more than

3% annual increase. Pakistan is being the 3rd

largest goat and 12th

sheep producing

47

country of the world (Economic survey, 2016-17; Afzal, 2010). The majority of sheep

and goats are produced on small farms, evenly distributed throughout the country. The

production system is nomadic, sedentary and transhumant (Ishaque, 1993). The farmer

of the country is mostly poor with limited resources and deficient management system.

The awareness about the outbreak of infectious diseases, vaccine schedule and control

strategies at farmer level is not satisfactory. The small ruminant is exposing to various

harsh climatic conditions, infectious and non-infectious diseases. As a sub-tropical

region of South Asia, Pakistan has favorable environmental condition for the growth of

various infectious agents like bacteria which are pathogenic for livestock population.

Theses pathogens are resultant in several outbreak of respiratory diseases including

CCPP, which causes heavy economic losses in southern and northern parts of the

country (Banaras et al., 2016; Awan et al., 2012).

In Pakistan, Mmc was for the first time reported in goats suffering from CCPP

by using different biochemical tests (Khan et al., 1989). Later on seroprevalence of

Mmc in small and large ruminants was investigated (Rahman et al., 2006). With the

development and introduction of advanced techniques, molecular identification of

different species was conducted. In the recent era CCPP diagnosis by the use of PCR

has greatly improved even directly from clinical sources like lungs tissue and nasal

discharge. The PCR using 16-S rRNA gene analysis accurately confirmed the detection

of Mmc. In Pakistan, the CCPP infection was considered to be caused by Mmc by using

various conventional techniques (Rahman et al., 2003). Later on PCR based

confirmation of Mmc was done by Shahzad et al. (2012) in Punjab and in Khyber

Pakhtunkhwa (Sadique et al., 2012), then in Baluchistan (Awan et al., 2012; Hira et al.,

2015). In a large scale international collaborative study, the seroprevalence of CCPP

caused by Mccp was 2.7% and 44.2% in Gilgit and Diamer Districts of Norther

Pakistan and 10.1% in the Shuro-Obod District of Tajikistan (Peyraud et al., 2014).

Similarly the seroprevalence of CCPP caused by Mccp was reported 8.52% in different

districts of Punjab, Pakistan (Shahzad et al., 2016).

No detail published data is available about the molecular confirmation of Mccp

and other non-cluster species like M. putrefaciens and M. agalactiae in the other

provinces particularly Khyber PakhtunKhwa of Pakistan.

48

2.24 Study area: Khyber Pakhtunkhwa, Pakistan

Pakistan is an agro-livestock based economy having livestock and dairy sector

as the main segment of agro- economy in all provinces. The country is consisted of four

provinces namely Punjab, Sindh, Balochistan, and Khyber Pakhtunkhwa (KP),

Northern regions of Gilgit-Baltistan and Federally Administered Tribal Areas (FATA).

The Northern zone adjacent with Bajawar Agency, while central zone with the border

along with Mohmand Agency and southern zone having borders with Kurram, North

and South Waziristan Agencies. KP is located in the North-West of the country. It

borders the Federally Administered Tribal Areas to the West, Gilgit–Baltistan to the

North-East, Azad Kashmir to the North-East, Punjab and the Islamabad Capital

Territory to the East, Afghanistan to the North-West and China to the North.

Strategically, it is very important province having a famous Khyber Pass a gate-way for

foreign invadors to Asia. The province mainly divided into three climatic zones named

as Northern, Central and Southern. The northern zone is extremely cold with heavy

snow and rainfall. The weather is extremely cold in winter and pleasant in summer. The

central zone consisted of beautiful valley of Peshawar surrounded by mountains having

hot hummid environment. The southern zone is arid and hot climate with scanty

rainfall. The three zones consisted of twenty six districts. The provincial capital and

largest population city is the Peshawar.

2.25 Sheep and Goats in Khyber Pakhtunkhwa, Pakistan

Total small ruminants population in Pakistan is 102 million in which KP

contribute 4.6 (15.4%) and 11.5 (16.7%) million sheep and goat respectively to the

national resources (Pakistan Economic Survey, 2016-17). Goat farming is a very

popular, profitable and incredible business model for lower economy class in

Pakistan. Goats can easily manage with other livestock animals and small feed

resources. Rearing of goats is very easy and simple; children and women can easily

raise and take good care of them. Sheep and goat farming in Pakistan is very common

and popular among the farmer community. Many people of KP prefer the goat and

sheep farming business, because it require comparatively less labor and management

and also relatively cheaper to buy and sell than cattle. Goats are known as “poor

man’s cow” because of their small size and having good capacity of producing milk

http://en.wikipedia.org/wiki/Federally_Administered_Tribal_Areas

http://en.wikipedia.org/wiki/Gilgit%E2%80%93Baltistan

http://en.wikipedia.org/wiki/Azad_Kashmir

http://en.wikipedia.org/wiki/Punjab_(Pakistan)

http://en.wikipedia.org/wiki/Islamabad_Capital_Territory

http://en.wikipedia.org/wiki/Islamabad_Capital_Territory

http://en.wikipedia.org/wiki/Afghanistan

http://en.wikipedia.org/wiki/Peshawar

49

and meat. There are certain breeds of goat which are easily maintained and having

high market value due to low fat contents. Marketing goat products is very easy,

because sheep/goat product has a huge demand in the local and global market. There

are approximately 30 sheep and 25 goat breeds in Pakistan. Khyber Pakhtunkhwa

provide good habitat for the different sheep and goat breeds. The different climatic

zones of the province provided good grazing pasture for the livestock. The farmer

holding small, medium and large herd of small ruminant consisted of sheep and goat

mostly in the farm of mix herd throughout the province.

50

III. STUDY-1

ISOLATION AND MOLECULAR IDENTIFICATION OF

PATHOGENIC MYCOPLASMA SPECIES FROM NATURALLY

INFECTED SMALL RUMINANT OF KHYBER PAKHTUNKHWA

51

ABSTRACT

Ruminant mycoplasmosis is an important highly fatal disease, causing

significant health issue and heavy economic losses in small ruminant production

throughout the world. The study was carried out to identify and characterize the

pathogenic member of Mycoplasma mycoides cluster and non-cluster species in small

ruminants of three different climatic regions of Khyber Pakhtunkhwa Pakistan. A total

of 1980 samples consisted of nasal discharge (n=1500), tracheal swab (n=300), lungs

tissue (n=147) and pleural fluids (n=33) were collected from animals exhibiting

respiratory sings suspected for contagious caprine pleuro pneumonia (CCPP). The

samples were taken in PPLO transport media then sub cultured in modified Hayflick

media and incubated at 37 °C with 5% CO2 for 7-10 days. Out of total samples, 737

(37.22%) were positive for Mycoplasma growth showing mass turbidity, whirling

movement in culture broth and typical fried egg colonies in agar media. The results

revealed that disease was significantly (P˂0.001) higher in northern (43%) followed by

southern zone (34.6%). Similarly, significantly higher (P˂0.01) frequency of isolates

was recovered from goats (58.8%) as compared with sheep (41.2%). The positive

cultures were further identified through biochemical assay and 592 (29.82%) were

identified as mycoides cluster and non-cluster species. The positive cluters were further

subjected to molecular analysis for identification of specific specie of mycoides cluster

and non-cluster. A total of 553 (27.92%) were confirmed as Mycoplasma with species

distribution of 13.53%, 5.5% and 7.97% for Mycoplasma mycoides subsp. capri (Mmc),

Mycoplasma capricolum subsp. capripneumoniae (Mccp) and Mycoplasma

putrefaciens (Mp), respectively. The highest isolates were confirmed from pleural

fluids (63.6%) followed by lungs tissues (58.5%), and least from tracheal swabs (21%).

It was revealed from the results that higher prevalence of mycoplasmosis was recorded

in the northern region followed by southern and central regions. These results for the 1st

time confirmed the presence of three pathogenic Mycoplasma species in the tudy area.

52

3.1 Introduction

Ruminant mycoplasmosis is caused by both Mm cluster and non-cluters

pathogenic species infecting different system of the host. It causes direct losses due to

serious outbreak in the form of high mortality, decrease milk and meat production,

reduce carcass weight, and indirect losses including treatment, vaccination and

managemen cost, etc. Due to its high pathogenicity and causing huge economic losses

to the livestock industry it is characterized as list-B disease (OIE, 2014). The country

suffering from the outbreak of this disease faces great hardship in export of meat and its

products due to trade embargo by the worid regime. In Pakistan, the ruminant

mycoplasmosis is known by one of the most important disease called contagious

caprine pleuropneumonia (CCPP) with a long history of causing havoc in the farming

community (Shahzad et al., 2013; Sadique et al., 2012). The disease is widely

distributed in the country lead to several outbreaks and causes heavy losses in small

ruminants (Awan et al., 2009; Rahman et al., 2006; Khan et al., 1989).

Ruminant mycoplasmosis is important bacterial disease poses a serious health

threat to the ruminant population and responsible for huge economic losses (Banaras et

al., 2016; Sadique et al., 2012). Among the different Mycoplasma infection CCPP is

extremely lethal disease caused by six pathogenic species called mycoides cluster

(Manso-Silvan et al., 2007). The disease for the 1st time was reported in Algeria in

1873 and later on in many countries of East Africa, Asia, Europe and Middle East

(Atim et al., 2016; Tigga et al., 2014). In Pakistan the disease was for the first time

confirmed that Mccp is the causative agent of CCPP in Baluchistan by Awan et al.

(2010). Recently in the international collaborative study Mccp was confirmed in the

northern Pakistan and Tajikistan (Peyraud et al., 2014). The disease was also reported

in different areas of central Punjab, Pakistan (Shahzad et al., 2016). However, the

classical form of disease is caused by Mccp that chiefly restricted to the chest cavity

(OIE, 2014; Manso-Silvan et al., 2007; Thiaucourt and Bolske, 1996).

The Mm cluster species are responsible for most significant devasting disease

called CCPP. The classical findings of CCPP are consisted of pyrexia, acute respiratory

distress, grunting (Zinka et al., 2013). The other important features of the disease

comprises of sero-fibrinous pneumonia, lungs hepatization, straw coloured fluid and

53

pleural effusion accompanied by high mortality (Abbas et al., 2013; Mondal et al.,

2004).

In Pakistan, a lot of work has been conducted for the diagnosis of pathogenic

Mycoplasma species using various conventional tests. But little work has been

conducted on molecular characterization of the local isolates of Mycoplasma species.

Mostly the conventional methods of diagnosis are failed to address the issue properly

because of its shortcoming. The isolation of Mycoplasma is very difficult because of its

fastidious nature, needs of special media growth requirements (OIE, 2013). The

serological and biochemical tests are usually failed due to sharing of common antigenic

epitopes by many species of Mycoplasma. Therefore, the advanced molecular

techniques like PCR and sequencing is the most accurate tool for identification and

confirmation of different Mycoplasma species (Woubit et al., 2004). It can confirm the

exact specie of microorganism even in mixed infection and directly from clinical

samples like nasal discharge and pleural fluids. The Mycoplasma having 16S-rRNA

genes allowed the identification of variable regions with both genus and species

specific primers to identify the particular species of Mycoplasma cluster (Kumar et al.,

2011; Manso-Silvan et al., 2007; Hotzel et al., 1996). Looking at the paucity of the

scientific literature on Mycoplasma in Pakistan the present work is carried out to

isolate, identify and characterize all the prevalent species associated with ruminant

mycoplasmosis in Khyber Pakhtunkhwa. This study will pave a way for researcher and

planner to design strategies for curbing this fatal disease. The study was designed with

following objectives;

1. Study on prevalence of CCPP in naturally infected small ruminants across the

three different climatic zones of Khyber Pakhtunkhwa.

2. Molecular characterization of the local isolates of Mycoplasma recovered from

small ruminants.

54

3.2 Materials and Methods

3.2.1 Sampling

For isolation of pathogenic Mycoplasma species, the samples were collected

from sheep and goats suffering from respiratory syndrome suspected for CCPP during

the period of December 2014 to May 2016. The study was approved by the faculty

ethical committee vid notification No. 2234/LM/UOA dated 03-12-2014, Faculty of

Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar.

The area was divided into three different climatic regions consisted of northern, central

and southern zone of Khyber Pakhtunkhwa, Pakistan (Fig. 3.1). A total of 1980

samples consisted of nasal (n=1500), tracheal discharge (n=300), lungs tissue (n=147)

and pleural fluids (n=33) were collected from small ruminants exhibiting the signs of

respiratory syndrome suspected for contagious caprine pleuropneumonia (CCPP). The

small ruminants were further divided into sheep and goats, and a total of 990 samples

were collected included 330 from each species in each zone. From each region, a total

of 660 samples (nasal n=500, tracheal n=100, lungs tissue n=49 and pleural fluids

n=11) were obtained collectively from sheep and goats.

In northern zone, samples were collected from district Abbotabad, Mansehra

Swat, Buner, Shangla and Dir upper. In central zone Peshawar, Nowshahra, Charsadda,

Mardan and Swabi were selected for samples collection. In southern zone, samples

were taken from Kohat, Karak, Bannu, Lakkimarwat, Tank and Dera Ismael Khan. The

area for collection of samples is presented in Fig. 3.1. A minimum representative 50

samples were collected from sheep and goats in each mentioned district of the three

climatic zones. To investigate the sex wise prevelance of disease, a total of 412 male

and 1568 female animals were sampled from all the the three regions of study area.

Similarly, the age effect was determined by taking samples from different age groups.

The different age groups of both species comprised of A, B and C that represented age

group of 1 to12 months, 13 to 24 months and 25 to 36 months, respectively. The

detailed history regarding animal was recorded on preformed questionnaire

(Annexure-1). The samples were taken by sterile cotton swab and then transfered to

the special transport media. Lungs tissue and pleural fluids were taken in sterile

container and kept in ice box. The collected samples were kept under refrigeration and

55

transported to the Pathology laboratory at Department of Animal Health, The

University of Agriculture Peshawar, Pakistan for onward processing.

Fig. 3.1 Map of Khyber Pakhtunkhwa, showing different climatic zones and districts of

samples collection.

3.2.2 Culturing of pathogenic Mycoplasma species

3.2.2.1 Processing of samples

For culturing of pathogenic Mycoplasma specie the samples were collected

from nasal discharge, tracheal swab, pleural fluids and lungs tissue under aseptic

condition and inserted in special transport medium as per standard protocol as

described by Miles and Nicholas, (1998).

3.2.2.2 Sterilization of Glass wares

All the glass wares used in the media preparation and research work like

graduated beakers, cylinders, conical flasks, glass jars, screw cape, test tubes, petri

56

plates and filtration assembly were properly washed and then dried. Washed clean and

dried glass wares were wrapped in diamond aluminum foils and then wrapped in paper,

were sterilized at a temperature of 121 °C for 15 minutes at 15 lb. pressure in autoclave

(Hiclave TM

HVE-50, Japan). All the activities of culturing were carried out in biosafety

cabinet Level-II (ESCO, USA).

3.2.2.3 Modified Hayflick Medium for Mycoplasma growth

The modified Hayflick media consisted of two parts, autoclavable and filterable.

Medium was prepared according to standard procedure of OIE, (2014).

3.2.2.3a Part A (Autoclavable Part)

Bacto PPLO (pleuropneumonia-like organisms) broth (HIMEDIA, India) (21g)

was dissolved properly in 700 mL of distilled water then adjusted to pH 7.8 and

autoclaved at 121 ºC for 15 minutes.

3.2.2.3b Part B (Membrane-filtered part)

Horse serum (210 mL) inactivated at 56 °C for 30 minutes was mixed with

100mL of fresh yeast extract (Biotech, Canada ) then added 8 mL of 25% sodium

pyruvate, 4 mL of 10% glucose (Biotech, Canada), 150mg Fluconazole® (antifungal),

Benzyl penicillin® + Sulbactam® (Antibiotics) and 4 mL of 0.5% phenol red. The pH

was adjusted by pH meter (Jenway, M-3505 U.K) to range of 7.6-7.8 by adding 5%

sodium hydroxide (NaOH) or hydrochloric acid (HCl). Then properly mixed all the

added components and then filtered through 0.2 μm membrane filter (Corning®, NY

14831, Germany) using sterilized glass filtration assembly (Sartorius, Germany).

Both parts A and B were mixed aseptically at 40 °C. To avoid any

contamination during the medium preparation all the process was performed inside the

safety cabinet (BSC, level-II ESCO, USA). The composition of Hayflick media is

enlisted in Annexure-2.

For preparation of solid medium (agar) the above procedure was followed along

with addition of 0.9% agarose in Hayflick agar base (HIMEDIA, India) to broth media.

57

3.2.2.3c Media storage

The prepared PPLO broth medium was poured into 3-5 mL capacity screw cape

glass test tubes. These tubes were tightly caped and placed for sterility test in CO2

incubator at 37 ºC for 72 h. After sterility assurance tubes that showed no turbidity or

color change were considered sterile, and placed in refrigerator at 4 °C till further use.

For preparation of solid medium, petri dishes were poured with 20 mL Hayflick agar

media to a depth of approximately 4-5 mm (Awan et al., 2010). Petri dishes were

wrapped by using wrapping papers and aluminum foil. These plates were stored at 4 °C

till further use.

3.3 Isolation and identification

All the collected samples were incubated in anaerobic incubator (New

Brunswick, Galaxy 48-S UK) with 5% CO2 at 37 °C for 3-10 days. The incubated test

tubes were examined daily for presence of mass turbidity, whirling movement and

change in color. The positive growths were sub-cultured on Hayflick agar media for the

appearance of nipple like or fried egg Mycoplasma colonies. The positive colonies were

taken by sterile loop and re-cultured three times for obtaining pure culture as per

standard protocol of (OIE, 2013).

3.3.1 Morphological Identification

Identification of isolates was made by specific morphological characteristic of

Mycoplasma colony grown on solid media as described by Mondal et al. (2004).

Typical characteristic colony having fried egg or nipple like appearance, tinny, smooth,

and 0.1-1 mm in diameter with dense elevated centers embedded in media were

suggestive of Mycoplasma species.

58

3.4 Identification and confirmation of isolates


Biochemical assay of the local isolates was carried out for prelimanry

identification of the Mycoplasma cluster and non-cluster specie as per standard protocol

of (Poveda and Nicholas, 1998). A volume of 0.5µL from each isolate was diluted in 5

mL of Hayflick broth and subjected to different biochemical tests like glucose

fermentation, serum digestion, tetrazolium reduction (aerobically and anaerobically),

casein digestion and arginine hydrolysis test for the identification of desired

Mycoplasma species.

3.4.2 Molecular confirmation and characterization

All isolates that showed turbidity and produced typical Mycoplasma colonies

followed by identification through biochemical assay were subjected to PCR for further

confirmation.

3.4.2.1 DNA extraction

The positive culture was subjected for DNA extraction. DNA was extracted by

using a commercially available tri reagent (Trizol®, Thermo Fisher, Scientific, USA)

according to the manufacturer’s direction. A volume of 1.5 mL of positive culture with

adequate growth was taken in eppendorf tube and centrifuged at 14000 rpm at 4 °C for

15 minutes using high speed refrigerated centrifuge machine (Z-216, HERMLE,

Germany). After completion the supernatant was discarded and the pellet was re-

suspended in 1000μl sterile phosphate buffer saline (PBS) and repeat the same

procedure twice for washing of pellet (Annexure-3). Then the pellet was again

resuspended in 1 mL PBS along with 1 mL of the Trizol® in a sterile eppendorf tube

inside the bio safety cabinet level-II (ESCO, USA). Then it was incubated at the room

temperature for 5 minutes and added 200µL chloroform and shake tube vigorously for

45 seconds followed by reincubated at room teperature for 10 minutes. The tubes were

centrifuged at 14000 rpm for 15 minutes at 4 °C, carefully taken the inter phase which

containing DNA and transferred to a new sterile eppendorf tube (1.5 mL). Precipitation

of the DNA was done by mixing pellet with 300μL of 100% ethanol and incubated for

59

three minutes at room temperature. After centrifugation discard the supernatant and

pellet DNA was washed two times by adding 1.0 mL of 0.1 M tri sodium citrate

solution follow by incubation at room temperature for 30 minutes with periodic shaking

and mixing. Then again it was centrifuged at 13500 rpm for 5 minutes at 4 °C. The

pellet was mixed with 75 % ethanol, and incubates at room temperature for 20 minutes

with periodic shaking and mixing. The tube was again centrifuged at 13500 rpm for 12

minutes at 4 °C, supernatant was discarded and the pellet was makes air dried by

placing the tubes opened inside the bio safety cabinet. At final step DNA pellet was

dissolved and suspended in 150µL of 8 mM NaOH in the sterile eppendorf tube and

stored at -20 °C till further use (Shahzad et al., 2013; Miserez et al., 1997).

3.4.2.2 Quantification of extracted DNA

The extracted DNA of 10µl was taken and added 2µl loading dye and gently

pipetted. Prepared 1% agarose gel and stained it with ethidium bromide. A 5µl of 1 kb

DNA ladder was loaded in the first well and extracted DNA in the reaming well. The

electrophoresis was carried out at 120 mV for 40 minutes (PS300-B, Hoefar, Inc.

USA). The gel was then analyzed in UV illuminator for the DNA band visualization.

The extracted DNA was further quantified for concentration and purity (spectra

260/280) by NanoDrop-2000 spectrophotometer (Thermo Fisher, Scientific, USA).

3.4.2.3 Polymerase Chain Reaction

The polymerase chain reaction (PCR) was performed for the detection of

Mycoplasma species by using following sets of primers the Mycoplasma cluster, specie

specific and non cluster (Table 3.1). These primers targeted the 16S-rRNA gene of

Mycoplasma with an amplicon size of 548, 316, 196 and 540 bp for Mycoplasmas

mycoides cluster, Mycoplasma capricolum sub specie capripneumoniae Mycoplasma

mycoides subsp. capri and Mycoplasma putrefaciens respectively.

60

Table 3.1 List of different PCR primer, sequence annealing temperature and

expected amplicon size of 16S-rRNA gene for confirmation of

Mycoplasma species. Species Primer name Oligonucleotide sequence 5’-3’ Tm

(°C)

amplicon

size (bp)

Source

Mycoplasma mycoides

cluster

Mm-F (CGA AAG CGG CTT ACT GGC

TTG TT)

52 548 Azevedo et al., 2006.

Mm- R (TTG AGA TTA GCT CCC CTT

CAC AG)

56

Mycoplasma

capricolum subsp.

capripneumoniae

Mccp.spe-F (ATC ATT TTT AAT CCC TTC

AAG )

54 316 Woubit et al., 2004

Mccp.spe-R (TAC TAT GAG TAA TTA TAA

TAT ATG CAA)

54

Mycoplasma mycoides

subsp. capri

P4-F (ACT GAG CAA TTC CTC TT)

56 196 Hotzel et al., 1996

P6-R (TTA AAT AAG TTT GTA TAT

GAA T)

56

Mycoplasma

putrefaciens

SSF1-F (GCG GCA TGC CTA ATA

CAT GC)

58 540 Shankster et al., 2002

SSR1-R (AGC TGC GGC GCT GAG TTC

A)

56

Mycoplasma

agalactiae

MAG-F (CCT TTT AGA TTG GGA TAG

CGG ATG)

54 360 Azevedo et al.,

2006

MAG-R (CCG TCA AGG TA TTCCTA C) 56

3.4.2.4 PCR conditions

PCR amplification reaction was carried out in a final volume of 25µl containing

DNA template and 10µl commercially available PCR master mix (PyroStart™ Fast

PCR Master Mix (2X), Fermentas, Canada (Annexure-4). The primers were used at a

concentration of 10 ρmols µl-1. Amplification was carried out in a thermocycler

(BIORED T100 USA) under the following conditions. Initial denaturation at 94 °C for

3 minutes followed by 35 cycles of denaturation at 94° C for 30 seconds, primer

annealing at 56 °C for 30 seconds, 72 °C for 45 seconds, polymerization at 72 °C for 5

minutes and then final extension at 12 °C for 10 minutes to polymerize all remaining

single strand DNA fragments (Annexure-5). After completion of the reaction the PCR

product was stored at 4 °C till further use (Hotzel et al., 1996).

61

3.4.2.5 Gel Electrophoresis

The amplified DNA was visualized by gel electrophoresis as described by

Hotzel et al. (1996). The gel was prepared by placing the gel caster on the level surface.

Agarose at 1% concentration was prepared in 50mL of 1X TBE buffer in a conical

flask and heated for 60 seconds in a microwave oven (Keenwood, Japan). After boiling

the gel was cool down to 35 °C then added 2µL of ethidium bromide (Sigma, Aldrich

Germany), gently shaked the gel and poured in caster having 15 teeth comb. Gel was

solidified completely after 15 minutes, remove comb carefully and placed the gel in

electrophoresis tray that filled with 1X TBE buffer (Annexure-6). DNA ladder 1 kb

(5µL) was loaded in the first well and 10µl of PCR product mixed with 3mL of 6X

DNA loading dye TM (R0611, Fermentas) that loaded in the remaining wells using

micropipette (Gilson, Germany). The tray voltage was adjusted to 110 mV, 500mA and

run for 40 minutes. After completion the gel was placed in Gel Doc system (Unitec,

BXT-26.M. UK), for visualization and images were captured.

3.5 Homology and phylogenetic analysis

The gel product of specific amplicon size was taken and submitted for

sequencing. The obtained sequences were subjected to NCBI BLAST to search for

homologous sequences for phylogenetic relation of the local isolates of Mmc Mccp and

Mp with other available sequences at gene data bank. Sequences of the isolates were

downloaded from NCBI and were multiple aligned through BioEdit version 7.0.5.2

(Hall, 1999). Furthermore, phylogenic tree topology was constructed for the obtained

sequences using software MEGA version 7.2 for evolutionary study and to build

correlation with the strains of different regions of the world (Tamura et al., 2011).

3.6 Statistical analysis

Data were compiled in Microsoft Excel sheet and analyzed through Chi-square

test using SPSS version 19. The Chi square test was used to check statistical association

between isolates and different climatic zones and also between specie, gender and age.

Z-test was used to check significant proportion (percent) difference between the

different Mycoplasma species and comparison between different sources of samples for

recovery of Mycoplasma isolates.

62

3.7 Results

Out of total samples 660 numbers were collected each from northern, central

and southern zone of the Khyber Pakhtunkhwa, Pakistan. Clinical investigation of the

diseased animals like body temperature, conjunctiva examination, lacrimation,

coughing, nasal discharge, dullness, diarrhea, urine color, nervous signs were recorded.

Detail history of dead animals was recorded from owner on preformed questionaire.

The postmortem was conducted for recording pathological lesions in different visceral

organs. Samples from lungs tissue and pleural fluids were collected for isolation of

Mycoplasma. Similarly, the animals in advanced stage of disease were purchased from

owner then slaughter for recording lesions and collection of samples for

histopathological examination and culturing. Field isolates of Mycoplasma were

identified by morphological appearance on modified Hayflick media, biochemical

assay and finally by molecular characterization.

3.7.1 Isolation of Mycoplasma

Out of total 1980 samples, 737 (37.22%) showed mass turbidity and whirling

movement in modified Hayflick broth identifired for the growth of Mycoplasma (Plate

3.1, 3.2). The zone wise distribution of positive isolates were 317(43.03%), 165(22.4%)

and 255(34.6%) on culture media for northern, central and southern zone, respectively.

On statistical analysis of data significantly (P˂0.001) higher frequency of isolates were

obtained from northern zone followed by southern zone (Table 3.2). The positive

growths were further recultured on modified Hayflick agar for development of

charectertic colony. The gross visible growth was developed on day three of post

incubation as presented in (Plate 3.3, 3.4. 3.5). On microscopic examination at 4X and

10X typical nipple like and fried egg colonies were appeared on day 3rd

to 7th

post

incubation (Plates 3.6 3.7, 3.8, 3.9, 3.10, 3.11). To obtained pure culture a

characteristic single colony was taken from agar media and re-cultured in modified

Hayflick broth for 24-48 h at 5% CO2. A pure culture of local isolates is presented in

Plate 3.12, 3.13.

63

Table 3.2 Result of Mycoplasma growth on culture media isolated from small

ruminants suffering from respiratory syndrome suspected for (CCPP) in

three different climatic zones

Climatic Zones Positive Percentage Chi-sq P-value

Northern

317/737

43.0%

Central 165/737

22.4% 75.7 0.001

Southern 255/737

34.6%

Statistical analysis (χ2) showed significant association (P˂0.001) between the isolates and climatic

zones.

Table 3.3 Comparative isolation of Mycoplasma from sheep and goats suffering

from respiratory syndrome suspected for CCPP.

Species Positive Percentage Chi-sq P-value

Sheep 304/737 41.2%

Goat 433/737 58.8% 35.9 0.001

Statistical analysis (χ2) showed significant association (P˂0.001) between the isolates and specie of

animal, df=1. Total numbers of positive isolates were 737.

The species based isolation on culture media revealed that out of total, 990

samples 304 (41.2%) and 433 (58.8 %) were recovered for sheep and goats,

respectively. On statistical analysis (χ2) significantly (P˂0.001) higher isolates were

recovered from goats as compared with sheep (Table 3.3). The prevalence of disease

was also investigated in sheep and goats across different climatic zone. The statistical

analysis (χ2) showed that significant (P˂0.001) association was present between the

positive isolates and climatic zone. The highest isolation was recovered from northern

zone 50.6%, 44.7% followed by southern zone 32.3% 33.2 % from goat and sheep,

respectively (Table 3.4).

64

Table 3.4 Distribution of positive isolates on culture media collected from sheep

and goats across different climatic zones.

Sheep

Zones Positive Percentage Chi-sq P-value

Northern 136/304 44.7%

Central 67/304 22.0% 33.9 0.002

Southern 101/304 33.2%

Goat

Northern 219/433 56.6%

Central 74/433 17.1% 1.29 0.001

Southern 140/433 32.3%

Statistical analysis (χ2) showed significant association (P˂0.001) between the isolates from both species

and climatic zones, df=2.

Out of total 412 male and 1568 female samples, 125 (30.3%) and 612 (39.03%)

were positive for male and female, respectively. On analysis of data (χ2) significant

(P˂0.001) association was observed between the isolates and sex of animals. The

findings showed that highest prevalence of mycoplasmosis was observed in female

animals as compare to male suffering from respiratory syndrome in different climatic

regions. The result is presented in Table 3.5.

Table 3.5 Gender based isolation of Mycoplasma from sheep and goat suspected

for mycoplasmosis.

Sex Positive Percentage Chi-sq P-value

Male 125/737 17.0%

Female 612/737 83.0% 10.54 0.001

Statistical analysis (χ2) showed significant association at (P ˂ 0.001), df=1, total male 412 and female

1568 were sampled.

The samples were collected from animals of three age groups. In sheep the

recovered positive isolates were 33.3%, 27.8% and 31% from age group A, B and C,

respectively. Similarly in goats, 47.6%, 43.2% and 41.4% of positive culture was

obtained from group A, B and C. Analysis of data (χ2) showed non-significant

association (P > 0.05) between different age groups of sheep and goats (Table 3.6).

The findings revealed that all age animals of both species are equally susceptible to the

mycoplasmosis.

65

Table 3.6 Age wise distribution of Mycoplasma isolated from sheep and goats on

modified Hayflick media.

Age Groups

Species group A group B group C Chi-sq P-value

Sheep 97/291

(33.3%)

86/309

(27.8%)

121/390

(31%)

2.16 0.34

Goat 131/275 149/345 153/370 2.59 0.27

(47.6%) (43.2%) (41.4%)

Statistical analysis (χ2) showed non-significant association (P > 0.05), df=2

A= 1-12 months, B=13-24 months, C= 25-36 months

66

Plate 3.1 Mycoplasma positive culture in modified Hayflick broth, showing

turbidity at day 5th

post incubation collected from lungs tissue of goats.

Plate 3.2 The culture showed turbidity for Mycoplasma growth for nasal

discharge taken from Dera Ismael Khan, southern zone.

67

Plate 3.3 Mycoplasma putrefaciens gross colonies after 2nd

day post inoculation on

modified Hayflick agar isolated from nasal swab of sheep.

Plate 3.4 Small tiny (0.2-0.3 mm) Mycoplasma capricolum subsp.

capripneumoniae (Mccp) visible gross colonies at day 7th

post

inoculation on modified Hayflick agar medium.

68

Plate 3.5 Gross colonies of Mycoplasma mycoides subsp. capri after 3 days post

incubation isolated from lungs tissue of goat in southern zone.

Plate 3.6 Mmc colonies with nipple like appearance on day 3rd

post incubation on

modified Hayflick agar at 10X, isolated from the lungs tissue of

naturally infected goats.

69

Plate 3.9 Typical Mccp colonies showing nipple like appearance with

pleomorphism on modified Hayflick agar, isolated from pleural fluids of

goat.

Plate 3.8 Mycoplasma cluster colonies with

nipple like appearance on day 5th

post incubation on modified Hay

flick agar at 4X, isolated from the

pleural fluid of naturally infected

goats

Plate 3.7 Mycoplasma capricolum subsp.

capripneumoniae colony with typical

fried egg colony on the 7th post

incubation in modified Hayflick agar

at 10X, isolated from the lungs tissue

of naturally infected goats.

70

Plate 3.10 M. putrefaciens colonies having pleomorphism on day 2nd

post

incubation isolated from nasal discharge of sheep at 10X.

Plate 3.11 Mycoplamsa mycoides subsp.capri colony with typical nipple like

appearance on day 3rd

post incubation in modified Hayflick agar isolated

from the nasal discharge of goats at 10X.

71

Plate 3.12 Modified Hayflick broth showing turbidity for pure growth of

Mycoplasma with 5% CO2 at 37° C on day 5th

post incubation. (Tube-1) growth recovered from trachea swab, (Tube-2) growth recovered from nasal

discharge, (Tube-3) growth recovered from pleural fluid, (Tube-4) negative control,

Hayflick media (Tube-5) growth recovered from lungs tissue

Plate 3.13 The tube in center showed turbidity for pure Mycoplasma growth after

3rd

passage collected from pleural fluid of goat, negative control tubes

having 0.5% phenol as an indicator.

1 2 3 4

5

72


All the positive culture showing Mycoplasma colonies on agar medium were

subjected to biochemical assay for the identification of pathogenic Mycoplasma species.

The biochemical assy consisted of glucose fermentation, serum digestion, casein

hydrolysis, tetrazolium reduction test (aerobically and anaerobically) and arginine

hydrolysis. The result of all the analysis for identification of Mm cluster and non-cluster

species is presented in Table 3.7. The results of biochemical assay revealed that Mm

cluster and other non-cluster species were prevalent in the animals of different climatic

zones of Khyber Pakhtunkhwa, Pakistan.

The detail of different biochemical assay is described as follow. In glucose

fermentation assay there was change in color in media was observed on day 4th

of

incubation. The color was changed from pinkish red to yellow. The result of positive

culture and control is presented in the Plate 3.14.

Plate 3.14 Result of Glucose fermentation test with yellow color represent positive

for Mmc while red color in center is negative control.

73

Table 3.7 Result of positive isolates identified through biochemical assay.

Mycoplasma species Positive Percentage Total

Mycoides cluster 450 22.7% 1980

Non cluster 142 7.1% 1980

For casein analysis a volume of 10uL of diluted Mycoplasma culture was placed

and allowed to spread on agar plates having casein, then incubated and observed on 5th

day. There was digestion of medium along the line of growth revealed Mycoplasma

growth. Casein digestion of the medium is shown in Plate 3.15.

Plate 3.15 Mycoplasma culture positive for casein hydrolysis test, showing growth

along with line of culture.

For serum digesion a 30 uL of diluted Mycoplasma culture was put and allowed

to spread from one side of the plate to the other. Plates were than incubated and

observed on day 5th

post incubation. The formation of channels along the line of growth

which filled with the gelatinous fluid consider positive for Mycoplasma (Plate 3.16).

74

Plate 3.16 Mycoplasma culture showing digestion of serum along the line of

growth of culture.

On arginin hydrolysis analysis medium change in color was observed on day 4th

of incubation. The color was changed from red to yellow consider positive for the

growth of Mycoplasma. The result of positive and control sample is presented in the

Plate 3.17 and 3.18.

Plate 3.17 Arginine hydrolysis test (aerobic) the tube on both side showed positive

result in center control tube.

75

Plate 3.18 Arginine hydrolysis test (anaerobic) tubes showing positive for

Mycoplasma culture and negative control in center.

In Tetrazolium test medium the color was change after 48 hours post

incubation. The medium color was changed to brick red indicating positive for

Mycoplasma growth. The result of positive and control sample is presented in Plate

3.19.

Plate 3.19. Tetrazolium reduction test, control tube (uninoculated) in the center with

Tetrazolium anaerobic positive in the right side and Tetrazolium aerobic

positive tube in the left.

76

3.7.3 Molecular identification and characterization of local isolates

The positive isolates were sub cultured three times in modified Hayflick broth

to obtain pure culture of Mycoplasma. All these pure culture were subjected to DNA

extraction for PCR analysis. Out of total sample 553 (27.92%) were confirmed as

Mycoplasma by using different set of primers. The zone wise distribution of confirmed

isolates were 247 (44.7%), 130 (23.5%) and 176 (31.8%) in northern, central and

southern zone, respectively. On analysis of data significant association (P ˂ 0.001) was

observed between the PCR confirmed isolates and climatic zone (Table 3.8). The

finding revealed that highest isolates were recovered from northern zone. Different

primers were used for the confirmation of mycoides clusters and non cluster species.

On gel electrophoresis of the PCR product an amplicon size of 548, 316, 194 and 540

bp were obtained that confirmed the Mycoplasma mycoides cluster (Mm cluster),

Mycoplasma capricolum sub-sp. capripneumoniae (Mccp), Mycoplasma mycoides

subsp. capri (Mmc) and Mycoplasma putrefaciens (Mp), respectively (Plate 3.20, 3.21,

3.22). However no Mycoplasma agalactiae was detected in the culture.

Table 3.8 PCR based confirmed isolates of Mycoplasma across the species in

different climatic zones.

Status

Zones

Total

Chi-sq P- value

Northern Central Southern

Positive Count 247/553 130/553 176/553 553/1980

52.3

0.001 % 44.7% 23.5% 31.8% 27.9%

660 animals were sampled in each climatic zone, total number of samples were 1980

77

Plate 3.20 PCR result of Mccp and Mycoplasma myocoides cluster with an

amplicon size of 316 and 548 bp in samples collected from goat. (A) M= 1Kb DNA ladder, C+ = positive control, samples=1, 2, 3.

(B) M= 1Kb DNA ladder, samples=1, 2, 3, N= negative control, C+ = positive

control.

Plate 3.21. PCR gel product of Mmc with an amplicon size of 194 bp, isolated from

lungs tissue of goat with respiratory syndrome. M= 1kb DNA ladder, C+ = positive control, Sample=1, 2, 3.

78

Plate 3.22 PCR products of M. putrefaciens with an amplicon size of 540 bp in

sample collected from animals, exhibiting signs of respiratory

complication. M=1 Kb DNA ladder, Sample=S1, S2, S3, N= negative control, C+ = positive control.

Out of total sample 395 (19.94%), 268 (13.5%), 109 (5.5%) and 158 (7.97%)

was confirmed as Mm cluster, Mmc, Mccp and Mp, respectively in samples collected

from small ruminants suspected for CCPP across different climatic zones (Table 3.9).

The overall prevalence of different pathogenic Mycoplasma species is presented in Fig

3.2. The finding showed that mycoides cluster, Mmc and Mccp were highly prevalent in

northern zone followed by southern zone of the province. However, the Mp were

showing high prevalence in southern zone followed by central and least in northern

zone (Fig 3.3). The prevalence of different spcies of Mycoplasma in sheep was 14.04%,

9.89%, 2.20% and 9.19% of Mm cluster, Mmc, Mccp and Mp, respectively. Similaraly,

the distribution of Mm cluster, Mmc, Mccp and Mp was 24.83%, 17.17%, 8.78% and

6.76% in goats suffering from respiratory syndrome (Fig. 3.4). On analysis of data by

Z-test a significant (P < 0.05) difference was found in the prevalence of pathogenic

species of Mycoplasma between the three climatic zones (Table 3.9). The prevalence of

Mm cluster and its proportion difference between the three different zones are displayed

in Table 3.10. It is evident that, in comparison to all three zones, maximum prevalence

of Mm cluster was found in northern zone followed by southern zone, while minimum

was recorded in central zone. On statistical analysis significantly (P < 0.05) lower

79

prevalence was recorded in the central zone by comparing with the northern and

southern zone. However, no significant difference was found between central and

southern zone of Khyber Pakhtunkhwa, Pakistan.

Table 3.9 Molecular identification and prevalence of pathogenic Mycoplasma

species from animals suspected for CCPP across different climatic

zones.

Pathogenic

Mycoplasma species Climatic Zones (n=1980)

Northern n=660 (%)

Central n=660 (%)

Southern n=660 (%)

Total PCR

confirmed

isolates

Molecular

prevalence (%)

Mm cluster 179 (27.12) 93(14.09) 123(18.6) 395 19.94

Mmc 106 (16.06) 71(10.75) 91(13.7) 268 13.5

Mccp 68 (10.30) 14(2.12) 27(4.09) 109 5.5

M. putrefaciens 33 (5) 43(6.51) 82(12.24) 158 7.97

Table 3.10 PCR result for confirmation of Mycoplasma Mycoides cluster and

proportional difference using Z- test analysis in different climatic zones.

Pairs Prop. Difference Z- value P- value

Northern vs Central 0.130 5.85 0.000***

Northern vs

Southern 0.085 3.67 0.002**

Central vs Southern -0.045 -3.23 0.026*

***Highly significant, * Significant, NS= Non significant

The prevalence of Mmc across the three zones was also analyzed and is

presented in Table 3.11. It is evident that in comparison to all three zones, maximum

prevalence was recorded in northern zone followed by southern zone, while minimum

prevalence was observed in central zone. On data analysis through Z- test, significantly

(P < 0.05) lower prevalence of Mmc was found in central as compared to northern

zone. However, no significant difference was found in the prevalence of Mmc in

between northern versus southern and central versus southern zone of Khyber

Pakhtunkhwa, Pakistan.

80

Table 3.11 PCR result for confirmation of Mmc and proportional difference using

Z- test analysis in different climatic zones

Pairs Prop.

Difference Z- value P- value

Northern vs

Central

0.053 2.83 0.005***

Northern vs

Southern

0.023 1.16 0.246

NS

Central vs

Southern

-0.030 -1.68 0.093

NS

***Highly significant, NS= Non significant

The proportional difference of PCR results for Mycoplasma capricolum subsp.

capripneumoniae (Mccp) in different zones was also analyzed and the findings are

displayed in Table 3.12. It is evident that in comparison to all three zones, maximum

prevalence was observed in northern followed by southern zone, while minimum was

recorded in central zone. On analysis of data significantly (P < 0.05) lower prevalence

was obsereved in central and southern versus northern zone.

Table 3.12 PCR result for confirmation of Mccp and proportional difference using

Z- test analysis in different climatic zones

Pairs Prop.

Difference Z- value P- value

Northern vs

Central

0.082 6.16 0.001***

Northern vs

Southern

0.062 4.37 0.000***

Central vs

Southern

-0.020 -2.060 0.053

NS


Similarly the proportional difference obtained from PCR results of Mycoplasma

putrefaciens (Mp) in three zones was also tested (Table 3.13). It is noted that maximum

prevalence was recorded in southern zone followed by central zone and northern zone.

On analysis of data revealed that the prevalence of M. putrefaciens was significantly (P

< 0.05) lower in northern as compared to southern zone. However, no significant

difference was found between northern and central zone of Khyber Pakhtunkhwa.

81

Table 3.13 PCR result for confirmation of M. putrefaciens and proportional

difference using Z- test analysis for different climatic zones


Northern vs Central -0.015 -1.18 0.237NS

Northern vs

Southern -0.074 -4.78 0.000***

Central vs Southern -0.059 -3.67 0.002***


The isolation of different species of Mycoplasma was carried out from different

sources of samples including pleural fluids, lungs tissue, nasal discharge and tracheal

swab. The isolates were confirmed through PCR by using selective set of primers. The

main aim of colllcetion of samples from diffrernt sources was to explore the best site

for Mycoplasma isolation and identification. The PCR results revealed that 63.6%,

58.5%, 25.5% and 21% isolates were recovered from pleural fluids, lungs tissue, nasal

discharge and tracheal swab, respectively (Table 3.14). The highest isolates were

recovered from pleural fluids followed by lungs tissue across the climatic zone (Fig

3.5). The data was analyzed by Z- test to check the level of significance between the

PCR result for confirmation of Mycoplasma and sources of samples obtained from

diseased animals (Table 3.15).

Table 3.14 Confirmation of Mycoplasma species by PCR from different clinical

samples of animals in three climatic zone

Sample Nasal=500 Tracheal 100 Lungs 49 Pleural=11 Total

Northern 174(34.8) 27 (27) 37 (75.5) 09 (81.8) 247

Central 89(17.8) 16 (16) 20 (40.8) 130

Southern 120(24) 20 (20) 29 (59.1) 07 (63.6) 176

Total

%

383/1500

(25.5)

63/300

(21)

86/147

(58.5)

21/33

(63.6)

553/1980

(27.92)

Total nasal discharge (1500), Tracheal swab (300), Lung tissue (147), Pleural fluid (33) across the three

climatic zones.

The proportional difference of PCR results for confirmation of CCPP from

different source of samples from diseased animals was also analyzed by Z- test. The

results revealed that maximum confirmed isolates of Mycoplasma were recovered from

pleural fluids followed by lungs tissue and minimum from tracheal swabs. On data

82

analysis it was predominant that significantly (P < 0.05) lower isolates were recovered

from nasal and tracheal swabs as compared to lungs and pleural fluids. However, non

significant difference was observed between the results of of Mycoplasma isolates of

nasal versus tracheal and lungs versus pleural fluids (Table 3.15).

Table 3.15 PCR result from different sources of samples and proportional

difference using Z-test analysis across three climatic zones


Nasal vs Tracheal 0.027 1.66 0.971NS

Nasal vs Lungs -0.33 -8.45 0.000***

Nasal vs Pleural fluids -0.382 -4.91 0.001***

Tracheal vs Lungs -0.75 -7.9 0.000***

Tracheal vs Pleural fluids -0.42 -5.35 0.002**

Lungs vs Pleural fluids -0.31 -0.54 0.592NS


Fig 3.2 Overall molecular prevalence (% age) of different pathogenic

Mycoplasma species in small ruminants across three climatic zones.

83

Fig 3.3. Overall molecular prevalence (% age) of pathogenic Mycoplasma species in

different climatic zones.

Fig 3.4. Comparative species based prevalence of pathogenic Mycoplasma

species in small ruminants.

84

Fig 3.5 PCR confirmed Mycoplasma isolates recovered from different source of

clinical samples.

3.7.4 Homology and phylogenetic analysis

The PCR confirmed local isolates were processed for sequencing and the

sequence of the PCR product obtained through specie specific primers showed

maximum sequence homology 99% of 16S-rRNA gene of Mccp with the strains of

neighboring countries. The phylogenetic tree was constructed by using software MEGA

version 7.2 and compared with 08 available sequences in NCBI gene data bank. The

constructed tree indicated that the local isolated field strain is different from the strains

of USA and France but having close similaraties with the strain of neighbour countries

like India and China (Fig 3.6). Similarly on sequencing of local isolates of Mmc it was

showed maximum sequence homology with Mm LC strain of Switzerland (Fig. 3.7).

85

Fig 3.6 Phylogenetic relationship of the Mycoplasma capricolum subsp.

capripneumoniae sequence obtained (Swat, Pakistan) comparing with

other eight isolates available sequences in NCBI. Sequences of the

isolates were downloaded from NCBI and were aligned through Bio Edit

multiple alignment. The phylogenetic tree was constructed by neighbor-

joining algorithm using the software MEGA version 7.2.

Fig 3.7 Phylogenetic relationship of the Mycoplasma mycoides subsp. capri

sequence obtained (Kamal & Sadique, Peshawar, Pakistan) comparing

with other ten isolates available sequences in NCBI. Sequences of the

isolates were ownloaded from NCBI and were aligned through Bio Edit



gi|83283139|gb|CP000123.1|:109457-109726: USA

gi|672893522|emb|LM995445.1|:124934-125204: France

gi|675241189|emb|LN515398.1|:124882-125152: Switzerland

gi|677282260|emb|LN515399.1|:124945-125214: Switzerland

gi|755906250|gb|CP006959.1|:124992-125261: China

gi|531624|emb|Z33099.1|:697-967: USA

gi|45511562|gb|AY529462.1|:4935-5205: France

gi|675153077|gb|KM000056.1|: India

1955120: Swat, KP, Pakistan

86

On sequencing of 16S rRNA gene of the local isolates of Mp it was indicated

that it showed homology with the strain of USA (Fig. 3.8).

Fig 3.8 Phylogenetic relationship of the Mycoplasma putrefaciens sequence

obtained (Kamal and Sadique, Kohat, Pakistan) comparing with other

three isolates available sequences in NCBI. Sequences of the isolates

were downloaded from NCBI and were aligned through BioEdit



87

3.8 Discussion

Small ruminant population plays significant role in the world economy by

contributing the 2nd

largest number in the total livestock population (FAO, 2015). In

Pakistan, small ruminants are contributing largest number of about 102 million to the

total livestock population of 191 million (Economic survey, 2016-17). Small ruminant

provides milk and quality meat for consumers and raw materials in the form of good

quality wool, hair and skin to the textile and leather industries. In developing countries

the majority of livestock owners belong to lower class and they generate their income

from animal resources (Abbas et al., 2013). Among small ruminants, the goats gain

importance in the rural economy of Pakistan by providing milk to the poor community

where the cattle are not manage easily. Due to this unique characteristic of goats it is

also called poor man cow (Rahman et al., 2003). However, this huge population of

ruminant facing various challenges in the form of intense hot and cold climate, shortage

of feedstuffs, poor husbandry practices and various infectious diseases. Amongst

various infectious diseases, the mycoplasmosis is a major threat to small ruminant

population causing high morbidity and mortality. Mycoplasmosis is multi systemic

disease referred to the infection collectively caused by various pathogenic Mycoplasma

species. The most important pathogenic Mycoplasma infections are consisted of avian

mycoplasmosis, bovine mycoplasmosis and caprine mycoplasmosis. Caprine

mycoplasmosis is prevalent throughout the world particularly in the developing country

of south East Asia and Africa and inflicting heavy economic losses to the small

ruminant industries (Tigga et al., 2014; Ongor et al., 2011; Srivastava et al., 2010).

This important disease is widely prevalent in Pakistan and causing huge economic

losses in the northern and southern regions of the country (Banaras et al., 2016;

Shahzad et al., 2013; Sadique et al., 2012).

Ruminant mycoplasmosis is caused by both Mm cluster and non-cluters

pathogenic species infecting different system of the host. It causes direct losses in

serious outbreak in the form of high mortality, decrease milk and meat production,

reduce carcass weight and indirect losses including treatment, vaccination and

managemental cost. Due to its high pathogenic nature and causing high economic

losses to the livestock industry it is characterized as list-B disease (OIE, 2014). The

country suffering from the outbreak of this disease faces great hardship in export of

88

meat and its product due to trade embargo by the world regime. In Pakistan the

ruminant mycoplasmosis is known by one of the most important disease called

contagious caprine pleuropneumonia (CCPP) with a long history of causing havoc in

the farming community (Shahzad et al., 2013; Sadique et al., 2012). The disease is

widely distributed in the country lead to several outbreaks and causes heavy loses to

small ruminant population (Banaras et al., 2016; Awan et al., 2009; Rahman et al.,

2006).

The CCPP is caused by six different pathogenic Mycoplasma species called as

mycoides cluster (Manso-Silvan et al., 2009). Some other non-cluster pathogenic

species like M. putrefaciens, M. ovipneumoniae and M. agalactiae are also reported in

mixed type of infections involving different systems (Banaras et al., 2016; Ejaz et al.,

2015). In Pakistan a single specie vaccine is available and used as prophylactic

measures throughout the country. However, inspite of vaccination regular outbreaks of

the disease have been reported (Sadique et al., 2012). The failure of single specie

vaccine might be due prevalence of other members of Mm cluster and non-cluster

species. The other species like Mccp was 1st time reported in southern Pakistan by

Awan et al. (2010). Later on the same specie was also confirmed in the northern and

central regions of country (Shahzad et al., 2016; Peyraud et al., 2014). The present

study was designed to investigate the various pathogenic Mycoplasma species in the

study area and adopt different therapeutic and prophylactic measures to effectively

control the ruminant mycoplasmosis in small ruminant population.

For identification of the exact specie of Mycoplasma different diagnostic

techniques are used worldwide with different outcomes. The isolation of pathogen is

essential to identify and characterize it through morphological and genomic studies.

The study was conducted in three different climatic zones of Khyber Pakhtunkhwa,

Pakistan to investigate the prevalence of pathogenic Mycoplasma species mainly

responsible for mycoplasmosis especially the contagious caprine pleuropneumonia

(CCPP). For isolation of Mycoplasma, different media are being used with various

success rates. In the present study, samples were taken in transport media and were

grown on modified Hayflick media for culturing and isolation of the causative agent.

Out of total 1980 samples, 737 (37.22%) showed mass turbidity and whirling

movement in Hayflick broth, while 667 (33.68%) were positive for the growth of a

89

charateristic colonies across different climatic zones. The Hayflick media was used by

many researchers for the isolation of Mycoplasma (Ongor et al., 2011). The

Mycoplasma being a fastidious organism difficult to grow on ordinary media and need

special media enriched with cholesterol and glucose (Nicholas et al., 2003; Waites and

Robinson, 1999). The Hayflick media consisted of horse serum, sodium pyruvate and

glucose, which provide the nutritive requirements of the Mycoplasma for obtaining

optimum growth (Woubit et al., 2007; Thiaucourt et al., 1992).

The bacterial contamination and fungus growth is the common hazard restricted

by adding thallium acetate and penicillin in the culture media. In the present study

excellent and contaminated free culture were obtained by adding the above reagents in

the media. The positive samples showed mass turbidity and whirling movement in

broth as also reported by OIE, (2014). The positive growth on broth was sub-cultured

on agar medium which produced typical fried egg and nipple like colonies on day 7 to

9th

post incubation under condition of 5% CO2 at 37 °C. These positive isolates were

re-cultured 3-5 times to obtained maximum growth and pure culture. Such observations

and results were also reported previously (OIE, 2014; Nicholas et al., 2009; Thiaucourt

et al., 1992). The series of culturing increase the adaptive capability of Mycoplasma

that turned to grow fast in short period. After 5th

passage of culture typical Mycoplasma

colonies were obtained on day 2nd

, 3rd

and 7th

post incubation representing the growth

of M. putrefaciens (Mp), Mmc and Mccp respectively. The findings revealed that Mp

and Mmc grow fast while Mccp was slow growing organism produced characteristic

colonies late as compared with other pathogenic Mycoplasma species. These findings

are supported by the results of Schumacher et al. (2011) who reported that Mmc

produced typical colonies at 48 hours post incubation. These findings are justified by

the results that the Mccp colonies were observed on day 7th

of post incubation (Kabir

and Bari, 2015; Noah et al., 2011). In another study, characteristics typical colonies of

Mccp were observed on day 5-6th

post incubation in agar media (Houshaymi et al.,

2002). Similarly, Mmc colonies with typical fried egg appearance having size of 1-2mm

were also observed in solid media (Wang et al., 2014).

M. ovipneumoniae has similar pattern of growth and obtained maximum

colonies on 4-6th

day post incubation (Gonçalves et al., 2010). The slow growth pattern

of Mccp is supported by several researchers (Azevedo et al., 2006; Hernandez et al.,

90

2006). It is observed that growth of Mccp is obtained late in incubation, however

prolong incubation also increases the chances of contamination. To overcome this issue

of contamination it is recommended that after obtaining the 1st culture it must be

processed through a series of passages to obtain a pure culture. The findings of this

study suggested that most of the pathogenic Mycoplasma species produced colonies

between 2 to 7 days post incubation in Hayflick media.

Different conventional and advanced techniques are used for preliminary

identification and confirmation of exact species of Mycoplasma. The morphological

appearance cannot confirm the exact species of Mycoplasma because of its

pleomorphic nature. Majority of Mycoplasma species attained a typical fried egg or

nipple like colony with different sizes. However, different biochemical tests comprising

glucose fermentation, gigitonin sensitivity, serum digestion, casein hydrolysis,

tetrazolium reduction test are in practice for identification of some of the important

species with varying degree of success (OIE, 2004; Mekuria et al., 2008; Eshetu et al.,

2007; Adehan et al. 2006). Inspite of its limitation the biochemical test were used for

preliminary screening and identification of species of Mycoplasma in small ruminants

(Nicholas et al., 2008). In the present study out of total samples, 592 (29.8%) were

identified as Mm cluster and non- cluster species.

All the local isolates were positive for serum digestion, glucose fermentation

test, casein digestion test and tetrazolium reduction test while negative for arginine

hydrolysis test. The result revealed the Mm cluster possibly positive for Mccp,

MmmLC, Mmc and M. putrefaciens and negative for M arginine, M. agalactiae and

Mcc. The findings of the study are supported by the results of several researchers

(Awan et al., 2009; Nicholas et al., 2008; Mondal et al., 2004). Arginine hydrolysis is

specific for Mcc among the other members of Mm cluster. The statement is justified by

the findings of Nicholas and colleagues (Nicholas et al., 2008). None of the isolates

showed positive result for arginine hydrolysis test that exclude the species of M.

arginini. The serum digestion test can differentiate the mycoides clusters from non-

clusters species of Mycoplasma of small ruminants. All members of Mm cluster digest

serum except Mccp is justified by the guidelines of (OIE, 2014). The failure of these

biochemical tests are testified by the fact that some of these test show same result for

more than two species of mycoides clusters. The findings of this study revealed that

91

biochemical tests cannot confirm the exact species of Mycoplasma clusters and non-

clusters.

The failure of biochemical tests to identify the exact species of Mycoplasma

compelled the researcher to explore advance techniques for accurate and specific

diagnosis. The DNA extraction and PCR analysis make it possible to characterize the

organism and enable the researcher to design strategies for effective control of the

disease. The PCR provide rapid, accurate and specific diagnosis of a disease

(Dominique et al., 2004). It has been observed that mixed type infection occurs in small

ruminants during outbreak of CCPP that can be answered only through molecular

characterization of the agents. The indiscriminate use of antibiotics in the field

condition and sharing of epitopes of Mycoplasma clusters restrict the use of

conventional methods like isolation and serological analysis. The introduction and

development of specie specific primers has enabled the application of this advance

technique to apply directly on clinical materials like nasal swabs and tissue samples

(Lorenzon et al., 2008; McAuliffe et al., 2005). In Pakistan, the conventional methods

are being used for identification of mycoplasmosis in small ruminants, which is the

main hindrance in control of this devastating disease. In such cases PCR is the only

choice which overcomes the cross-reactivity and variability that usually occurred in

biochemical and serological analysis. PCR can provide the opportunity and play

significant role in the surveillance of mycoplasmosis. The PCR has been used

successfully for preliminary identification and characterization of different pathogenic

Mycoplasma species by several researchers (Sadique et al., 2012; Manso-Silvan et al.,

2009; Hotzel et al., 1996). In the present study out of the total samples, 553 (27.92%)

were confirmed as Mm clusters and non-cluster specie, the M. putrefaciens, through

PCR. The highest prevalence (32.12%) was recorded in northern followed by southern

(31%) and least in central zone (20.6%) of Khyber Pakhtunkhwa, Pakistan. This

variation in the prevalence of disease across the three zones might be due to difference

in climatic condition, husbandry practices, stock density, pastoral practices and porous

boundaries with neighboring countries. Similar study was also conducted in eight

different districts of Afar region Ethiopia and revealed 10-36% prevalance of CCPP in

goats (Regassa et al., 2010).

92

The northern zone is densely goat populated area, nomadic in nature and harsh

cold climatic conditions that predispose the animal to immunosuppression, resultantly

succumb to Mycoplasma infection. The findings are supported by the facts that

management and production system, agro-ecological, population density, carrier

animals in the area play significant role in the magnitude of disease among the small

ruminants population (Sherif et al., 2012). The northern zone is popular for snow fall

during winter accompanied by extreme cold and the lower temperature was recorded

between 5 to -9 °C in various areas. The extreme cold and intense climatic conditions

and nomadic husbandry practices of farmer produce severe stress which effect livestock

especially the small ruminant population. The statement is an agreement with the

findings that humidity, temperature and extreme cold weather are the risk factors for

sheep pneumonia (Knowles et al., 1995). These results were further strengthen by the

observations that indictated the associated risk factor including age, husbandry system,

flock size and agro-climatic conditions of the area influence the prevalence of CCPP

(Yousuf et al., 2012).

The nomads across the country play important role in spreading of disease

among different climatic zones. The regular movements of animals from one place to

another cause stress, which lead to decrease immune status and make the animals

vulnerable to multiple infections. The other possible reasons of high prevalence of

disease in the northern zone are mountainous area, heavy snow fall and prolong rainy

season. The seasonal outbreaks of CCPP claimed by the farmers in the study area

during onset of rainy season are also agreed by the previous reports in southern

Ethiopia (Mekuria et al., 2008). Secondly, nomads frequently used different routes of

northern zone for shifting their animals to alpine pastures from neighboring districts.

These practices play significant role in the dissemination and propagation of this lethal

pathogen among the small ruminant population. During this displacement in search of

posture provide an opportunity for chronic carrier of CCPP to disseminate and transmit

the infection leads to emerging and reemerging of diseases. This statement is supported

by the findings that such type of practices play significant role in ruminant

mycoplasmosis (Sadique et al., 2012; Mekuria et al., 2008; Gelagay et al., 2007).

Similar findings were also reported in various studies that animal movement and

grazing habit can contribute in the transmission of CCPP (Bekele et al., 2011; Gelagay

et al., 2007).

93

The southern zone of the province is comprised by long built of terrestrial and

sandy Plato with low rain fall, hot and humid condition. During intense cold season the

nomads migrate their animals from northern to the southern part of the province that

carries the carrier animals responsible for transmission of disease. Scarcity of fodder

and intense climatic condition of the southern zone are the contributing factors of poor

health status and immunosuppression that prone the animal to infection. High intensity

of disease during harsh and cold climatic conditions has also been reported by Mekuria

et al. (2008). The findings supported by the facts that high prevalence of

mycoplasmosis was recorded in hilly areas of Pakistan (Shahzad et al., 2012).

Several pathogenic Mycoplasma species are responsible for mycoplasmosis in

small ruminants. The CCPP is highly contagious disease of small ruminants caused by

Mycoplasma clusters comprised of six different species. However mixed infection by

non-cluster pathogenic Mycoplasma species may be notice. The classical form of CCPP

is caused by mycoides clusters associated with respiratory syndromes and other multi

systemic involvement (Samiullah, 2013; Sadique et al., 2012; Nicholas et al., 2008;

Laura et al., 2006). However, the recent finding revealed that the CCPP is caused only

by Mccp restricted to thoracic cavity (OIE, 2014). The non-cluster species like M.

agalactiae, M. putrefaciens are commonly occurring along with mycoides clusters

associated with multiple complications. In Pakistan the Mmc was consider responsible

for CCPP in small ruminants (Sadique et al., 2012, Waseem et al., 2012; Rahman et al.,

2003). However, later on some other species of Mycoplasma clusters like Mccp were

reported (Peyraud et al., 2014; Awan et al., 2010). In the current study the overall

prevalence of different pathogenic Mycoplasma species were Mm cluster (19.94%),

Mmc (13.53%), Mccp (5.5%) and M. putrefaciens (7.97%). Mycoplasma agalactiae

was not confirmed in the present study. The milk and synovial fluids samples were not

included in the study that may the possible reason for failure of M. agalactiae isolation

and confirmation. It is justified by the facts that M. agalactiae has tissue tropism to

mammary and joints fluids are supported by the previous findings (Abtin et al., 2013;

Azevedo et al., 2006). Among all the confirmed isolates the Mmc was highest in

proportion and its distribution pattern was 16%, 10.75% and 13.7% in northern, central

and southern zones, respectively. The results are in agreement with the findings of

several researchers that Mmc is widely prevalent pathogenic specie across the country

(Banaras et al., 2016; Shahzad et al., 2012; Sadique et al., 2012). Similarly, Mmc was

isolated from goats in many parts of the world by several researchers (Wang et al.,

2014; Schumacher et al., 2011; Laura et al., 2006; Mondal et al., 2004; Greco et al.,

2001; DaMassa et al., 1992). On DNA sequencing the local isolates showed maximum

94

sequence homology of 16S-rRNA gene of Mmc with the Mmc and MmLC strains of

other countries. The phylogenetic tree was constructed by using software MEGA

version 7.2 and compared with 10 available sequences in NCBI gene data bank. The

sequence results (Kamal and Sadique) reveled that local strains showed similaraties

with MmLC strain of Switzerland and distance from Mmc and MmLC strain of France.

Mycoplasma putrefaciens (Mp) was the second most prevalent pathogenic

specie in the study area. It was highly prevalent in the southern zone (12.4%) followed

by central (6.5%) and least in northern zone (5%) in the study area. It is justified by the

facts that sheep are commonly reared in the southern part of the country and provide

raw material in the shape of wool to the wool industries. The statement is justified by

the findings of prevalence of Mp (6.7%) in the sheep in southern part of Pakistan

(Awan et al., 2009). These results are further supported by another study which claims

5% prevalence of Mp in sheep in Quetta, Baluchistan the southern region (Banaras et

al., 2016). It was observed that sheep were more susceptible (9.19%) to infection of this

organism as compared to goats (6.76%). Similarly, higher prevalence of Mp was

recorded in sheep (5%) followed by goats (0.5%) in Khanozai District Pishin,

Baluchistan (Ejaz et al., 2015). Very limited published data are available on molecular

prevalence of Mp in the study area as well as in other parts of the country. The wide

spread prevalence of this pathogenic specie is 1st time reported that will provide base

line data for researchers to devise strategies for effective control of mycoplasmosis in

the small ruminants. The gel product of Mp with amplicon size of 540 bp was

processed for sequencing. BLAST result was obtained through NCBI which shows

homology with different isolates. Phylogenetic relationship of the Mp sequence

obtained (Kamal and Sadique, Kohat, KP, Pakistan) was compared with other three

available sequences in NCBI. The phylogenetic tree was constructed that indicated the

local isolates of Mp showed homology with the strains of USA.

The Mccp is chiefly responsible for CCPP causing high morbidity and mortality

in small ruminants across the world (OIE, 2014). This species was 1st time isolated and

confirmed in the study area and negate the previous report of presence of only one

specie i.e. Mmc. The overall prevalence of the Mccp was confirmed 5.5% with zonal

distribution of 10.3%, 2.12% and 4.1% in northern, central and southern zones,

respectively. These findings are in agreement with the prevalence of Mccp in

Baluchistan, Pakistan by Awan et al. (2010). It is further justified by findings of an

95

international collaborative study that confirmed the sero-prevalence of CCPP caused by

Mccp was 2.7% and 44.2% in Gilgit and Diamer districts of Northern Pakistan

(Peyraud et al., 2014). Similar findings were also reported about sero-prevalence of

Mccp 8.52% in different District of Punjab, Pakistan (Shahzad et al., 2016). This

species of Mycoplasma is worldwide in distribution predominantly present in south

Asia, central Asia, North and South Africa, Middle East and Europe (Atim et al., 2016;

Peyraud et al., 2014; OIE, 2014; Abbas et al., 2013; Sherif et al., 2012; Manso-Silvan

et al., 2011; Noah et al., 2011; Chu et al., 2011; Ingle et al., 2008; Adehan et al., 2006).

On sequencing of the amplified DNA of local isolates (Swat, KP. Pakistan) showed

maximum sequence homology 99% of 16S-rRNA gene of Mccp with the strains of

neighbor countries. The phylogenetic tree was constructed by using software MEGA

version 7.2 and compared with eight available sequences in NCBI gene data bank. The


of USA and France but closely related with the strain of neighbor countries like India

and China. The sequence results reveled that strains of neighbor countries resemble

similar genetic structure and possibly may cause similar manifestation.

The specimen collection from diseases animal play an important role for initial

isolation and identification of the primary causative agent. The Mycoplasma has

selective in tissue tropism and the successful isolation is possible by collecting samples

from definite site of the host (Whitford, 1994). Furthermore different species of

Mycoplasma has predilection site of host for multiplication and colonization that

determine the success of isolation and culturing. The CCPP caused by Mm cluster

mainly infected the respiratory tract of the small ruminant lead to respiratory syndrome.

The respiratory complication especially pneumonia develops when antibacterial

defense mechanism of lungs breaks down and bacterial proliferation occur (Bruere et

al., 2002). In this disease the preliminary isolation are carried out from various site of

respiratory tract with different outcome (OIE, 2014; Zinka et al., 2013; Thiaucourt and

Boleske 1996). In the present study four different sites were selected consisted of nasal

discharge, tracheal swab, lungs tissue and pleural fluids for sample collection to

investigate and explore the best site for obtaining maximum growth. The highest

isolation were confirmed from pleural fluids (63.6%) followed by lungs tissue (58.5%),

nasal discharge (25.5%) and tracheal swab (21%). It revealed that the pathogens mainly

target the respiratory tract of the host and maximum isolates were recovered from

96

pleural fluids and lungs that indicated heavy load of pathogen. Similar observations

were made in a study that maximum isolates (83.78%) of Mycoplasma were recovered

and confirmed from pleural fluids (Noah et al., 2011). These findings are supported by

the fact that the Mycoplasma having tissue tropism to lung tissues and lower respiratory

tract, where the receptors for its antigenic epitope are abundantly present. The antigenic

protein having lipoglycan that stimulate the acute inflammatory response in the host

tissues leads to maximum exudation and pleural effusion (Rosendal, 1993).

The Mmc have the characteristic to invade the lower respiratory tract mainly the

lungs tissue. The results are supported by the findings of previous studies (Sadique et

al., 2012; Awan et al., 2010; Thiaucourt et al., 1994), who also reported maximum

Mmc isolation form lung tissues. However, in case of Mccp infection maximum

isolation was confirmed form pleural fluids. These findings are strongly supported by

several researches (Samiullah, 2013; Noah et al., 2011; Adehan et al., 2006; Nicholas

et al., 2002). The cluster and non-cluster specie like Mp and M. agalactiae were also

isolated from nasal discharges and tracheal fluids. Similar finding indicated that Mmc

was confirmed from milk, ocular and nasal discharge by using multiplex-PCR (Greco

et al., 2001). Similarly, several other pathogenic Mycoplasma species like M.

ovipneumoniae (16.9%) were isolated from nasal discharge from small ruminants in

Bosnia and Herzegovina (Zinka et al., 2013). In chronic cases the disease spread to

multiple organs due to systemic manifestation. The isolation and identification of

different Mycoplasma species from nasal and tracheal swabs were also previously

reported (Kabir and Bari, 2015; Kumar et al., 2011). The results are further supported

by the findings that M. ovipneumoniae (29.5%) were isolated form nasal discharge of

goats in Eastern Turkey (Ongor et al., 2011). The findings are in accordance with the

results that isolated Mm cluster and Mp from nasal swab samples collected from sheep

in Baluchistan, Pakistan (Ejaz et al., 2015). Similarly, the isolation of Mycoplasma

from tracheal and nasal discharge were also reported by several researchers (Banaras et

al., 2016; Liljander et al., 2015; Sadique et al., 2012; Nicholas, 2002).

The isolation from upper respiratory tract is justified by the facts that as the

disease is progressed and get chronic the purulent pulmonary discharge containing the

pathogens come along with coughing to upper respiratory tract. Therefore, the tracheal

secretion and nasal discharge containing heavy load of pathogen in advance stage of

97

disease. Secondly, the upper respiratory tract has also receptor for Mycoplasma

adherence and subsequent proliferation. The presence of pathogens in the upper

respiratory tract and nasal passage are helpful for the isolation of Mycoplasma from

living animals. The nasal discharge is an important clinical signs showing involvement

of respiratory system in mycoplasmosis and provide easy site for sample collection

from live animals. Although maximum isolation was recovered from lungs and pleural

fluid but it could only be possible from dead animal at postmortem. It was concluded

from the present findings that all the three species were successfully isolated from nasal

discharge and tracheal fluids of the infected animals. However, maximum growth of

Mmc and Mccp were recovered from lung tissues and pleural fluids of the dead

animals.

CCPP is primarily the disease of goats but it can also infect sheep and wild

ruminants (Arif et al., 2007; Madanat et al., 2001). Mostly the small ruminants are kept

together in small and large herds in the developing countries providing an equal

opportunity of the disease transmission among the different species. In Pakistan a

normal herd consisted of sheep and goats of different age, sex and breed. In the country

most farmers are adapted mixed farming and keep sheep and goats together that

increases the chances of dissemination of disease among the two species. It was

revealed in the present study that the CCPP is highly prevalent in goats (58.75%) as

compared with sheep (41.24%). The findings are supported by the facts that

mycoplasmosis is highly prevalent in goats (65%) and 35% in sheep (Al-Momani et al.,

2006). Both sexes are susceptible to CCPP however high morbidity and mortality is

reported in female animals due to lactation and pregnancy stress. The results of the

present study indicated that prevalence of disease was 39% in female and 30.33% in

male. Similar observations were made in a study that the prevalence of CCPP was high

in female (33.03%) than in male (29.2%) animals (Sherif et al., 2012). These findings

were further justified by the work that high prevalence of mycoplasmosis was recorded

in female (16.9 %) as compared to male (8.4 %) in Spanish ibex of Spain (Verbisck-

Bucker et al., 2008). In another study it was recorded that female (16.1%) goats were

more affected than male (10.7%) bucks (Abegunde et al., 1981). The high prevalence

of disease in female animals may be due to various factors including lactation, gestation

and estrus cycle responsible for development of stress that in turn targeted the immune

mechanism and predispose the animal to opportunistic pathogens like Mycoplasma.

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Pathogenic Mycoplasma species are normal inhabitant of respiratory system and target

easily the immune compromised animals (Blood et al., 2007). However, the results are

contradictory with the findings of Yousuf et al. (2012), who observed low prevalence

of CCPP in female goats (4.67%) as compared with bucks (5.32%). Similarly in

another study high prevalence was recorded in bucks (24.08%) as compared to female

goats (6.66%) in Afar region of Ethiopia (Regassa et al., 2010). However these findings

were not in accordance to the study conducted in Tanzania and in Ethiopia, which

documented that sex has not affected the epidemiology of CCPP (Yousuf et al., 2012;

Mekuria and Asmare, 2010; Hadush et al., 2009; Kusiluka et al., 2000). All these

mentioned findings revealed that variation and prevalence of disease based on sex may

be due to male female ratio in the herd, immune status, herd size, biosecurity and

different locality.

All the ages are susceptible to caprine mycoplasmosis; however, young kids are

severely infected with high morbidity and mortality. The lymphoid organs are in

growing stage in the young kids, which are unable to encounter effectively the invading

pathogens. As the animal grows they are exposed and experienced to variety of

pathogens that ultimately developed stronger immune system in the host. The

secondary lymphoid organs are also developed with age and make the host capable to

encounter the invading pathogen effectively. In the present study high prevalence of

disease was recorded in young kids up to one year of age of both species of small

ruminants consisting (33.3%) in sheep and (47.6%) in goats. These results are

supported by the finding of the study that high seroprevalence of CCPP caused by

Mccp was recorded in goat kids of 1-180 days of age (Shahzad et al., 2016). Similarly

the statement was further supported by the findings of another study that high

prevalence upto 90% of disease caused by Mmc specie was reported in goat kids up to

four months of age (Nascimento et al., 1986). These findings are further justified by the

results of many researches who reported that age contributes significant role in

prevalence of mycoplasmosis among different ages (Sherif et al., 2012; Tesfaye et al.,

2012; Regassa et al., 2010).

It was recorded by Verbisck-Bucker et al. (2008) that M. agalactiae causes

severe infection in young Spanish ibex. In the country the farmers keeping animals of

various age groups together for short and long period. It is well established that CCPP

99

is highly contagious in nature and can be easily spread by direct contacts, aerosol and

by contaminated feed and drinking water among different age groups. The old animals

that recovered from acute phase of infection become chronic carrier for the rest of life

and serve as constant source of spreading of disease (Mekuria et al., 2008; Gelagay et

al., 2007; Thiaucourt et al., 1996). It is justified the communal grazing, watering and

marketing play important role in the spreading of infection from infected to healthy

animals. Uncontrolled and regular movement of small ruminants due to seasonal

grazing practices, marketing and sacrifice festival are some common practices that lead

to dissemination of disease among different age groups. However, some other findings

are not agreed with the results of present study, which and were in the view that high

prevalence of CCPP was recorded 30% in age group above four year than 16.93%

below four year in goats (Regassa et al., 2010). Similarly, it was reported that high

prevalence of disease was recorded in old and adult age than young goats (Sherif et al.,

2012). This contradiction might be due to husbandry practices, small herd size, agro-

ecological zone, spices of the Mycoplasma and immune status of the infected animals.

3.9 Conclusions

The three pathogenic Mycoplasma species Mmc, Mccp, and M. putrefaciens

were 1st time isolated and confirmed in KP, Pakistan.

The results revealed that mycoplasmosis was highly prevalent in goats (58.75%)

as compared to sheep (41.24%).

The young kids were more susceptible to the disease as compared with adult

animals.

High prevalence of mycoplasmosis was recorded in female (39%) than in male

(30.3%) in suspected diseased animals.

High prevalence (43%) of CCPP was recorded in northern zone followed by

southern zone (34.6%) of KP.

The highest isolates of Mycoplasma were recovered from pleural fluids (63.6%)

followed by lung tissues (58.5%) and nasal discharges (25.5%) from animals

showing signs of respiratory syndrome.

100

The phylogenetic study of Mccp revealed that it was different from the strains of USA

and France, however having close similarity with the strain of neighboring countries

such as India and China. The phylogenetic analysis of Mmc and Mp also revealed close

similarities with the strains of Switzerland and USA, respectively.

3.10 Recommendations

1. Farmers are needed to improve managemental practices to reduce climatic stress

in extreme cold areas especially northern regions of the country.

2. Phylogentic analyses of the whole genome of the local isolates are needed to be

conducted for any mutational changes.

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IV. STUDY -2

STUDY THE PATHOGENESIS OF CCPP IN NATURALLY

INFECTED SMALL RUMINANTS OF KHYBER PAKHTUNKHWA

102

ABSTRACT

Ruminant mycoplasmosis is an important contagious disease of small

ruminants, which is causing respiratory syndrome and multi-systemic manifestation.

Different pathogenic species of Mycoplasma mycoides cluster and non-cluster are

responsible for the disease with severe clinico-pathological outcomes. In the current

study, a total of 1800 diseased animals were surveyed for recording of the clinico-

pathological picture of diseases in naturally infected sheep and goats. Similarly, 180

dead animals were examined on postmortem examination for gross and

histopathological study. The clinical manifestation of disease revealed that respiratory

signs were more prominent in diseased animals followed by other systemic

involvement. Out of total examined animals, pneumonia was recorded in (61.55%)

followed by pyrexia (58.2%), coughing (56.83%), watery nasal discharge (52.22%) and

lacrimation (40.77%). The other clinical findings consisted of diarrhoea (22.33%),

mastitis (3%), nervous signs (1.6%) and abortion (1.27%). The overall mortality was

recorded (15.72%) in infected animal population. Pathomorphological study revealed

that majority of the animals exhibited lesions in the respiratory system followed by

gastero-intestinal tract, urinary and nervous system. The most frequent lesions were

recorded in the lungs (53.88%), followed by trachea (37.7%) and pleural effusion

(18.33%). The multisystemic involvement of the disease was the frequent feature in

lesions distribution comprising of nephritis (18.33%), hepatitis (17.22%), enteritis

(13.33%) and pericarditis (12.2%). The histopathological findings of lungs revealed

atelectasis, sloughing of alveoli, thickening of interlobular sepat and extensive

leukocuytic infilteration. The kidneys, liver, spleen and intestine showed necrosis and

accumulation of infilmatory cells. The gross and histopathological scoring of the

disease revealed that maximum lesions were observed in lungs and trachea followed by

liver, kidneys, spleen, intestine and brain. It was concluded from the findings that most

frequent signs were observed in the form of respiratory distress, coughing, nasal

discharge and pyrexia. The gross and histopathological lesions scoring revealed the

high pathogenic nature of infection and multisystemic involvement justified the

prevalence of several pathogenic Mycoplasma species in study area.

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4.1 Introduction

Ruminant mycoplasmosis is an important contagious disease of small ruminants

causing respiratory syndrome and multi-systemic manifestation. Different pathogenic

species of Mm cluster and non-cluster are responsible for the disease with severe

clinico-pathological outcomes. Several pathogenic members of Mm cluster and non-

cluster are responsible for disease development in various tissues of the host. The Mccp

and Mmc having tissue tropism to lungs tissue and the clinical findings and lesions are

restricted to respiratory tissues. Some other cluster member like MmLC, Mcc, M.

agalactiae and M. putrefaciens produced lesion in the respiratory tissues accompanied

by multi-systemic involvement. In many outbreaks mixed infection also reported with

the involvement of cluster and non-cluster Mycoplasma species.

The major syndrome associated with pathogenic Mycoplasma species is

pneumonia in small ruminants (Hernandez et al., 2006). The pathogenic species of the

Mm cluster comprising of six different members mainly responsible for disease in small

ruminant called CCPP (Laura et al., 2006). Some other non- cluster pathogenic species

like M. putrefaciens, M. ovipneumoniae and M. agalactiae are also reported in mixed

type of infections involving different system. This multi-systemic and typical

manifestation is called MAKePS (Mastitis, Arthritis, Keratitis, Pneumonia and

septicemia) syndromes (Egwua et al., 2001; Thiaucourt and Bolske, 1996). Some

Mycoplasma species also cause different systemic and inflammatory condition like,

cervical abscesses, hepatitis, peritonitis, spleenitis and in rare cases meningitis and

abortion (Schumacher et al., 2011; Madanat et al., 2001; Jubb et al., 1985).

Mycoplasma is the smallest genus of Mollicutes that can invade both the

phagocytic and non-phagocytic cells of the infected host. It is the normal inhabitant of

respiratory and urogenital tract epithelial lining and can also invade tissues (Razin et

al., 1998). Most of the pathogenic Mycoplasma species have tissue tropism to lungs and

respiratory tissues. Therefore typical signs like cough, pneumonia, painful respiration

and pyrexia are main clinical findings in many infections. Due to multi-systemic

involvement the lesions are also observed in other body tissues (Sadique et al., 2012;

Laura et al., 2006). The Mycoplasma having surface antigenic protein, called the

lypoglycan, plays role in acute inflammatory process in the host tissues that leads to

maximum exudation and pleural effusion (Rosendal, 1993). It has been reported that

104

Mmc and Mccp produce a peroxide free radicals in tracheal tissues of the experimental

animal that is an important factor for the pathogenesis of CCPP (Howard, 1984; Cherry

and Taylor, 1970). The main histopathological lesion in affected lungs is comprised of

atelectasis, sloughing of alveoli, severe necrosis and polymorph nuclear neutrophil

infiltration in alveolar spaces (Sadique et al., 2012; Riaz et al., 2012; Mondal et al.,

2004).

The other tissues like trachea, liver, kidneys, intestine and spleen also showing

moderate to severe abnormality at cellular level in the form of hemorrhages, necrosis

and polymorph infilteartion (Sadique et al., 2012; Laura et al., 2006; Wesonga et al.,

2004; Gutierrez et al., 1999). The classical form of disease caused by Mccp that is

confined to the thoracic cavity and characterized by pyrexia, unilateral or bilateral sero-

fibrinous pleuropneumonia with severe pleural effusion and hepatization (Kabir and

Bari, 2015; Mondal et al., 2004). In some acute cases the pleural cavity contains an

excessive straw colored fluid with fibrin flocculations (Abbas et al., 2013; Sadique et

al., 2012). This disease is one of the major Mycoplasma infections responsible for

immunosuppression that make animals susceptible to various other viral and bacterial

infections.

The present study was aimed with the following objectives:

Study the pathogenesis of CCPP in small ruminants of Khyber

Pakhtunkhwa.

Recording of gross and histopathological lesions in naturally infected

small ruminants.

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4.2.1 Clinico-pathological picture of ruminant mycoplasmosis

To study the clinical picture of the disease, a total of 1800 small ruminants

exhibiting signs of respiratory syndrome were surveyed. The detail of clinical signs

ofdisease and involvement of various body systems were thoroughly recorded in

predesigned Questioniare (Annexure-1). Different clinical parameters such as

coughing, pneumonia, nasal discharge, lacrimation, conjunctivitis, breathing, diarrhoea,

dysponea, mastitis, arthritis and pyrexia were documented.

4.2.2 Necropsy

To study the pathogenesis of CCPP, a total of 180 animals with equal numbers

of sheep and goats were examined on post mortem examination. Similarly those

animals exhibiting severe signs of respiratory disease suspected for CCPP were

purchased and sacrificed for recording pathological lesions. On post mortem

examination, detail lesions were recorded in thoracic, abdominal cavity, joints and on

the surface of meninges. The detail lesions scoring were recorded in different organ of

each necropsied animal (Wesonga et al., 2004). Tissues samples were collected from

trachea, lungs, liver, kidneys, intestine, spleen and brain, and preserved in neutral

buffered formalin (10%) for histopathological examinations (Annexure-7).

Pleural fluids and lung tissues were also collected in aseptic condition in sterile

container under refrigeration for isolation of Mycoplasma. All the collected samples

were properly labelled and transported to the Pathology laboratory, Department of

Animal Health, the University of Agriculture, Peshawar for further processing.

4.2.3 Gross lesions and scoring

On post mortem, the gross lesions were recorded in all visceral organs like

lungs, pleura, liver, heart, kidney, spleen, trachea, small intestine and mediastinal

lymph node. Similarly the joint and skull were opened for record of lesions. Gross

lesions were recorded in different organs and the scoring was made on the basis of

severity of lesion. The severity level was 0, 1, 2, 3 and 4 that represent normal, mild,

moderate, severe and higly severe, respectively (Sadique et al., 2012).

106

4.2.4 Histopathology

Tissues of about 20-30 gm were taken in 10% neutral buffered formalin and

stored at room temperature for onward histopathological examinations. Samples were

processed with slight modification according to the standard protocols as adopted by

Bancroft and Gamble, (2007).

4.2.4.1 Procedure for Histopathology

Tissues of about 1-2 cm size were taken from Trachea, lungs, liver, intestine,

spleen, kidneys and brain and placed for overnight washing in running tap water. The

tissues were placed in such a way that water did not touch it directly to avoid tissue

damage. After washing tissues were processed for dehydration by placing it in

ascending grade of alcohol in automatic tissue processor with automatic time control

(Tissue-Tek® Sakura, Japan). The detail of tissue processing is summarized as follows;

i. Dehydration

30% alcohol 3.5 h

50% alcohol 2.5 h

70% alcohol 1h

80% alcohol 2 h

95% alcohol 1.5 hrs

Absolute alcohol I 1hr

Absolute alcohol II 45 min

ii. Clearing

Alcohol + Xylene 40 min

Xylene I 30 min

Xylene II 20 min

iii. Impregnation

For impregnation tissue samples were placed in paraffin melted at 72 ºC.

Paraffin I 2 h

Paraffin II 2h

107

iv. Embedding

After impregnation of tissues the blocks were prepared by using automatic

tissue embedding assembly (Tissue-Tek® TEC

™ Sakura). Blocks were made by pouring

carefully melted paraffin over the placed tissue in plastic cassettes. Blocks were then

shifted and placed in cold chamber of Tissue Tek® and were allowed to solidify.

v. Sectioning

Paraffin tissue blocks were sectioned with thickness of 5 µm by using

microtome (Accu-Cut® SRM

™ 200 Sakura, Japan). The cut fine sections were placed in

water bath (M-1450 Sakura) at 56 ºC, so that it floats over the surface of water and

folds were removed. For proper sticking of sections, egg albumin was applied on clean

glass slides. Section was mounted over the slide and placed on slide drying hot plate

(Mod. 1452, Sakura) for 30-40 minutes for drying followed by placing in hot air oven

(Mod. LDO-060E, Daihan Lab Tech. Co. Ltd, Korea) for 2-3 hours for drying and

removal of extra paraffin.

4.2.4.2 Staining

Slides that having fine, complete and good sections of tissue were placed for

staining after final drying. For staining of slide section Hematoxylin and Eosin (H & E)

stain were used. Automatic slide stainer (Tissue-Tek® DRS

™ 2000 Sakura, Japan) was

used for staining process. Staining was performed according to a standard protocol as

follows.

i. Removal of Paraffin

Box # Reagents Time duration

1. Xylene 3 min

2. Xylene 3 min

3. Xylene 3 min

ii. Removal of Xylene with alcohol

4. Ethyl alcohol 100% 1 min

5. Ethyl alcohol 100% 1.30 min

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21. Tap water 2 min

25. Distilled water 2 min

iii. Principal Dye

24. Hematoxylin (Annexure-8) 6 min

22. Tap water 2 min

iv. Decolorization

7. Acid alcohol (Annexure-9) 2 dips

23. Tap water 1 min

v. Mordanting the tissue sections

8. Amino alcohol (Annexure-10) 5 min

21. Tap water 1 min



vi. Counter staining

11. Eosin (Annexure-11) 1 min

vii. Dehydration





viii. Clearing

18. Xylene 1.30 min

17. Xylene 1 min

16. Xylene 1.30 min

109

ix. Mounting of cover slip

After completion of staining process the stained slides were cleaned properly,

DPX (Scharlau) was droped on slides and cover slips were placed in such a way to

avoid bubbles formation.

4.2.4.3 Slide Reading

For studying of microscopic lesions slides were studied under 10X and 40X

(Wesonga et al., 2004). Slides with desired pathological changes were photographed by

digital CCTV camera (Olympus DP71, U-CMAD3, Japan).

4.3 Microscopic lesions scoring

Histopatholgical lesions were documented in different organs and the scoring

was made on the basis of severity of lesion. The severity level was 0, 1, 2, 3 and 4 that

represent normal, mild, moderate, severe and higly severe, respectively.

4.4 Statistical analysis

Data were compilied in Microsoft excel sheet for calculating clinical signs,

lesion and scoring. To find out the clinical signs and post mortem lesion of slelected

animals, the results were expressed in terms of counts and percenatges. Similarly, to

check the overall severity of lesion scoring, average lesion score was calculated and

compared with the five categories of lesion scoring.

110

4.5 Results

4.5.1 Clinical findings

A total of 1800 animals, suspected for mycoplasmosis exhibiting signs of

respiratory syndrome were surveyed for recording of clinical signs of the disease.

Clinical investigation of the diseased animals like body temperature, conjunctiva

examination, lacrimation, coughing, nasal discharge, dullness, diarrhoea, urine color,

nervous signs were recorded. The respiratory signs were common feature of all the

infected animals. Out of total examined animals, 1108 (61.55%), 1023 (56.83%), 940

(52.22 %%) and 734 (40.77%) showing pneumonia, coughing, watery nasal discharge

and lacrimation, respectively. Along with upper respiratory symptoms the lacrimation

(40.77%), conjunctivitis (30.61%) and corneal opacity (7.7%) was also recorded. The

other systemic involvement revealed diarrhoea (22.33%), mastitis (3%), arthritis

(2.66%) and nervous signs (1.6%) in animals. Most of the animal exihibiting signs of

high ferver (58.2%) with anorexia and weight loss. There was high morbidity and

mortality (15.72%) in all surveyed animals (Table 4.1). It was observed that the

animals in advanced stage of disease were reluctant to move with obducted fore limb

and finally lie down on the ground with lateral recumbancy. In some animals the

nervous signs like circling and ballowing were obsereved at the terminal stage of

disease. Abortion was also recorded in the pregnant animals. Mastitis and arthiritis

were also obsereved in few cases. The graphic presentation of clinical signs are

exhibited by the animals during survey are presented in Figure 4.1. Some of the

important clinical signs of the diseased animals are shown in Plate 4.1.

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Table 4.1 Occurrence (% age) of clinical signs in naturally infected small

ruminants suffering from respiratory syndrome.

S.No. Clinical signs (n=1800) Showed signs Signs (%) 1 Pyrexia 1047 58.2

2 Cough 1023 56.83

3 Pneumonia 1108 61.55

4 watery nasal discharge 940 52.22

5 Mucopurulent nasal discharge 425 23.61

6 Lacrimation 734 40.77

7 Conjunctivitis 551 30.61

8 Corneal opacity 140 7.7

9 Dysponea 699 38.8

10 Diarrhoea 402 22.33

11 Mastitis 54 3

12 Pyuria 68 3.77

13 Weight loss 366 20.33

14 Arthritis 48 2.66

15 Nervous signs 29 1.6

16 Abortion 23 1.27

17 Mortalities 283 15.72

A total of 1800 animals were examined for recording clinical signs of mycoplasmosis.

112

Plate 4.1 Important clinical signs in small ruminants suffering from respiratory

syndrome suspected for CCPP. 1= Nasal discharge, 2= Goat kid with severe depression, 3= Nasal discharge,

4= Mucopurrulent discharge, 5= keratoconjunctivitis and lacrimation

6= Nasal discharge along with sample collection, 7= Sheep with inflamed udder/

mastitis, 8=Sheep extended head, 9= Buck with weight loss

4.5.2 Gross Pathology

Necropsy was carried out on 180 animals consisted of 90 sheep and 90 goats

across the different climatic zones of Khyber Pakhtunkhwa. On post mortem

examination majority of animals presented lesions in the thoracic cavity comprising of

pneumonia, enlarged mediastinal lymph node, pleural effusion, pericarditis and

tracheitis. Out of total, 97 (53.88%) animals were recorded showing pneumonia

followed by tracheitis in 68 animals (37.7%) and pleural effusion in 33 animals

(18.33%). On abdominal incision the lesions were also recorded in liver, kidneys,

spleen and intestine with different degree of involvement. The distribution of these

lesions consisted of nephritis (18.33%), hepatitis (17.22%) and spleenitis (6.11%) in the

total necropsied animals. In few cases, synovitis (3.33%) and meningitis (1.66%) were

also observed (Table 4.2).

113

Table 4.2 Occurence (% age) of gross lesions in different body tissues in naturally

infected animals.

S. No. Lesions No. of animals necropsied

(n=180)

lesion recorded (%)

1 Tracheitis 68 37.7

2 Purulent exudation in trachea 37 20.5


4 Pleural effusion 33 18.33

5 Hepatitis 31 17.22

6 Enteritis 24 13.33

7 Enlargement of mediastinal lymph node 26 14.44

8 Pericarditis 22 12.22

9 Pericardial fluid accumulation 14 7.77

10 Splenitis 11 6.11

11 Nephritis 33 18.33

12 Synovitis 6 3.33

13 Meningitis 3 1.66

Total number of animal necropsied = 180

The mycoplasmosis has the tendency to produced lesion in several domestic and

wild ruminants across the world. However the disease is mainly affected sheep and

goats with the production of severe lesion in various tissues. In both species the

prominent lesions were recorded in the respiratory system accompanied by multi-

systemic involvement. The lesions in goats were more severe in nature comprising

puemonia (69.77%) followed by tracheitis (45.5%), purulent exudate (27.7%) and

pleural effusion (22.2%) in all necropsied animals. The other lesions consisted of

nephritis (21.1%), enlargement of mediastinal lymph node (17.1%), enteritis (16.6%),

pericarditis (13.33%) and splenitis (7.7%). The least observed lesions were synovitis

(3.3%) followed by meningitis (2.2%). Lesions in lungs were mostly bilateral and

restricted to middle and apical lobes. However, the ilateral lesions in the lungs were

also recorded and in few cases the the caudal lobe was also affected. In 23.3% of

animals, trachea showed hemorrhages and fibrinous exudates in lumen while 58.8%

animals reflected mild hemorrhages. In most of the cases the heart was found normal in

size and texture; however, in few cases there was pericarditis and accumulation of

pericardial fluid (7.8%) in observed animals. The kidneys of infected animal were

114

presenting lesions with varying degree of congestion, hemorrhages and necrotic foci. In

few cases pus was observed in pelvis of kidneys reflecting pyelonephritis.

Hepatomegaly was observed in animals with charactristic lesion of pale coloration and

necrotic foci on its surface. In the digestive system lesions were observed in intestine in

the form of mild hemorrhages accompanied by enlarged mediastinal lymph nodes in

17.7% animals (Plate 4.2).

Plate 4.2 Gross lesion in various organs of animals at postmortem examination

suffering from CCPP. (1) Severe pneumonia with hemorrhages and hepatization of lungs, (2) Enlargement of

mediastinal lymph node, (3) Lungs with pus and plural adhesion, (4) Nodules on liver surface

(5) Pericardial fluid accumulated in pericardial sac, (6) Trachea showing exudate in lumen and

hemorrhages.

A total of 90 sheep died from respiratory syndrome suspected for CCPP were

necropsied for recording of lesions in various organs. It was revealed that pattern of

lesions were mild in nature and less in percentage as well as in severity as compared to

goats. Howevere the distribution of lesions were almost similar in different organs as

observed in goats. The most frequent lesions were recorded in the respiratory tract

consisted of pneumonia (40%), followed by tracheitis (30%), pleural effusion (14.44%)

and purulent exudates in trachea (13.3%). The most distinct feature of the lesion was

involovement of liver more in severity as compared to goats. The hepatitis was

recorded in 21% of necropsied sheep. The other lesions consisted of nephritis (15.5%),

enlargement of mediastinal lymph node (11.1%), pericarditis (11.1%) and spleenitis

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(4.4%). Meningitis (1.1%) and synovitis (2.2%) are least observed lesions among the

recorded animals.

The organ wise distributions of lesions were comprised of consolidation of

lungs with unilateral or bilateral involvement. The middle and apical lobes were more

frequently infected as compared to caudal and intermediate lobe. The tracheal lesions

were comprised of mild to moderate hemorrhages accompanied by fibrinous and

catarrhal exudates in lumen (Plate 4.3).

Plate 4.3 Gross lesion in various organs of animals at postmortem examination

suffering respiratory syndrome suscepted for CCPP. (1) Lungs of sheep showing hemorrhages, (2) Goat lungs showing consolidation in acute

mortality (3) Goat lungs with odema, emphysema and exudation, (4) Mastitis inflamed teet, (5)

liver with focal necrosis and abscessation, (6) CCPP suspected lungs with pleuropenumnia and

hemorrhages.

The heart lesions were recorded in the form of mild enlargement with

accumulation of pericardial fluid. The kidneys were almost normal in majority of

animal however some mild congestion and necrotic foci were recorded over the

paranchymal surface. Liver was severly inflamed in 15 (15.5%) animals showing pale

coloration, petechial hemorrhages and necrotic foci on it surface. The mediastinal

lymph nodes were slightly enlarged and congested in 10 (11.1%) animals. The detail of

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lesions recorded in different organs of both the species including sheep and goats are

presented in Table 4.3. The findings revealed that goats are most susceptible and

produced more severe pathological changes in different body tissue as compared to

sheep. The comparative analysis of distribution of lesions between the two species is

shown in Figure 4.1.

Table 4.3 Occurrence (% age) of gross lesions in different body tissue in naturally

infected sheep and goats.

S.No. Gross Lesions (n=90)

Sheep

lesion (%) Goat lesion

(%)

1 Tracheaitis 30 45.5

2 Purulent exudation in trachea 13.3 27.77


4 Pleural effusion 14.44 22.22

5 Hepatitis 21 14.44

6 Enteritis 10 16.66

7 Enlargement of mediastinal lymph node 11.11 17.77

8 Pericarditis 11.11 13.33

9 Pericardial fluid 6.66 8.78

10 Splenitis 4.44 7.77

11 Nephritis 15.55 21.11

12 Synovitis 3.33 3.33

13 Meningitis 1.11 2.22

In sheep and goats, a total of 90 numbers of observations for each species were recorded for gross

lesions.

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Fig. 4.1 Comparative distribution of gross lesions in variuos organs of sheep and

goats at post mortem examination. Blue color represent sheep and red lining goat.

4.5.3 Histopathology

The tissues samples of trachea, lungs, liver, kidney, intestine, spleen and brains

were collected in 10% buffered formalin and were processed for histopathological

studies. The following lesions were recorded in different organs.

4.5.3.1 Trachea

Majority of the tracheal sections of both species of sheep and goats exhibited

moderate to severe microscopic changes. Erosion of epithelium lining of trachea is

commonly observed, submucosal layer showing hemorrhages and infiltrated with

polymorph nucleated cells. Muscular layer was showing edema and hyperplasia of

goblet cells. However, the lesion scoring revealed severe nature of lesions in goats as

compared to sheep. The lesions of trachea are presented in Plate 4.4.

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4.5.3.2 Lungs

The classical lesions in CCPP were observed in lungs comprising of

emphysema, atecectasis, thickning of alveolar wall and interstitial layer with extensive

leukocytic infiltration. In some cases the alveoli were filled with fibrin exudates and

some get ruptured and coalase together to form a bullae. The epithelial linings of

bronchi were disrupted and the interlobular septa were thickened with extensive

leukocytic infiltration. Hemorrhages, congestions and necrotic areas are surrounded by

pyogenic band and granulation tissues with scattered inflammatory cells were also

found. In few cases chronic inflammatory reaction was observed in the form of

granulomatous inflammation consisted of fibrotic core, macrophage, giant calls and

plasma cells (Plate 4.5 and 4.6).

Plate. 4.4 Tracheal section of goat showing sloughing of epithelial layer (arrow

head) and polymorph leukocytic infilteration (long arrow) (H & E stain

400X).

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Plate 4.5 Lungs of goat suffering from respiratory syndrome showing sloughing

of ciliated epithelium in bronchioles (long arrow) and leukocytic

infiltration (arrow head) (H & E stain, 100X).

Plate 4.6 Lungs of goat suffering from respiratory syndrome showing emphysema

(long arrows) and rupture of aveoli (short arrow) suffering from CCPP

(H & E stain 400 X)

4.5.3.3 Intestine

The intestinal sections of animal showed erosion and sloughing of villi and

lining epithelium. There are hemorrhages, hyperplasia of mucus secretory cells and

aggregation of inflammatory cells (Plate 4.7).

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Plate- 4.7. Intestine of goat showing sloughing of villi and leukocytic infilteration

(H&E stain 400X).

4.5.3.4 Kidneys

The sections of kidneys showed congestion, degeneration in the glomeruli and

necrosis in tubular epithelium accompanied by leukocytic infiltration. Some urinary

tubules were filled with cast and cell showed necrosis. In the present study three

pathogenic Mycoplasma species especially Mmc were confirmed that may possibly

target the urinary tissue. Tissue section of majority of the animal exhibited signs of

pyelonephritis with leukocytic infiltration. Few kidney sections were also recorded for

loss of glomeruli (Plate 4.8, 4.9).

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Plate 4.8 Histo-micrograph of kidney of the goat infected with Mycoplasma,

degenerative changes in glomerulus (short arrow) and tubular epithelial

cells (long arrow). The disappreance of glomeruli (star). The brush

boarder of tubules showing (long arrow) (H&E stain, 400X).

Plate 4.9 Histo-micrograph of kidney of the sheep infected with Mycoplasma,

showing severe degeneration in tubular epithelium (triangular),

leukocytic infiltration (long arrow) and deposition of cast (star) tubular

epithelial cells (H & E stain, 100X).

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4.5.3.5 Spleen

Mild hemorrhages, congestion and leukocytic infiltration in parancyma of

spleen were observed. In few cases there were microabscsses in the splenic parenchyma

(Plate 4.10, 4.11).

Plate 4.10 Spleen of sheep suffering from respiratory syndrome showing

congestion and extensive leukocytic infiltration (H & E stain, 40X).

Plate 4.11 Spleen of goat suffering from respiratory syndrome showing mild

leukocytic infiltration at (H & E stain, 100X).

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4.5.3.6 Liver

Liver sections in majority of the animals showed area of congestion, local

necrosis and hemorrhages. Hepatocytes revealed hydropic degeneration and

condensation of nuclei. Extensive leukocytic infiltration around bile duct and central

vein was the common feature of hepatic lesion (Plate 4.12, 4.13, 4.14).

Plate 4.12 Liver of goat infected with CCPP showing swollen hepatocytes (arrow

head), congestion is seen in central vein filled with blood (long arrow)

(H&E stain, 100 X).

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Plate 4.13 Liver of sheep infected with mycoplasmosis showing extensive

leukocytic infiltration (arrow) around and congestion (star) in central

vein (H & E stain 100X).

Plate 4.14 Liver of sheep infected with mycoplasmosis showing swollen

hepatocytes (arrow) (H & E stain 400X).

125

4.5.3.7 Brain

Section of brain was showing mild inflammatory condition like slight

congestion and inflamed meninges. There were few polymorph leukocytic infiltrations

seen in the meninges. However most of the brain section was presenting normal

morphology and architectural detail (Plate 4.15, 4.16).

Plate 4.15 Brain of goat from respiratory syndrome showing mild congestion (long

arrow) and few inflammatory cells (arrow head) (H & E stain, 100X).

Plate 4.16 Brain of sheep suffering from respiratory syndrome showing normal

histological structure (H & E stain, 400X).

126

4.5.4 Gross lesions scoring

The scoring was carried out to record and evaluate the severity of Mycoplasma

infection in various organs of sheep and goats. Out of total lungs lesion scoring of 24

the distribution of lesion score were 13 and 19 in sheeps and goats respectively. It

revealed that the lungs of goats were more severly infected as compared to sheep.

However, the lesion scoring of liver in sheep presented different picture as compared

with goats. The lesion was more severe by counting 8 in sheep than goats where the

lesion scoring was 5. The overall lesion scoring revealed more severe nature of disease

in goat as compared to sheep. The results of gross lesions scoring is presented in Table

4.4.

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Table 4.4 Scoring of gross lesions in naturally infected sheep and goats suspected

for CCPP across different climatic zone.

Tissue Gross lesions Scoring

Sheep Goat

Conjunctiva Congestion

Pus Exudates

2

0

2/8 3

1

4/8

Trachea Mucous exudates

Hemorrhages

2

2

4/8 2

3

5/8

Lungs Hemorrhages

Consolidation

Abscess

Alveolar exudation

Fibrin layer

Pleural fluids

3

2

1

2

3

2

13/24 3

3

2

3

4

3

19/24

Liver Hepatomegaly

Congestion

Focal necrosis

3

3

2

8/12 2

2

1

5/12

Intestine Odematous

Hemorrhages

blood stained ingesta

2

2

1

5/12 2

3

2

7/12

Heart Inflammation

Pericardial fluids

2

1

3/8 2

2

4/8

Spleen Splenomegaly

Focal abscessation

1

2

3/8 2

3

5/8

Kidneys Nephritis

Hemorrhages

Pus in pelvis

2

1

2

5/12 3

3

2

8/12

Mediastinal lymph

node

Enlargement

Hemorrhages

2

0

2/8 3

1

4/8

Joints Bone swelling

Exudates

1

0

1/8 2

1

3/8

Brain Meningitis

Hemorrhages

1

0

1/8 2

0

2/8

Involvement of tissue on the bases of severity, 0= Normal, 1 = Mild, 2= Moderate, 3 = Severe, 4=

Highly severe

4.5.5 Microscopic lesions scoring

The microscopic lesions scoring were carried out to eveluate the intensity and

pathogenecity of disease and involvement of various organs. Out of total lesion scoring

of 32 of lung tissue the sheep and goat presented a scoring of 17 and 26, respectively.

The scoring indicated that the lungs of goats were more severly affected as compared to

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sheep. The lesion scoring of liver in both species present almost similar picture of

disease as described in gross lesion scoring. The liver lesions score revealed 15 and 11

in sheep and goats, respectively. The overall microscopic lesions scoring revealed that

in goat the severity of lesion was moderate to severe as compared to sheep where mild

to moderate lesions were observed (Table 4.5).

Table 4.5 Scoring of microscopic lesions in naturally infected sheep and goats

suspected for CCPP across different climatic zone.

Tissue Microscopic lesions Scoring

Sheep Goat

Trachea Sloughing of epithelium

Hemorrhages

leukocytic infiltration

2

2

2

6/12 3

2

3

8/12

Lungs Emphysema

Atelectasis

Rupture of alveoli

Micro vessel thrombi

Abscess

Alveolar exudation

Fibrosis

Leukocytic infiltration

2

1

2

2

3

2

2

3

17/32 4

3

3

2

4

3

3

4

26/32

Liver Congestion

Hydropic degenration

hepatocytes necrosis

Focal necrosis

Leukocytic infiltration

3

3

3

4

2

15/20 2

2

2

2

3

11/20

Intestine Sloughing of villi

Hemorrhages

Necrosis of epithelium

3

1

2

6/12 3

2

3

8/12

Spleen Splenomegaly

Leukocytic infiletration

2

0

2/8

2

2

4/8

Kidneys Nephrosis

Hemorrhages

Pus in pelvis

Tubular necrosis

2

2

1

2

7/16 3

2

2

3

10/16

Brain Meningitis

Hemorrhages

1

0

1/8 2

0

2/8

Involvement of tissue on the bases of severity, 0 = Normal, 1= Mild, 2 = Moderate, 3= Severe, 4=

Highly severe

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4.6 Discussion

Pathogenesis is the progressive development of a disease that taking specific

time in a particular tissue to produced abnormality. For successful infection and

manifestation of disease various factors including the entry of pathogen to the host,

tissue tropism, adherence, invading the target cell followed by multiplication,

colonization and then dissemination to various organs (Smith, 2009). Various

pathogens have different tissue tropism and incubation period to develop infection in a

particular tissue. In the respiratory tract, several physical and biochemical defense

mechanisms existed to protect the animal against invading pathogens. This protective

mechanism includes intact epithelium, mucociliary apparatus, surfactant, surfactant

proteins and alveolar macrophages. However, when this immune system is overcome

by the invading pathogen the infection become established and lead to development of

disease.The other predisposing factor like immunosuppression, age, sex, managemental

practices and poor health status also play role in making the host susceptible to various

infections. Mycoplasma is the normal inhabitant of respiratory and urogenital tract

epithelial lining but rarely invade tissue (Razin, 1999). The mechanisms of the disease

development of CCPP are exactly unknown, but it is well established that most of

Mycoplasma species adopted complex strategies to enter into the host tissue showed the

tissue and then establish the infection at cellular level in the predilection site followed

by pathological alteration at gross level with multiple clinical complications.

The incubation period of the disease is variable that normally takes 5-15 days.

The fate of disease is fatal in per acute cases, animal may die within one to three days

with minimal clinical signs (OIE, 2008). In chronic cases, the infection persisted for

longer period of time from weeks to months and the animal act as chronic carrier.

Different pathogenic Mycoplasma species are responsible for disease production in

various tissues of the infected animals. For example Mm cluster can invad both the

phagocytic and non-phagocytic and causes multisystemic manifestation. Several

pathogenic species of Mm cluster like MmLC and Mmc and non-cluster group like M.

putrefaciens and M. agalactiae causes septicemia and multiple systemic complications.

This multi-systemic manifestation is called MAKePS (mastitis, arthritis, keratitis,

pneumonia and septicemia) syndromes (Thiaucourt and Bolske, 1996; Egwua et al.,

2001). One of the most important member of mycoides cluster is Mccp that confined to

thoracic cavity is the principal cause of CCPP in small ruminants (OIE, 2014). The

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CCPP is characterized by high fever, dysponea, productive coughing, mucopurulent

nasal discharge, lacrimation, and in terminal stage animal lie on lateral recumbancy

(Laura et al., 2006; OIE, 2013). In some cases the animals exhibited signs of lameness

of the forelimb which is accompanied by severe diarrhoea (Sadique et al., 2012).

In the present study, the infected animals suspected for CCPP were surveyed for

recording of clinical signs of the disease and was revealed that the incubation period

was ranging from 5 to 15 days. The respiratory signs were the common feature of

infected animals characterized by pneumonia (61.55%) followed by pyrexia (58.2%),

cough (56.83%), watery nasal discharge (52.22%) and lacrimation (40.77%). Similar

respiratory signs accompanied by high fever and mortality were also reported by many

researchers (OIE, 2014; Shahzad et al., 2012; Chu et al., 2011). These findings are

further supported by the results that mycoplasmosis infected animals showed high body

temperature (40-43 °C), painful respiration and persistent violent cough (Mondal et al.,

2004). It is justified by the facts that most of the pathogenic Mycoplasma species have

tissue tropism to respiratory tract. Therefor, typical signs like cough, pneumonia and

pyrexia are developed due to involvement of lower respiratory tract. The Mycoplasma

having surface antigenic protein called the lypoglycan is responsible to stimulate the

acute inflammation in the host tissue that leads to maximum exudation and pleural

effusion (Rosendal, 1993). The high fever developed due to release of inflammatory

mediators strongly provoked by Mycoplasma protein and release of its toxin. Other

sings like nasal discharge, lacrimation and conjunctivitis are produced when upper

respiratory tract become involved. The Mmc and M. putrefaciens have the tendency to

infect the lower and upper respiratory tract along with other system of the host.

Majority of the diseased animals in the present study were showing the sings of

conjunctivitis, lacrimation and corneal opacity. The unilateral or bilateral conjunctivitis,

lacrimation accompanied by corneal opacity in mycoplasmosis is also reported by

(Dezfouli et al., 2011; Mondal et al., 2004).

The other signs of CCPP are comprised of lameness, diarrhoea, inability to

move, abducted forelimb, stiff neck and in advance cases the animal lie down on

ground (OIE, 2014). In multi-pathogenic infection the clinical signs are also observed

in various organs. Diarrhoea was observed in 22.33% animals followed by pyuria

(3.7%) and mastitis 3% in the examined animals. Frequent fluid losses occur in acute

131

diarrhoea lead to severe dehydration and electrolytes imbalance in turn causes

hemoconcentration result to kidney failure. This statement is justified by the findings of

several researchers (Sadique et al., 2012; Laura et al., 2006; Gutierrez et al., 1999),

they reported diarrhoea and pyelonephritis. Nervous signs (1.6%) and abortion (1.27%)

were recorded in few animals. The nervous system are rarely affected by pathogenic

Mycoplasma species, however some cluster member causes acute septicemia

accompanied by high fever and meningitis. Similar findings were also reported in a

study that goats exhibited sings of bellowing and circling suffering from Mmc infection

(Sadique et al., 2012). The neurologic signs like circling, seizures and nystagmus were

also observed in 2 year old female goat infected by Mmc (Schumacher et al., 2011).

Severe stress and involvement of urogenital tract may cause abortion in animal.

Anorexia is the common feature of septicemic diseases like CCPP that

ultimately lead to poor weight gain in the infected animal. In the present study 20.33%

animals were found weak and emaciated due to anorexia and decreed feed intake.

These results are in accordance with the findings that mycoplasmosis causes weight

loss in goat kids (Sadique et al., 2012). The overall mortality in the study area was

15.72% however high morbidity was observed. The morbidity and mortality in the

animals suffering from CCPP varies due to multiple factors including pathogenic

species of Mycoplasma, agro-ecological zone, managemental practices and immune

status of the animals. Similarly different mortality rate in the goats suffering from

CCPP in various parts of the world is reported. In a study it was reported that 9.17%

mortality was occurred in Beetal goats in Punjab, Pakistan (Riaz et al., 2012).

However, high mortality up to 32.9% was recorded in goats of West Bengal, India

(Mondal et al., 2004). Similarly, in another study the morbidity and mortality were

32.14 and 15% respectively in naturally infected Black Bengal goats in Bangladesh

infected by mycoplasmosis (Kabir and Bari, 2015).

Pathological lesions play vital role in the diagnosis of a disease and provide a

clue for the physician to evaluate the severity of the infection and devise therapeutic

intervention. The lesions are starting at ultra-structural level and then adopted at

microscopic and gross level. The different pathogenic species of Mycoplasma has the

ability to produced lesions in various tissue, organs and system of the body (Mondal et

132

al., 2004; Leach et al., 1993). Lesions are also helpful to take desirable tissue samples

for successful isolation and identification of the causative agents.

Several factors including age, sex, pathogenicity and virulencey of the causative

agent are playing significant role in the development of mild and severe lesions in the

host body. The species of Mycoplasma cluster causes CCPP in small ruminant may

decide the nature and site of lesions in the host. In the present study necropsy was

carried out on 180 animals consisted of 90 each from sheep and goats across the

different climatic zones of Khyber Pakhtunkhwa. It was revealed that majority of the

animals exhibited lesions in the respiratory system. The prominent lesions were

consisted of congested trachea in (37.7%) of animals with varying degree of

hemorrhages and frothy exudation in the lumen. These observations were similar to the

findings that indicated tracheal hemorrhages and purulent exudate in caprine

mycoplasmosis (Sadique et al., 2012; Mondal et al., 2004). The Mycoplasma has tissue

tropism to respiratory tract and provoked acute inflammation increased the extra

vasation of blood lead to local hemorrhages. The local inflammatory reaction causes

hypertrophy of the goblet cells and increases its exudation. Accumulation of exudates is

initially catarrhal in nature that turned into fibrino purulent in the advance stage of

disease. These exudates lodged in the trachea causes hindrance in the respiration and

lead to a prominent signs of dysponea in Mycoplasma infection. The mixed type lesions

in various tissues in Mycoplasma infection have been reported by many researchers

(Sadique et al., 2012; Goncalves et al., 2010; Balikci et al., 2008). Similar findings like

catarrhal exudates in the nasal and tracheal passages along with hemorrhages were

observed in mycoplasmosis infected goats (Gelagay et al., 2007).

The lungs lesions were recorded in 53.88% of animals comprising of

consolidation, congestion and hemorrhages, focal abscessation, pleural adhesion and

accumulation of straw colored fluids in the pleural cavity. The lesions recorded in the

present study are supported by the findings of many researches who described that

CCPP is mainly the disease of respiratory system that produced lesions like exudates in

trachea, consolidation and hepatization of lungs, hemorrhages, focal abscessation and

presence of sero fibrinous fluid in the thoracic cavity (Sadique et al., 2012; Wesonga et

al., 2004; Gutierrez et al., 1999; Rodriguez et al., 1996). The unilateral and bilateral

involvement of lungs was the predominant feature of the study. These findings are in

133

agreement with the results of a study that described that majority of the animals

suffering from CCPP showing the involvement of bilateral or unilateral infection of

lungs (Sadique et al., 2012; Laura et al., 2006). Similar observations were also recorded

by the findings of a study that right lung were severely infected with grey hepatization

in goats suffering from CCPP (Riaz et al., 2012). The Mccp infection is restricted to

thoracic cavity and the lesions are mainly confined to the lungs tissue. The classical

lesions developed during its infection comprised of massive hepatization of lungs,

pleuritis and excessive pleural effusion (OIE, 2014). In acute stage of disease some

animals developed immunity followed by resolution and recovery of the animal.

However, in chronic cases the pleura became thickened along with fibrin deposition

and adhesions to the chest wall (Kabir and Bari, 2015). The results are in close

conformity with the findings of some previous reports (Chu et al., 2011; Laura et al.,

2006), they reported consolidation and massive hepatization of lungs, pleuritis and

pleural effusion. The involvement of lungs with severe pleuritis and pleural effusion is

justified by the study that reported mortality in sheep suffering from respiratory

syndrome (Al-Momani et al., 2006). Our results are further strengthen by the findings

that reported the lungs showed massive hepatization, covered with yellowish material,

excessive fluid of upto 160 mL (Abbas et al., 2013).

The Mmc cause multisystemic infection and the lesions are developed in

different organs of the infected host (Sadique et al., 2012; Laura et al., 2006). In the

present study three different pathogenic species of Mycoplasma were isolated from the

animals suffering from respiratory syndrome suspected for CCPP. Therefore, the

lesions were recorded in other visceral organs along with respiratory system. The

multisystemic lesions were hepatitis (18.33%), nephritis (17.22%), pericarditis

(12.22%), pericardial fluids (7.7%), enteritis (13.33%) and enlargement of mediastinal

lymph node in 14.44% in animals. The multisystemic manifestation of the disease is

supported and justified by the findings that Mmc and Mmc LC infections causes wide

spread lesions in different organs characterized by consolidation of lungs, enteritis,

hepatitis, splenitis, nephritis, arthritis and enlargement of mediastinal and mesenteric

lymph nodes (Sadique et al., 2012; Riaz et al., 2012; Goncalves et al., 2010; Mondal et

al., 2004; Gelagay et al., 2007). In mixed infection of Mm cluster the animals on post

mortem examination revealed lesions in different organs including pericarditis and

accumulation of pericardial fluids (Nicholas et al., 2008; Mondal et al., 2004).

134

Similarly, in Mccp infected goats that mainly restricted to the thoracic cavity exhibited

lesions like unilateral fibrinous pleuropneumonia (88.3%), pleural effusion (49.1%),

hydrothorax (84.8%), and pericardial fluids in 1.7% (El-Manakhly and Tharwat, 2016).

Similar findings were also made that indicated an excessive straw colored plural fluid

with fibrin flocculation (Rurangirwa and McGuire, 2012). Mixed type infection with

involvement of three pathogenic species including Mccp, M. arginini and M.

ovipneumoniae were also reported by (Abbas et al., 2013).

Some pathogenic species of Mm cluster are causing hyper pyrexia, septicemia

with severe consequences. The nervous system signs are occasionally developed in

peracute infection characterized by convulsion, circling and nystagmus. The

Mycoplasma is the smallest mollicute having the ability to invade body cavities and can

cross blood brain barrier and causes inflammatory condition in the nervous tissue. The

other possible reason of the pathogen entry to the brain is through ear canal

(Schumacher et al., 2011). The nervous lesions characterized by meningitis were

present in a few animals 1.6% in the natural outbreak of CCPP. The presence of

meningeal lesion were also reported by Schumacher et al. (2011) who investigated two

year old female goat infected by Mmc and noted creamy-colored pus in subarachnoid

spaces. However, no nervous lesion and signs were observed in sheep population

during post mortem findings. The findings revealed that the goats are more susceptible

than sheep and presented more severe manifestation of disease. The statement is

supported by the findings that domestic goats are primary host of CCPP (Arif et al.,

2007). It is confirmed and isolated three different pathogenic species of Mycoplasma

from small ruminant population responsible for multisystemic infection in the small

ruminants. The results revealed wide range of lesions distributed in the different vital

organs of the body. In this study, the combined action of the few Mycoplasma strains

may have been responsible for the severe lung lesions. Damaging combinations of two

or more pathogenic agents have frequently been described in lung infections

(Rodriguez et al., 1996; Jones, 1989). This wide spread nature and distribution of

lesions is justified by the findings of several researchers (Kabir and Bari, 2015; Riaz et

al., 2012; Nicholas et al., 2008; Laura et al., 2006; Mondal et al., 2004). However these

findings are contrary to the results that in CCPP the lesion are restricted to respiratory

tract (OIE, 2014; Wesonga et al., 2004). The non-agreement could be justified by the

facts of difference of specie of Mm cluster. The Mccp has the characteristic to infect the

135

respiratory tract only, while the other species of Mycoplasma cluster have wide range

of tissue tropism leads to multisystemic lesions.

Some pathogenic Mycoplasma species like Mmc also provoke the important

bleeding disorder called disseminated intravascular coagulation (DIC). Due to

septicemic nature of disease they cause damage to the endothelial surfaces. Massive

platelets adhesion occurred at the damage endothelial surface that causes excessive

utilization of platelets and leads to hemostatic defects due to reduction of clotting time.

These abnormalities trigger coagulative disorder like thrombocytopenia, neutropenic

leukopenia, increased in prothrombin time and antithrombin III. Collectively, these

bleeding disorder promote the DIC scattered throughout the body. These findings are

supported by the findings that indicated that in Mycoplasma infection a series of

changes occurred that alter the coagulation system of blood that finally leads to fatal

DIC (Sadique et al., 2012; Gutierrez et al., 1999; Rosendal, 1993; Bolske et al., 1989).

In the era of advance molecular techniques the histopathological tools are still

playing an important role in the diagnosis of some important fetal diseases. The

histopathological changes in different visceral organs and tissue are dependent on the

involvement of species of Mycoplasma that causes CCPP infection in small ruminants.

Some pathogenic species are causing acute infections that showing early vascular and

cellular changes while other are chronic in nature and produce lesions late in the form

of fibrosis, granuloma and hyperplasia. The upper respiratory tract are most frequently

infected by the Mycoplasma and developed lesions in the form of tracheaitis,

desquamation of epithelial cells, hyperplasia of goblet cells and leukocytic infiltration.

There was sloughing of epithelium, hyperplasia of goblet cells, scattered hemorrhages

in submucosal layer infiltrated with polymorph nucleated cells. During the infection the

secretary glands became hyperactive to produce more mucous for encountering and

flushing of the infection. The lesions recorded in the present study are supported by the

findings that reported tracheal hemorrhages, desquamation of upper epithelial layer

(Sadique et al., 2012; Laura et al., 2006; Mondal et al., 2004). The sloughing of

epithelium is commonly seen in most of the respiratory tract infection due to the initial

attachment of pathogen then its colonization and subsequent production of lethal

product at the site.

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On histopathological examination of the lungs sections the majority of animal

tissues exhibited lesions like atelectasis, emphysema, thickening and rupture of alveoli

with leukocytic infiltration. The interlobular septae were thickened infiltrated with

leukocytes and some lobules were filled with inflammatory exudates, sloughing and

desquamation of ciliated epithelium in bronchioles was the predominant feature of the

findings. Such type of lesions were also reported by the study that the lungs tissue of

goats died from CCPP revealed marked thickness of pleura, excessive fibrin deposition

in alveoli and abundantly infiltrated neutrophils and few lymphocytes (Abbas et al.,

2013). These observation are with close conformity with the findings of many

researchers (Sadique et al., 2012, Riaz et al., 2012; Mondal et al., 2004), they reported

that lungs alveoli were filled with proteinaceous exudates with abundant macrophages,

fibroblast and lymphocytes. In few section of lungs micro vascular thrombi,

hemorrhages and fibrosis were also recorded. The micro thrombi in lung tissues of kids

died from Mmc infection were frequently observed with perivascular cuffing and

leukocytic infiltration (Gutierrez et al., 1999).

The septicemic nature of Mmc infection alter the hemostatic condition of the

circulatory system and prone the host to disseminated intravascular coagulopathy. The

statement is justified by the results of the study that the platelets depletion are

frequently observed in Mmc infection (Gutierrez et al., 1999; Rosendal, 1993; Nayak

and Bhowmik, 1988; Thigpen et al., 1981). The fibrosis is important pathological

manifestation commonly observed in chronic inflammation. Several pathogenic

Mycoplasma infections like MmcLC, Mmc have the ability to modulate itself to survive

within the host tissue for longer period of time and causes chronic inflammation. Few

lungs sections revealed chronic inflammatory changes in the form of macrophages,

plasma cell and accumulation of fibroblast. Similar observations were also reported by

the results that fibroblast and macrophages were abundantly present in chronic

Mycoplasma infection (El-Manakhly and Tharwat, 2016). In another study the lungs

section of chronic infected diseased goats contain chronic inflammatory cells including

macrophages, fibroblast and plasma cells (Riaz et al., 2011).

The sections of kidneys showed congestion, extensive degeneration in the

glomeruli and necrosis in tubular epithelium accompanied by leukocytic infiltration.

Some urinary tubules were filled with cast. In the present study three pathogenic

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Mycoplasma species especially Mmc were confirmed that may possibly target the

urinary tissue. Few kidney sections were also recorded for loss of glomeruli. The

findings are in accordance with the results that presented glomerulonephritis; tubules

showed deposition of cast and disintegration recorded in the glomerular tufts (Mondal

et al., 2004). Medullary micro abscessation, congestion and extensive inflammatory

cells also reported by Gutierrez et al. (1999). These findings are also correlated with the

findings of several researches (Sadique et al., 2012; Laura et al., 2006; Rodriguez et al.,

1996; DaMassa et al., 1992), they observed congestion, urinary cast, necrosis of tubules

and polymorph infiltration in kidneys of Mmc infected goats.

Most of the liver sections showing congestion and swelling of hepatocytes that

represent the acute changes. Some hepatocytes were condensed that indicated the early

stage of necrosis and cell death. Inflammatory cells especially polymorph nucleated

were present around the hepatic triade. These results are supported by the findings that

showed congestion and hyperemia of central vein accompanied by necrotic foci in the

liver parenchyma (Sadique et al., 2012; Mondal et al., 2004). The Mmc infection causes

multisystemic disease in small ruminants and produced lesions in different organs

including GIT. In the present findings majority of the intestinal sections showed

erosion and sloughing of villi and lining epithelium. There were hemorrhages in

submucosa invaded by aggregation of inflammatory cells. In most sections of spleen

were showed moderate hemorrhages and leukocytic infiltration. In few cases there was

microabscsses in splenic parenchyma. Such microscopic lesions in wide spread organs

including liver, intestine and spleen were reported in the form of multifocal

hemorrhages in spleen and liver and desquamation of villi (Riaz et al., 2012; Sadique et

al., 2012; Laura et al., 2006; Mondal et al., 2004; Gutierrez et al., 1999).

Few sections of brain showed mild congestion and inflamed meninges. There

were few lymphocytes and neutrophil seen in the meninges. However most of the

animals were presenting normal histology of brain. A brain was rarely affected by

Mycoplasma infection. The nervous tissue lesion recorded in the present study might be

due to Mmc isolated and confirmed in the goats suffering from respiratory syndrome in

the natural outbreak. The lesion of brain characterized by diffuse congestion and

infiltration of macrophages, lymphocytes and plasma cell were reported in a goat

suffering from CCPP (Schumacher et al., 2011). The meningeal lesions are further

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supported by the findings that limited meningeal inflammation accompanied by

perivascular leukocytic infiltration were recorded in brain of goat died from

Mycoplasma infection (DaMass et al., 1992). Similarly, it was reported that various

types of lesion were recorded in different organs including brain of goats during the

natural outbreak of Mycoplasma infection with Mmc (Bajmocy et al., 2000; Hernandez

et al., 2006). It was also reported that M. bovis has the potential to cause nervous

infection and the lesion were recorded in brain of calf (Stipkovits et al., 1993).

4.7 Conclusions

Goats are more susceptible species suffering from CCPP as compared to sheep.

Among the clinical manifestation, pneumonia (61.55%) was the predominant

clinical sign observed in the diseased animals.

The multisystemic involvement in the study animals reflecting mixed infection

of Mycoplasma mycoides cluster and non-cluster Mycoplasma species.

Lesions scoring revealed severe manifestation of the disease in the form of

pneumonia followed by moderate types of lesions in kidneys, intestine, liver,

heart and spleen.

Gross and microscopic lesions scoring revealed more severe nature of disease in

goats as compared to sheep.

Involvement of nervous system revealed septecimic form of disease suspected

for Mmc infection.

4.8 Recommendation

1. Immunohistochemical study needs to be conducted to detect Mycoplasma

antigen in different tissue for confirmation of tissue tropism of various species

of Mycoplasma for devising effective treatment protocol.

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V. STUDY -3

CHEMOTHERAPEUTIC TRIAL OF COMMONLY USED

ANTIMICROBIAL AGENTS AND INDIGENOUS MEDICINAL

PLANTS FOR THE TREATMENT OF CCPP

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ABSTRACT

A study was conducted to investigate the efficacy of commercially available

antimicrobial agents and indigenous medicinal plants against the local isolated

pathogenic Mycoplasma species. Five different commercially available antimicrobial

agents like tylosin, oxytetracycline, enrofloxacin, gentamycin and ceftofer sodium, and

three medicinal plants including Calotropis procera, Azadirachta indica and Artemisia

herba-alba were tested in-vitro as disc diffusion assay and broth micro dilution. The

results revealed that maximum zone of inhibition 19.00±0.71, 18.00±0.71 and

17.00±0.45mm was produced by enrofloxacin against Mmc, Mccp and Mp,

respectively. Gentamycin was moderately effective with zone oh inhibition 11.00±0.45,

12.00±0.55 and 12.40±0.51mm against Mmc, Mccp and Mp, respectively. The isolates

showed resistance against oxytetracycline and ceftofer sodium which produced zone of

inhibition 3.00± 0.32 mm and 0.00± 0.00 mm, respectively. The antimicrobial effects

were further investigated by broth micro dilution method against all the local isolates of

Mycoplasma. The results revealed that enrofloxacin exhibited strong antibacterial

activity with minimum inhibitory concentration (MICs) value of 0.001, 0.001 and

0.01mg/mL against Mycoplasma mycoides subsp. capri (Mmc), Mycoplasma

capricolum subsp. capripneumoniae (Mccp) and Mycoplasma putrefaciens (Mp),

respectively. Interestingly, all the isolates showed resistance against tylosin,

oxytetracycline and ceftofer sodium with high MICs values. Among the tested

methanolic plant extracts, A. herba-alba showed maximum zone of inhibition

16.33±0.33, 14.00±0.44 and 15.4±0.12mm at 30.0mg against Mmc, Mccp and Mp,

respectively. It was concluded that local isolates developed resistance to the commonly

used antimicrobial agent like tylosin, oxytetracycline and ceftofer sodium. However,

enrofloxacin was found the most efficacious drug for the treatment of caprine

mycoplasmosis. Among the tested medicinal plants A. herba-alba was showing high

anti-mycoplasmal activity to all local isolates of Mycoplasma.

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5.1 Introduction

Ruminant mycoplasmosis is an important respiratory tract infection causes

heavy economical losses in the world. The disease is prevalent in many countries of

Africa and Asia and widespread in Pakistan, causing heavy losses to the small ruminant

population especially in the northern and southern regions of the country (Samiullah,

2013; Shahzad et al., 2013; Sadique et al., 2012). Pakistan is 3rd

largest goat and 12th

sheep producing country providing a quality protein source and engine for boosting the

economy of the poor farmer and contributing an ample amount in the national GDP in

the form of meat, wool and hide export (Economic Survey, 2016-17). However this

large animal inhabitant is facing various threats in forms of harsh climatic conditions,

poor husbandry practices, infectious and non-infectious diseases. Among the various

bacterial infectious diseases the mycoplasmosis get significant role by posing serious

threat to this large population of livestock. This disease is responsible for acute

respiratory syndrome and usually terminated in chronic complications which cause

heavy economic losses in the form of decreased production, treatment cost, high

mortality and decreased export of animal products (Sadique et al., 2012).

Among the ruminant mycoplasmosis the CCPP is highly fatal disease of small

ruminant inflicting high mortality and production losses. The treatment is mainly

carried out by traditional available antimicrobial agents with varying degree of success.

The most commonly used antibiotics comprise of tylosin, gentamycin, enrofloxacin,

oxytetracycline, kanamycin, chloramphenicol and nalidixic acid. However, due to their

indiscriminate use and improper dose in the field condition, drug resistance issue has

been recently developed that usually leads to therapeutic failure (Scott and Menzies,

2011; Laura et al., 2006). The other important reason for the therapeutic failure and

development of bacterial resistance in the under developed countries is the quackery

practices, poor drug quality, improper therapeutic duration and rapid replacement of

antimicrobial agents (Canton et al., 2013; Mathew et al., 2007). The use of plants in

treating various diseases is as old as civilization and traditional medicines still provides

a major share in treatment of different maladies (Alviano and Alviano, 2009; Fabricant

and Farnsworth, 2001). The development of drug resistance issues of antibiotics to

various pathogens further signifies the role of herbal medicines. Now a day, due to

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historical and cultural reasons, folk medicine is still important in developing countries

due to poverty and scarce health services.

Therefore, medicinal plants has extensively used in Unani, Ayurveda and

Homeopathic medicine (Kausik et al., 2002). It is estimated that only 1% out of 0.26

million flowering plants on earth has been studied for their bio-active compounds as a

medicinal use (Verpoorte, 2000; Cox et al., 1994). The plant extracts are used for their

antibacterial, antifungal and antiparasitic properties. Use of herbal medicines are

continuously rise up due to their rich source of bioactive compounds, less side effects

and also no known resistance issue (Aburjai et al., 2001). Screening of medicinal plants

for animals infections especially for caprine anti-mycoplasmal activity are neglected

chapter. The phyto-chemical compound after manipulation provides new and improved

drugs for the treatment and management of these infectious diseases. Plants are

naturally available at every land on the earth thus provide cheaper and easily available

source for the development of new drugs discovery. The Khyber Pakhtunkhwa and

northern regions of Pakistan are gifted with large reservoirs of flora having high scope

for herbal and medicinal plants.

Azadirachta indica commonly known as “Neem” in subcontinent belong to the

family Meliaceae. The leaf, seed, bark and oil are well knwon for antiviral,

antibacterial, antifungal and antimalarial activities (Biswas et al., 2002). The U.S.

National Academy of Science in a scientific report in 1992 declared “Neem a tree for

solving global problem”. About 135 active phyto-chemical compounds like flavonoids,

terpenoids, tannins and steroids has been isolated from different parts of Neem having

strong antibacterial activity (Emran et al., 2015; Hoque et al, 2007; Talwar et al.,

1997). Calotropis procera commonly known as milk weed belong to family

Asclepiadaceae, consisted of 280 genera and 2000 species. Different active compounds

such as triterpinoids, alkaloids, resins, calotropin, anthocyanins and proteolytic

enzymes in latex, flavonoids, tannins, saponins, mudarin, sterol and cardiac glycosides

(Ali et al., 2014; Nenaah, 2013). Artemisia herba-alba belongs to the family

Asteraceae commonly known as white wormwood, consisted of 500 species are mainly

found widely in the northern hemisphere (Bremer and Humphries, 1993). Only about

30 Artemisia species are investigated for phytochemical analysis for their medicinal

uses. The plant of Artemisia is dwarf shrub, commonly grow in northern regions of

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Pakistan and also in the Western border of Pakistan including the major areas of

Afghanistan. Some important compounds like terpenin, camphor, davonone, herbalbin,

flavonoides, acetate and borneofl has been isolated from having antibacterial and

antifungal properties (Mohamed et al., 2010; Soković et al., 2010; Willcox et al., 2009;

Bakkali et al., 2008; Baser et al., 2002; Aburjai et al., 2001).

Antibiotics are normally used to treat human and animals diseases. Ruminant

respiratory pneumonia is one of the common problems and treats with variety of

antibiotics. The indiscriminate use of such antibiotics also developed drugs resistance

in multiple species of bacteria. Therefore, prior to evaluate resistance to a number of

isolated Mycoplasmas from small ruminant in Pakistan it was necessary to compare

different tests including broth micro dilution and agar well diffusion, to determine

which one would perform best and was the easy to apply for testing field isolates for

effective control. The minimum inhibitory concentration (MIC) is best assay for the

determination of efficacy of antibiotic because of its accuracy, easy to conduct and

gave quick results. It is the lowest concentration of antimicrobial agent that inhibits

visible growth of bacteria. In disc diffusion assay the antibiotics and plant extract

diffuses in agar media cultured with Mycoplasma and producing a clear zone of growth

inhibition around the disc. Therefore, the present study was aimed to probe the

therapeutic effects of different commercially available antimicrobial agents and the

indigenous medicinal plants against local isolates of Mycoplasma. The study was

consisted of two parts: to check most efficacious drug for the treatment of caprine

mycoplasmosis and to test the anti-mycoplasmal effect of indigenous medicinal plant.

The plant testing opens a new drug discovery of plant origin as an alternate source of

ruminant mycoplasmosis treatment.

The study was aimed with the following objectives;

1. Screening of commercially available antimicrobial agent for selection of most

efficacious antibiotic for the treatment of mycoplasmosis.

2. Testing of indeginous medicinal plants for anti-mycoplasmal activities against

the local isolates.

https://en.wikipedia.org/wiki/Camphor


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This study was designed to investigate the antibiogram study of commonly used

antimicrobial agents against local isolates of Mycoplasma species. Similarly, indiginous

medicinal plants were also tested for anti-mycoplasmal activities againt the local

isolates of mycoplasama of small ruminants.

5.2.1 Antimicrobial agents used in-vitro trial

The commercially available antimicrobial agents including tylosin,

oxytetracycline, gentamycin, enrofloxacin and ceftofer sodium (ICI and Ghazi

Brothers, Ltd. Pakistan) were selected for screening. The concentration of each agent

was calculated 10.0mg/mL by the formula

C1V1=C2V2

Where C1 is the weight of active ingredient, V1 is the required volume to be measured,

C2 is the desired weight of the active ingredient and V2 is the volume of PPLO broth in

which the agents are diluted.

5.2.2 Collection and identification of medicinal plants

Fresh leaves of C. procera, A. indica and aerial part of A. herba-alba were

collected from district Peshawar, Dera Ismael Khan and Abbottabad (Plate 5.4). The

plant species were identified by herbal taxonomist at Pakistan Forest Institute,

Peshawar. Leaves of collected plants were thoroughly washed with tap water followed

by final dip in distilled water. The washed clean leaves of each plant were shed dried

for 15 days separately.

5.2.3 Preparation of methanolic extract

The clean dried leaves were grinded to fine powder by electric grinder

(Moulnix, 600 W LM-240, France). Approximately, 200g grinded powder from each

plant was separately placed in 2000 mL of absolute methanol for two weeks with

regular shaking. The extracts were then filtered through muslin cloth followed by

Whatmann filter paper No.1. The methanol was removed in rotary evaporator

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(Heidolph, Laborota-4000 Germany). The dry extract kept in air tight container at 4 °C

till further use. The plant extracts (5.0 g) were dissolved in 10% DMSO (5.0 mL) in

falcon tube and mix properly with the help of vortex mixer (KMC-1300V, Korea). The

tube was then placed in water bath for 30 minutes at 40 °C to properly dissolve all

active compounds in stock solution. The final concentrations of working solution of

each plant extract were made as 05, 10, 20 and 30 mg/mL for in-vitro study.

5.2.4 Test organisms used in-vitro trial

The microorganisms used for in-vitro trial consisted of Mycoplasma mycoides

subsp. capri (Mmc) Mycoplasma capricolum subsp. capripneumoniae (Mccp) and

Mycoplasma putrefaciens (Mp) were isolated from naturally infected small ruminants.

All these isolates were previously confirmed by PCR followed by sequencing as

discribed in study-1.

5.2.5 Preparation of Mycoplasma culture

The bacterium inoculum of Mmc, Mccp and Mp was prepared as per standard

protocols of OIE, (2013). A pure and single colony of each isolate was taken from

modified Hayflick agar and then transferred into fresh Hayflick broth maintained in

sterile glass tube and incubated in CO2 (5%) at 37 °C for 24 hours. The culture growth

was adjusted to 1x104 CFU/mL for in-vitro trials as described by Hannan, (2000).

5.2.6 Determination of antibiogram assay

The antibacterial activities of the antibiotics against local isolates were carried

out by using broth microdilution method and disc diffusion assay. Five different

commonly used commercially available antibiotics comprising of enrofloxacin,

gentamycin, tylosin, oxytetracycline and ceftofer sodium were used to assess its

efficacy against the local isolates. Disc containing 10% DMSO was used as negative

control in the study.

5.2.6.1 Disc diffusion assay for antimicrobial agents

The PPLO agar plates were streaked with 10μL of Mycoplasma culture

containing 1×104

CFU/mL and allowed the plates to dry for 10-15 minutes. With the

146

help of sterile forceps each of antibiotic disc 10μg (Oxoid, England) was taken from the

dispenser and placed gently on agar plate at recommended distance of 24 mm apart

from each other and 12 mm from the edge of plate. The inoculated petri plates were

labelled and properly sealed with parrafilm. All these activities were carried out in

Biosafety cabinet level-II (ESCO, USA) to avoid contamination. The petri plates were

then incubated in 5% CO2 at 37 °C for 24-48 hours. The zone of inhibition was

recorded in millimeter (mm) around each tested disc after 24 and 48 hours. The

negative control was maintained by using disc containing 10% DMSO for comparison.

All the tested plates were made in triplicate for reproducible results.

5.2.6.2 Determination of minimum inhibitory concentration (MIC) for

antimicrobial agents and plants extract

The broth micro dilution method was conducted as described by (Hannan,

2000). The minimum inhibitory concentrations (MICs) of the different antimicrobial

were carried out in 96 well micro titration plate. The volume of PPLO media,

concentration of antibiotics and inoculum was used as described previously (Neal et al.,

2012). From the freshly prepared PPLO broth 200 μl was added to each well of micro

titration plate that were properly labelled and sterilized. Antimicrobial agents were

added to the micro titration plate in triplicate to make a final concentration of

10.0mg/mL in the first wells. Then it was serially 10 folds diluted to make the final

concentrations of 1, 0.1, 0.01, 0.001, 0.0001 mg/mL of each tested drug. In the same

way plants extract were added to the micro titration plate in triplicate to make a final

concentration of 30 mg/mL in the first wells and then serially 10 folds diluted to make

further concentrations i.e. 3.0 mg/mL, 0.3 mg/mL, 0.03 mg/mL and 0.003 mg/mL of

each extract. Enrofloxacin 1.0 mg/mL was used as positive control and added to three

designated well of the micro titration plate. Ten (10) µL of the PBS washed

Mycoplasma cell (OD600= 0.3 having 1x104 CFU/mL) was pipetted to each well of the

micro titration plate except the negative control which contained only PPLO broth. The

Optical density (Growth of the bacteria) was checked at 600 nm through ELISA reader

(Humareader Plus, 3700 Human, GmbH, Germany) before incubation (t = 0) and 48

hours (t = 48) after incubation at 37 °C with 5% CO2.

5.2.6.3 Agar well diffusion assay

The PPLO agar plates were streaked with 10.0μl of Mycoplasma culture

containing 1x104 CFU/mL. The inoculated agar plates were punched with sterile cork

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borer to make six different 6mm open well on agar surface with distance of 24mm apart

from each other and 12 mm from the edge of plate. These wells were filled with plant

extract having concentration of 05, 10, 20 and 30 mg. DMSO 10% was used as

negative control while enrofloxacin (20 μg) was used as a positive control. All these

activities were carried out in Biosafety cabinet level II to avoid contamination. The agar

plates were prepared in triplicate and properly sealed. The agar plates were then

incubated in 5% CO2 incubator at 37 °C for 24-48 hours. The zone of inhibition was

recorded in millimeter (mm) around each tested plant concentration after 48 hours. For

each plant extract three separate plates were prepared to obtained a robust data.

5.3 Statistical Analysis

Data was compiled in Microsoft Excel and analyzed through SPSS 19.0

software to check for statistical difference. Student-t test and one way ANOVA was

used to check statistical difference between different agents used as anti-mycoplasmal

assay (Steel et al., 1997). Least Significant Difference (LSD) test was used to separate

the means that were significantly different at P ≤ 0.05.

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5.4 Results

The PCR confirmed local isolates consisted of Mycoplasma mycoides subsp.

capri (n=60), Mycoplasma capricolum subsp. capripneumoniae (n=45) and

Mycoplasma putrefaciens (n=45) were subjected for chemotherapeutic trial. Five

different commercially available antimicrobial agents including enrofloxacin, tylosin,

gentamycin, oxytetracycline and ceftofer sodium were tested in-vitro as broth

microdilution and disc diffusion assay for minimum inhibitory concentration (MIC) and

zone of inhibition, respectively. The data was analyzed by Student t-test to check for

statistical significance of tested antimicrobial agents. The one way ANOVA was used

to determine level of significance among means of the tested agents.

5.4.1 Comparative efficacy of antimicrobial agents against local isolates of

Mycoplasma using disc diffusion assay

The antimicrobial effect was further investigated by disc diffusion assay against

all of the local isolates of Mycoplasma. To compare the efficacy of different

antimicrobial agents against the isolates, the data was analyzed by using one way

analysis of variance (ANOVA). The results revealed that maximum zone of inhibition

(19±0.71 mm) were produced by enrofloxacin followed by gentamycin (11±0.45 mm),

and tylosin 6.8±0.37mm against Mmc. However, it showed resistance against

oxytetracycline and ceftofer sodium which produced zone of inhibition 3± 0.32 mm

and 0± 0.00 mm (Table 5.1). In the same way antimicrobials were also tested against

Mccp. Enrofloxacin produced maximum zone of inhibition (18±0.71mm) followed by

gentamycin (12±0.55mm) and tylosin (7±0.45 mm). Oxytetracycline and ceftofer

sodium produces zone of inhibition of 4.6±0.45mm and 0±0.00 mm, respectively

(Table 5.2). Among the tested agents, enrofloxacin produced maximum zone of

inhibition of 17±0.45mm followed by tylosin 14±0.55 and gentamycin 12.4±0.51 mm

against Mp. Oxytetracycline and ceftofer sodium were least effective with zone of

inhibition 6±0.44mm and 2.4± 0.25mm (Table 5.3).

The findings revealed that enrofloxacin was most efficacious agent in-vitro against all

the three Mycoplasma species followed by gentamycin. The two local isolates Mmc and

Mccp developed resistance against the tylosin. However, it was moderately effective

against Mp. Oxytetracycline and ceftofer sodium were not effective against all the local

tested isolates.

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Table 5.1. Antimicrobial activity of commercially available agents against the local

isolates of Mmc using agar disc diffusion assay.

Antimicrobial Concentration

(μg)

Zone of inhibition

(mm)

DMSO

C-

Enrofloxacin 10 19.00±0.71a -

Tylosin 10 6.80±0.37c -

Gentamycin 10 11.00±0.45b -

Oxytetracycline 10 3.00±0.32d -

Ceftofer sodium 10 0.00±0.0 -

Means in column with different superscripts are significantly different at α =0.05 (LSD=1.20), Mmc =

Mycoplasma mycoides subsp. capri

Table 5.2. Antimicrobial activity of commercially available agents against the local

isolates of Mccp using agar disc diffusion assay.


(μg)

Zone of inhibition

(mm)

DMSO

C-

Enrofloxacin 10 18±0.71a -

Tylosin 10 7±0.45c -

Gentamycin 10 12±0.55b -

Oxytetracycline 10 4.6±0.46d -

Ceftofer sodium 10 0±0.0e -

Means in column with different superscripts are significantly different at α =0.05 (LSD=1.42), Mccp=

Mycoplasma capricolum subsp. capripneumoniae

Table 5.3 Antimicrobial activity of commercially available agents against local

isolates of M. putrefaciens using agar disc diffusion assay.


(μg)

Zone of inhibition

(mm)

DMSO

C-

Enrofloxacin 10 17±0.45a -

Tylosin 10 14±0.55b -

Gentamycin 10 12.4±0.51c -

Oxytetracycline 10 6±0.45d -

Ceftofer sodium 10 2.4±0.25e -

Means in column with different superscripts are significantly different at α =0.05 (LSD=1.3)

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5.4.2 MICs of antimicrobial agents using broth micro dilution method

The results of antibiogram assay revealed that enrofloxacin was found most efficacious

agent with lowest MICs value of 0.001, 0.001 and 0.01mg/mL against Mmc, Mccp and

Mp (Plate 5.1, 5.2, 5.3).

Plate 5.1. A 96- well micro plate showing MIC of different antimicrobial using broth micro dilution

method against Mycoplasma mycoides subsp. capri in PPLO broth. C- = row of negative

control having only PPLO broth, C+ = is positive control having PPLO broth +Mmcs

culture. The number 1,2,3,4 and 5 represent enrofloxacin, gentamycin, tylosin,

oxytetracycline and ceftofer sodium respectively. The antimicrobial dilutions were made

from top to down (arrow). MIC= the lowest antimicrobial concentration that inhibit

visible growth of Mycoplasma putrefaciens and color change from red to yellow.

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Plate 5.3 A 96- well micro plate showing MIC of different antimicrobial agents using broth micro

dilution method against Mycoplasma putrefaciens in PPLO broth. C- = row of negative

control having only PPLO broth, C+ = is positive control having PPLO broth

+Mycoplasma putrefaciens culture. A number 1,2,3,4 and 5 represent enrofloxacin,

gentamycin, tylosin, oxytetracycline and ceftofer sodium respectively. The antimicrobial

dilutions were made from top to down (arrow). MIC= the lowest antimicrobial

concentration that inhibit visible growth of Mycoplasma putrefaciens and color change

from red to yellow.

Plate 5.2 A 96- well micro plate showing MIC of different antimicrobial agents using broth micro

dilution method against Mycoplasma capricolum subsp. capripneumoniae in PPLO broth. C-

= row of negative control having only PPLO broth, C+ = is positive control having PPLO

broth +Mycoplasma capripneumoniae culture. A number 1,2,3,4 and 5 represent

enrofloxacin, gentamycin, tylosin, oxytetracycline and ceftofer sodium respectively. The

antimicrobial dilution were made from top to down (arrow). MIC= the lowest antimicrobial

concentration that inhibit visible growth of Mycoplasma putrefaciens and color change from

red to yellow.

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Plate 5.4 Indigenous medicinal plants: 1= Calotropis procera, 2= Artemisia

herba-alba, 3= Azadirachta indica.

The MICs of tylosin, oxytetracycline and ceftofer sodium against Mmc were 1,

10 and 10mg, respectively (Plate 5.1). The MIC against Mccp were 0.1, 10, 10mg for

tylosin, oxytetracycline and ceftofer sodium, respectively (Plate 5.2). The results

against Mp were recorded 0.1, 01 and 10 mg for the above three drugs, respectively

(Plate 5.3). The MIC of gentamycin was 0.01, 0.01 and 0.1mg against Mmc, Mccp and

Mp, respectively (Plate 5.1, 5.2, 5.3).

The average MICs of tested antibiotics were also calculated against all the three

local isolates of Mycoplasma. The average MIC of enrofloxacin was 0.004, 0.006 and

0.02 mg/mL against Mmc, Mccp and Mp, respectively. The MIC of gentamycin was

0.04, 0.04 and 0.28 mg/mL against Mmc, Mccp and Mp. The average MICs value of

tylosin was 2.62, 2.44 and 0.28mg/mL against the three isolates. Oxytetracycline and

ceftofer sodium were having higher MIC values of 10mg/mL against Mccp, fallowed

by 4.6mg/mL against Mmc and 8.2mg/mL against Mp. The results revealed that all the

local isolates were sensitive against enrofloxacin followed by gentamycin. The Mp was

moderately sensitive to tylosin. The isolates of Mmc and Mccp showed resistance

against ceftofer sodium with high MIC value of 10mg/mL. However, the stricking

finding of the present study was that the local isolates developed resistance against

tylosin, oxytetracycline and ceftofer sodium with high MICs value (Fig 5.1, 5.2, 5.3).

1 2 3

153

Fig 5.1 Average MICs value of different antimicrobial agents against the local

isolates of Mmc. Different letters showed significant difference at α=0.05.

Fig. 5.2 Average MICs value of different antimicrobial agents against the local

isolates of Mccp. Different letters showed significant difference at α =0.05.

a a

b

c

c

154

Fig. 5.3 Average MICs value of different antimicrobial agents against the local

isolates of M. putrefaciens.Different letters showed significant difference at α

=0.05.

5.4.3 Comperative efficacy of medicinal plants against local isolates using agar

well diffusion assay

Microbial resistance against commonly used antibiotics is one of the emergent

issues of the 21st

century which get a serious health concern throughout the world. The

development of drug resistance of antibiotics to various pathogens further signifies the

role of herbal medicines. Therefore, the anti-mycoplasmal activity of methanolic

extract of the three medicinal plants named as C. procera, A. indica and A. herba-alba

were tested as broth micro dilution and agar well diffusion assay against PCR

confirmed locally isolated pathogenic Mycoplasma species. By using disc diffusion

assay the zone of inhibition against Mmc was 10.0±0.58mm, 14.0±0.58mm and

16.3±0.33 mm at 30mg/mL for A. indica, C. procera and A. herba-alba, respectively

(Table 5.4).

155

Table 5.4 Anti-mycoplasmal activity of methanolic extract of A. indica, C. procera

and A. herba-alba using agar well diffusion assay against Mmc.

Plant

species

Concentrations

(mg/mL)

Diameter zone of inhibition (mm)

Zone of inhibition

(mm)

Enrofloxacin

C+

DMSO

C-

Azadirachta indica

05 2.3±0.33h

19±0.75

-

10 4.3±0.34g -

20 7±0.58ef

-

30 10±0.58d -

Calotropis procera

05 4±0.57g -

10 6±0.58f -

20 11.6±0.33c -

30 14±0.58b -

Artemisia herba-

alba

05 3±0.57gh

-

10 7.6±0.34e -

20 12±0.60c -

30 16.3±0.33a -

Means in column with different superscripts are significantly different at α=0.05 (LSD=1.43)

Similarly methanolic extract were tested against Mccp and maximum zone of

inhibition was recorded i.e. 12±0.32mm, 13±0.32mm and 14±0.44mm at 30mg/mL for

A. indica, C. procera and A. herba-alba, respectively (Table 5.5). It was evident from

the results that methanolic extract of all the three plants having anti-mycoplasmal

activities and produced zone of inhibition at different concentration levels and

indicating that maximum zone of inhibition was produced by using high concentration

of plant extract. The enrofloxacin was kept as positive control and produced maximum

zone of inhibition 19±0.75, 18±0.66 and 17±0.52 mm for Mmc, Mccp and Mp,

respectively. The DMSO (10%) was used as a negative control that produced no zone

of inhibition in this experiment. The A. herba-alba showed maximum zone of

inhibition 15.4±0.52mm at 30mg/mL followed by C. procera and A. indica with

14±0.58 mm and 11±0.72 mm respectively against Mp (Table 5.6).

156


and A. herba-alba using agar well diffusion assay against Mccp.

Plant species

Concentrations

(mg/mL)


Zone of inhibition

(mm)

Enrofloxacin

C+

DMSO

C-

Azadirachta indica

05 2.71±0.28f

18±0.66

-

10 5.28±0.29e -

20 8±0.33d -

30 12±0.44c -

Calotropis procera

05 3.14±0.26f -

10 5.43±0.37e -

20 12±0.31c -

30 13±0.32b -

Artemisia herba-alba

05 5±0.30e -

10 8±0.32d -

20 11.5±0.52c -

30 14±0.44a -

Means in column with different superscripts are significantly different at α =0.05 (LSD value 0.98)

157


and A. herba-alba using agar well diffusion assay against M.

putrefaciens.

Plant species

Concentration

(mg/mL)


Zone of inhibition

(mm)

Enrofloxacin

C+

DMSO

C-

Azadirachta indica

05 4±0.32h

17±0.60

-

10 5.8±0.37f -

20 11.6±0.51b -

30 14±0.58c -

Calotropis procera

05 2±0.44fg

-

10 4.4±0.51e -

20 6.8±0.37c -

30 11±0.72b -

Artemisia herba-alba

05 3±0.32gh

-

10 7.6±0.50d -

20 12±0.32c -

30 15.4±0.52a -

Means in column with different superscripts are significantly different at α=0.05 (LSD value=1.32)

5.4.4 MICs of medicinal plants extract using broth micro dilution method

Methanolic extracts of the tested plants were further analyzed by broth micro

dilution method to determine minimum inhibitory concentrations (MICs). The data was

analyzed by Student t-test to check for statistical significance of tested plants extract.

The one way ANOVA was used to determine level of significance among the tested

medicinal plants. The MICs value of the tested plant extracts were recorded 0.03, 0.3

and 03mg/mL for A. herba-alba, C. procera and A. indica, respectively, against M.

putrefaciens (Fig 5.4). The MICs values of extracts against Mmc and Mccp were 0.03,

0.3 and 0.3 mg/mL for A. herba-alba, C. procera and A. indica respectively (Fig 5.5,

5.6).

158

Fig 5.4 Anti-mycoplasmal activity of methanolic extracts of A. indica, C. procera

and A.herba-alba against the local isolates of M. putrefaciens using broth

micro dilution method. Different letters indicate statistically significant differences significant (at α =0.05).

Extracts from all plants having anti-mycoplasmal activity; however, A. herba-alba was the

most potent among tested plants with lowest minimum inhibitory concentration (0.03

mg.mL-1

).

159


and A.herba-alba against the local isolates of Mccp using broth micro

dilution method. Different letters indicate statistically significant differences (at α =0.05). Extracts from all

plants having anti-mycoplasmal activity; however, A. herba-alba was the most potent

among tested plants with lowest minimum inhibitory concentration (0.03 mg.mL-1

).

160


and A.herba-alba against the local isolates of Mmc using broth micro

dilution method. Different letters indicate statistically significant differences (at α =0.05). Extracts from all

plants having anti-mycoplasmal activity; however, A. herba-alba was the most potent

among tested plants with lowest minimum inhibitory concentration (0.03 mg.mL-1

).

161

5.5 Discussion

Bacterial pneumonia is a common and often life-threatening respiratory

problem in small ruminants. Among the bacterial infections the mycoplasmosis is the

major threat to the small ruminant population inflicting high mortality and reduce

animal production (Ongor et al., 2011; Sharif and Muhammad, 2009; Nicholas, 2002).

The Genus Mycoplasma consisted of several pathogenic species having respiratory

tissue tropism, which leads to severe respiratory syndrome in small ruminants (Manso-

Silvan et al., 2007). The disease is generally treated by different commercially

available antimicrobial agents with different degree of success (Laura et al., 2006). The

effectiveness of treatment depends upon timely response to the infection and selection

of accurate antimicrobial agent with proper dose and duration (Hirsh, 2000).

Mycoplasma has the ability to rapidly change its antigenic structure by mechanism of

switching on and off of certain surface lipoproteins. This rapid change in its antigenic

structure creates hindrance in the treatment and control of this fatal disease (Bradbury,

2005; Behrens et al., 1994). The formation of biofilm is another important feature of

Mycoplasma species that mask it and minimize the efficacy of therapy and get

resistance against antimicrobial chemotherapeutic agents (McAuliffe et al., 2006).

Therefore, many antibiotics not mitigated the infection properly and provide an

opportunity for the pathogen to persist for longer period of time.

The disease is mainly treated by commercially available antimicrobial agents

like enrofloxacin, tylosin, kanamycin, penicillin and oxytetracycline with various

degree of success (Laura et al., 2006). The microbial resistance against the common

antibiotics is an emerging issue of 21st century and poses a serious health concern

throughout the world. At the dawn of discovery of these antimicrobial agents most

pathogenic microbes were highly susceptible; however the efficacy of broad-spectrum

antibiotics has been decreased due to its indiscriminate use and acquisition of genetic

mutation in the susceptible microorganism (Gautier et al., 2002; Bradbury et al., 1994).

To address this important issue the present study was conducted to find out in-vitro the

efficacy of commonly used antimicrobial and indigenous medicinal plant against the

local isolates of Mycoplasma including M. mycoides subsp. capri (Mmc), Mycoplasma

capricolum subsp. capripneumoniae (Mccp) and Mycoplasma putrefaciens (Mp)

isolated from naturally infected small ruminants. The antimicrobial agents were tested

162

against the isolated pathogenic specie of Mycoplasma by broth microdilution method

for the determination of minimum inhibitory concertations (MICs) and disc diffusion

assay for the zone of inhibition.

The antimicrobial agents were tested by disc diffusion assay to record zone of

inhibition produced by each antimicrobial agent. The maximum zones of inhibition

recorded for enrofloxacin were 19±0.71, 18±0.71

and 17±0.45mm against Mmc, Mccp

and Mp, respectively. The gentamycin was moderately effective by using the two

assays against Mycoplasma species. Tylosin was considered a drug of choice and

extensively used in veterinary practice for the treatment of bovine, caprine and avian

mycoplasmosis since long time. The other agents like oxytetracycline and ceftofer

sodium were also used in past to treat several bacterial infections with different

outcome. However, it was found that some bacteria developed resistance against the

commonly used antibiotics. The Mycoplasma is among the few microbes that has the

ability to developed resistance to the commonly used antimicrobial agents due to its

rapid structural modulation and mutational changes. The striking findings of this study

revealed that all the local isolates of Mycoplasma were showing resistance against

tylosin, oxytetracycline and ceftofer sodium. However, tylosin was moderately against

the local isolates of Mp. This resistance to antimicrobial agents in the study area might

be due to continuous and indiscriminate use of these agents by the veterinary

professionals. The other possible reasons might be due to the poor quality of antibiotics

for animal health and the prevailing quackary practices in the country. The

oxytetracycline and ceftofer sodium produced lower zone of inhibition and having high

MIC value that indicated that the local isolates has developed resistance against these

antibiotics.

The finding of the present study revealed that enrofloxacin was the most

effective therapeutic agent with lowest MICs value of 0.001, 0.001 and 0.01mg/mL

against Mmc, Mccp and Mp respectively. These results are an agreement with the

findings of the study who reported that enrofloxacin was the most efficacious

antibacterial agent among the tested drugs against Mycoplasma infection (Hannan,

2000; NCCLS, 1999). Similar results were also illustrated in a study that enrofloxacin

being a broad spectrum antibiotic has wide range activity against several pathogenic

bacteria and Mycoplasma species of small ruminants and poultry (Ghaleh et al., 2008;

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Laura et al., 2006). In another study it was revealed that enrofloxacin was highly

effective against local isolated strain of M. bovis with lowest MIC value of 0.25-2

μg/mL (Siugzdaite et al., 2012). Similarly, the statement is further strengthen by the

findings of previous study (Al-Momani et al., 2006), who tested six antimicrobial

agents against local isolates of Mycoplasma and found the MICs value was 0.25 and

0.12 μg/mL for enrofloxacin against Mp and MmcLC, respectively. Similar findings

were also reported by Loria et al. (2003), who screened different antibiotics by broth

micro method against 24 local isolates of M. agalactiae and founded that enrofloxacin

was the most effective antibacterial agent with lowest MIC value of 0.25 μg/mL. In

other study it was revealed that enrofloxacin and its metabolites were effective against

some species of mycoides cluster like Mmc LC and Mcc (Antunes et al., 2007). The

findings of present study exhibited MICs value of 1, 10, 10mg/mL for tylosin,

oxytetracycline and ceftofer sodium, respectively. The findings of this study revealed

that broth micro dilution is a good method for the evaluation of antimicrobial agents.

This method had been successfully used for antimicrobial agents testing in-vitro by

many researchers (Jin et al. 2014; Mustafa et al., 2013; Hannan, 2000; Taylor-

Robinson and Bebear, 1997; Roberts et al., 1992).

In many countries of Europe microbial resistance is developed by different

pathogenic Mycoplasma species against oxytetracycline, tylosin and spectinomycin

(Ayling et al., 2005; Ayling et al., 2000). These results are supported by the findings

that the M. bovis has developed resistance to tilmicosin, oxytetracycline, spectinomycin

and florfenicol (Nicholas and Ayling, 2003). The major risk factors contributing in the

emergence of drug resistance in livestock population are self-medication, poor quality

and misuse of antibiotics accompanied by incomplete course of therapy (Bushra et al.,

2016; Canton et al., 2013; Mathew et al., 2007). The results are further defensed by the

facts that irrational and indiscriminate use of the commonly used drugs in the animal

practices may develop varying degree of resistance against the pathogenic bacteria

(Habila et al., 2013). However the results of the present study are contradictory with

the findings of (Al-Momani et al., 2006) who found that tylosin and erythromycin were

effective in-vitro with lowest MICs value of 0.03 μg/mL against the local isolates of

Mcc of northern Jordan. Similarly, in another study it was found that the long acting

oxytetracycline stopped mortality in CCPP infected goats soon after parenteral

administration (Giadinis et al., 2008). This disagreement of the results might be due to

164

the difference in pathogenic specie of Mycoplasma and difference in pathogens

suspected for CCPP.

The disease is treated by different commercially available antimicrobial agents

with different outcome. In the underdeveloped countries the indiscriminate use of

different antibiotics in veterinary practices are responsible for development of drug

resistance that ultimately leads to treatment failure (Canton et al., 2013; Mathew et al.,

2007; Gautier et al., 2002). To avoid and address the drug resistance problems the

medicinal plants therapy is getting importance in the recent era (Iqbal and Aqil, 2007).

Many naturally occurring phyto-compounds found in plants and herbs that possess

antimicrobial properties and act as antimicrobial agents against various pathogens

(Kumar et al., 2006). Development of new antimicrobial agents from plants could be

useful in combating the emerging resistant species of microbes with improved efficacy,

least side effects and high level of safety (Srivastava et al., 2000). Huge research

publications are available on antibacterial properties of many medicinal plants against

human and animals pathogens. However, very limited research data are available about

the antibacterial activities of herbal plants against pathogenic Mycoplasma species of

livestock. In the present study the methanolic extract of three medicinal plants were

tested against the local isolates of pathogenic Mycoplasma species including Mmc,

Mccp and Mp.

The C. procera showed moderate anti-mycoplasmal activities both in well

diffusion assay as well as broth micro dilution method. The maximum zones of

inhibition 11±0.33. 13±0.32 and 15.4±0.52 mm were produced against Mmc, Mccp and

Mp, respectively. These results indicated that high anti-mycoplasmal activity recorded

against Mp. The results are in agreement with the findings of Mako et al. (2012), who

tested leaf and root extracts of C. procera as an antibacterial agent at different

concentrations against various bacteria. The findings are further supported by previous

study (Kareem et al., 2008), who found that ethanolic extract of C. procera produced

widest zone of inhibition 14.1mm against E.coli. Similar observation were also made

by Shittu et al. (2004), who indicated that leaves of C. procera have stronger

antibacterial activity than roots against several bacteria. The results of this study is

further strengthen by the findings of (Mainasara et al., 2011) who reported that water,

ethanolic and methanolic extracts of C. procera having antibacterial activities against

165

several pathogenic bacteria including Salmonella typhi, Pseudomonas aeruginosa,

Streptococcus pyrogenes and Escherichia coli. The antimicrobial activities of C.

procera might be due to the presence of bioactive compounds like mudarin,

calotropains, saponin, glycosides, tannin, calactin and flavonoides (Tiwari et al., 2014).

The findings are supported by the facts that tannin react with cell membrane protein of

bacteria and form a stable compounds that leads to cell membrane damage which

ultimately result in killing of bacteria (Elmarie and Johan, 2001). The antibacterial

activities of flavonoids and alkaloids have been reported by other researchers (Aliero et

al., 2008; Yesmin et al., 2008).

The A. indica (Neem) has been extensively used in Homeopathic, Unani and

Ayurvedic medicine (Girish and Shankara, 2008). It has known for wide range of

therapeutic properties like antimalarial, anti-inflammatory, antifungal and antibacterial

(Hoque et al., 2007). The antibacterial and antifungal activities were reported by many

researchers against various bacteria and fungi but limited data available against

pathogenic Mycoplasma species (Adyanthaya et al., 2014). The findings of present

study indicated that A. indica at 30mg/mL produced maximum zone of

inhibition14±0.58mm against Mp. At the same concentration it was moderately

effective against Mmc and Mccp with zone of inhibition 10±0.58 and 12±0.44,

respectively. Similar results have been reported by Saba et al. (2011) who tested

metanolic and other solvent extract of A. indica against seven pathogenic bacteria with

variable antibacterial properties. Similarly, in another study it was revealed that the

bark extract showed antibacterial activities at all concentration used against

Pseudomonas aeruginosa, Corynebacterium diphteriae and Bacillus species (Yerima et

al., 2012). The findings strengthen by a study (Reddy et al., 2013), which tested

different parts of Neem and found that leaf has strong antibacterial activity at 0.5, 1.0

and 2.0mg/mL against several pathogenic bacteria.

The present results are supported by El-Hawary et al. (2013), who tested leaves

for antibacterial activity and found that it produced maximum zone of inhibition

14.3±2.1 and 13.33±1.33mm against Staph. epidermidis and Strep. pyogenes

respectively, while 11.5±1.2 and 13.5±1.4mm were recorded against Klebsiella

pneumonia and E.coli respectively. These results further supported by the findings of

166

Upadhyay et al. (2010) who reported that MICs value of tested Neem essential oil was

0.125 to 4.0 μl/mL against different Gram-negative and Gram-positive bacteria. Our

results revealed that A. indica methanolic extract exhibited minimum anti-mycoplasmal

activity at low concentration of 5mg/mL against Mycoplasma species. Similar findings

were also reported by Francine et al. (2015) that Neem extract with low concentration

not having antibacterial effect on S. aureus. The presence of various phyto-active

components in the leave of A. indica like azadirachtins, nimbidin, limonoids, azadirone,

nimbidinin, nimbinin, flavonoids, nimbidic acid, quercetin and β- sitosterol, alkaloids,

terpenoids, carotenoids and lipid might be responsible for strong antibacterial and

antifungal activity as compared with bark and seed (El-Hawary et al., 2013; Subapriya

and Nagini, 2005; Verkerk and Wright, 1993). The results are justified by the facts that

alkaloid, flavonoids and lipid were separated by TLC from aqueous extract of A. indica

and found more effective as antibacterial components against Escherichia coli and

Salmonella (Susmitha et al., 2013). The results of the present study were further

supported by a previous work (Vinoth et al., 2012), which isolated tannins, alkaloids,

flavonoids, saponins, terpenoids and glycoside from methanolic and ethanolic extract of

A. indica. They found that these phyto components having strong antibacterial activities

against several pathogenic bacteria including S. typhi, P. aeroginosa, E. coli and Staph.

aureus.

The findings of present study revealed that among the tested plants Artemisia

herba-alba was found the most effective anti-mycoplasmal agent as compared to other

tested plants. By using gel diffusion assay the A.herba-alba produced maximum zone

of inhibition 16.3±0.33, 14.0±0.44 and 15.4±0.52 mm against Mmc, Mccp and Mp,

respectively. The findings are in accordance with the results of Riachi et al. (2012) who

tested several plants of Algeria and found that hydroalcoholic extract of A.herba-alba

at 25mg/mL produced maximum zone of inhibition 13.66±3.21 and 19.00±1.00 mm

against Staphylococcus aureus and Pseudomonas aeroginosa, respectively. The anti-

mycoplasmal activities was further tested by broth micro dilution method to find out

minimum inhibitory concentration (MIC). The MICs were determined as the lowest

concentration of methanolic extracts of tested plants that preventing visible growth of

each Mycoplasma specie in the broth media. The lowest MICs value were 0.03, 0.03

and 0.3mg/mL for Mmc, Mp and Mccp respectively. The findings are supported by

167

Huda et al. (2005) who reported that methanolic extract of A. herba-alba having

stronger antibacterial activity at concentration of 16 mg/mL and produced zone of

inhibition 8, 10 and 12mm against E. coli, Pseudomonas aeruginosa and Staph. aureus,

respectively. The results are in agreement with the findings of (Al-Momani et al.,

2007), who reported that methanolic extract of Jordanian plants of A.herba-alba was

found with higher antibacterial activity against 32 local isolates of pathogenic

Mycoplasma species. Similar findings of lowest MICs value of A.herba-alba were 1.25

and 2.5 mg/mL against different gram negative and positive bacteria (Sbayou et al.,

2014). The antibacterial activities might be due to presence of phyto-active compounds

like herbalbin, davonone, flavonoides, borneol and acetate (Moufid and Eddouks, 2012;

Seddik et al., 2010). The results supported by the facts that some phyto-active

compounds like davonone, herbalbin, flavonoides, acetate and borneol have been

reported for antibacterial activity (Seddik et al., 2010; Baser et al., 2002; Yashphe et

al., 1987).

5.6 Conclusions

Among the tested antimicrobial agents, enrofloxacin was found the most

efficacious agent with lowest MIC value (0.001mg/mL) and maximum zone of

inhibition (19±0.71 mm) against Mmc.

Local isolates of Mycoplasma species has developed resistance against tylosin,

oxytetracycline and ceftofer sodium with high MICs value of 1, 10, 10mg/mL,

respectively.

Indigenous medicinal plants having anti-mycoplasmal activities, consider a

candidate for new drug development.

A. herba-alba having strong activity amongst tested plants having lowest MIC

value (0.03mg/mL) and maximum zone of inhibition 16.3±0.33 mm against

Mmc followed by Mp with lowest MIC value of 0.03mg/mL and 15.4mm

maximum zone of inhibition.



168

5.7 Recommendation

1. It is dire need of the day to establish national surveillance program for regular

testing of antimicrobial agents to establish the contemporary regimen for

effective treatment.

2. Quackary practices and indiscriminate uses of antibiotics should be discouraged

at national level.

3. Good quality of veterinary medicine should be ensured in the market for animal

health practices.

4. The indigenous plant should be screened for the isolation of phyto-active

compounds having anti-mycoplasmal activity to establish their chemical

structure for further drug development.

5. Toxicity studies should be conducted in-vivo to determine the safety indices.

6. It is essential to determine the synergetic effects of tested medicinal plants with

commonly used antimicrobial agents for effective treatment of mycoplasmosis.

169

VI. STUDY -4

TRIAL OF INDIGENOUS VACCINE DEVELOPMENT AGAINST

THE LOCAL ISOLATES OF MYCOPLASMA MYCOIDES SUB SP.

CAPRI (Mmc)

170

ABSTRACT

Immunization is an easy and effective way to control and prevent many

infectious diseases of livestock population. Mycoplasmosis is important respiratory

disease inflicting heavy losses in the small ruminant population. The present study was

aimed to prepare a saponized vaccine from the local isolates of Mycoplasma mycoides

subsp. capri (Mmc). The PCR confirmed local isolates of Mmc having 0.2mg/mL

protein content was used and inactivated with saponin at the dose rate of 3.0 mg/mL.

The indigenous saponized vaccine and commercially available lyophilized Mmc

vaccine were inoculated in experimental animals for evaluation and comparison of its

immunogenic potential. Two species of small ruminants, comprising of sheep and goats

were used for evaluation of safety and immunogenic potential of both vaccines. In 1st

trial, a total of 15 sheep were procured and divided into three groups A, B and C that

were vaccinated with indigenous saponized, lyophilized vaccine and normal saline,

respectively. Similarly, in second trial 18 goats were divided into three groups A, B and

C. The groups A and B were split further into two sub groups that served as

unchallenged and challenged groups. All animals were observed twice daily for any

clinical and physiological alteration throughout the experimental trial. The antibodies

titer was monitored by indirect haeme agglutination (IHA) for 75 days post vaccination.

In sheep the maximum antibodies titer was achieved with GMT value of 147.1 and 128

for saponized and lyophilized vaccine on day 35 post vaccination. The antibodies titer

with highest GMT value of 224 was recorded on day 28 post vaccination in a

challenged group vaccinated with saponized vaccine. However, comparatively low

GMT value of 192 was observed in challenged group vaccinated with lyophilized

vaccine. No abnormal clinical signs were observed in all experimental animals

throughout the experimental trial. It was concluded that saponin was successfully used

as inactivated agent and vaccine adjuvant for the preparation of indigenous Mmc

vaccine. It was also confirmed that the saponized vaccine prepared from local field

strain of Mmc confer better protection as compared to the commercially available

vaccine.

171

6.1 Introduction

In good husbandry practices prophylaxis is the effective tool to control and

prevent many infectious diseases of livestock. The vaccination against bacterial and

viral diseases is carried out globally with variabe success. Protection against CCPP was

shown to be possible more than a century ago when Hutcheon subcutaneously

inoculated goats with the lung extract from affected animals (McMartin et al., 1980).

Furthermore, goats vaccinated with an attenuated broth culture of Mccp the infection

from spreading (MacOwan and Minette, 1978). This clearly indicated that control

against Mycoplasma infection is possible. Since then a number of different preparations

have been produced which are reported to confer solid immunity up to one year. These

include a vaccine composed of sonicated antigens emulsified with incomplete Freund’s

adjuvant and another in which lyophilized Mccp is inactivated with saponin

immediately before immunization (Rurangirwa et al., 1987b). The saponin inactived

Mycoplasma vaccine has been successfully used in many parts of Kenya for the last

few years (OIE, 2014).

The CCPP is caused by six different species of Mm cluster and its distribution

varies in different area of the globe. Before designing control strategies it is pertinent to

know about the geographic distribution of different species of the cluster for effective

immunization and eradication. Previous research work reported that several pathogenic

species including Mcc, Mccp, Mmc, MmLC, M. agalactiae and M. putrefaciens have

been isolated from sheep and goats in Pakistan (Banaras et al., 2016; Hira et al., 2015;

Sadique et al., 2012). However limited research work has been carried out on vaccine

preparation for ruminant mycoplasmosis using different adjuvants (Ahmed, 2013;

Rahman, 2003). In the present era vaccine against Mycoplasma is available and carried

out in different areas but inspite of vaccination regular disease outbreak occurred at

every corner of the country. The failure of vaccine is justify by the facts that the

available one specie specific Mmc vaccine could not confer protection in small

ruminant population against mycoplasmosis. In a study saponin inactivated vaccine

prepared from the field isolates of Mmc has been used as prophylaxis (Shahzad et al.,

2012). In other study saponin adjuvanted inactivated M. bovis vaccine confer protection

in experimental challenged calves (Ahmad et al., 2013; Kesnil et al., 1991).

Vaccination against Mccp commercially produced in different countries of the world,

172

such as Pulmovac and capridoll (live) and CCPPV (killed) in Turkey and Ethiopia,

respectively (Samiullah, 2013).

Different adjuvants are used in the preparation of vaccines with various degree

of success. Among the inactivated agents saponin is preferred and used both as an

inactivant and adjuvant in the vaccine preparation. Saponin is an extract from the bark

of the South American tree Guillaia saponaria has been successfully used for

inactivation of Mycoplasma and also recommended for use in food animals (Ahmad et

al., 2013; Kensil et al., 1991; Mulira et al., 1988). The Mccp based vaccine is reported

to give solid immunity for 14 months against CCPP with a recommendation of a

booster dose after one year (OIE, 2014).

Therefor the present study was aimed with the following objective:

1. Preparation of saponized vaccine from the local isolates of Mmc.

2. Comparative study for the immunogenic potential of indigenous saponized and

commercially available lyophilized Mmc vaccine in experimental animals.

173


The study was aimed to prepare a saponised vaccine from locally isolated field

strain of Mycoplasma. It was further evaluated and compared for its immunogenic

potential with commercially available lyophilized Mmc vaccine.

6.2.1 Preparation of Mycoplasma vaccine

The locally isolated specie of Mycoplasma cluster the Mmc used as candidate

for indigenous vaccine preparation. The Mmc was confirmed through specie specific

primer followed by sequencing as described in (Study-1).

6.2.2 Culture preparation

The PCR confirmed culture of Mmc showing mass turbidity was sub cultured in

modified Hayflick media with 5% CO2 at 37 °C for 72 hours to obtained pure seed

culture (OIE, 2004). The pure culture of 20.0mL was taken and inoculated in 200 mL

of production media (Annexure-12). It was incubated at 37 °C with 5% CO for 5 days

to get desire turbidity and maximum growth. Culture was then taken aseptically in

biosafety cabinet and a smear was prepared on glass slide stained with Giemsa stain.

The slide was examined under microscope at 10X and 100X for the presence of any

contamination before processing to the next step (OIE, 2014).

6.2.3 Inactivation of Mmc antigen

The pure culture of 50.0 mL was taken in falcon tube and centrifuged at 12000

rpm for 15 minutes at 4 °C in refregirator centrifuge mechine (Model-2-16KC,

SIGMA, Germany). The pellet was resuspended in 0.1M phosphate buffer saline (pH

7.2) and washed three time in the same way. Supernatant was discarded and pellet was

re-suspended in 1/50th of its original volume in PBS. For the inactivation of the washed

cells, autoclaved saponin (S4521; Sigma, Aldrich®, Germany) was added at the dose

rate of 3.0mg/mL and incubated for eight hour at 37 °C (Ahmad et al., 2013; Nicholas

et al., 2004). The saponised cells were then stored at 4 °C. The vaccine was plated onto

blood agar plate and Hayflick agar to check for bacterial contamination and

Mycoplasma inactivation.

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6.2.4 Protein estimation of cultured cells

The titer of the washed cells was made to 1x108

CFU/mL as described by

Albers and Fletcher, (1982) and protein content was estimated as 0.2 mg/mL using

Bicinchoninic acid (BCA) assay (Sigma- Aldrich, Germany). A volume of 50µL of

known Bovine serum albumin (BSA) mixed with BCA reagent was taken as standard.

Similarly the test samples (Mmc pellet suspension) was also mixed with the BCA

reagent and incubated at 37 oC for 1hour. The Optical density (OD) of the standard

(BSA) and test samples were read under 562 nm wavelength through ELISA reader

(Humareader Plus, 3700 Human, GmbH, Germany). A protein concentration was

obtained from standard curve made by comparing the result of Mmc culture with

known concentration of BSA.

6.2.5 Quality control of saponized vaccine

6.2.5.1 Sterility testing of vaccine

To check sterility, the newly prepared whole cell saponized Mmc vaccine was

cultured on sensitive sterility test laboratory media like Sabourad dextrose agar,

tryptose soya broth and thioglycollate broth. Then smear was prepared on clean glass

side stained with Gram stain and examine under the microscope for bacterial

contamination.

6.2.5.2 Fluid thioglycollate medium

Thioglycollate Broth supports the rapid growth of a large variety of fastidious

anaerobe and aerobe microorganisms. It is commonly used laboratory media to check

contamination in bacterial and viral vaccine. The composition is summerized in

Annexure-13.

Method of preparation

Mix the pancreatic digest of casein, yeast extract, glucose, sodium chloride, L-

cystine, agar and water in the proportions specified above and heat until dissolved.

Dissolve the sodium thioglycollate in the solution. Add the specified quantity of

polysorbate 80 and adjust pH by adding 1.0 M of NaOH or to achive pH 7.1±0.2. If the

175

solution is not clear, heat to boiling but do not boil, and filter while hot through

moistened filter paper. Add the resazurin sodium solution and mix.

6.2.5.3 TSB Soybean-casein digest medium

The medium support a luxuriant growth of many fastidious organisms without

the addition of serum. Casein peptone and Soya peptone provide nitrogen, vitamins and

minerals. The natural sugars from soya peptone and glucose promote rapid growth of

organism. Sodium chloride is for the osmotic balance, while dipotassium hydrogen

phosphate is a buffering agent (Annexure-14).


Mix the ingredients, in the proportions specified above, warming slightly to

effect solution. Cool the solution to room temperature. Add the specified quantity of

polysorbate 80, add sufficient 1 M NaOH or 1M HCL so that after the solution is

sterilized its pH will be 7.3± 0.2. If the solution is not clear filter it through moistened

filter paper.

6.2.5.4 Mannitol Salt Agar (MSA) media

It is selective and differential media which detect the growth of several species

of staphylococcus. The composition is summarized in Annexure-15.


Add the measured ingredient in 1000 mL disttiled water and heated to disolvo

the components properly. Autoclaved at 15 Ibs at 121 °C for 15 minutes. Cool the

media to 45 °C and then gently poured a volume of 20-30mL into petri plates.

6.2.5.5 Sabourad dextrose agar media

It is commonly used laboratory media to detect any fungal contamination. In

media it detects the growth of yeast, dermatohytes and other fungi. It acidic pH inhibit

bacterial growth but promote the growth of yeast and filamentous fungi. The

composition of media is enlisted in Annexure-16.

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Suspend the measured ingredient in 1000mL of distilled water and heat to

dissolve all the components. Sterilized it by autoclaving.

6.2.6 Safety of vaccine

For safety evaluation of freshly prepared whole cell saponized Mmc vaccine

four rabbits (RI, R2, R3 and R4) were kept at experimental rabbit house VRI,

Peshawar. Three rabbits R1, R2, R3 were subjected subcutaneously with different level

of 1.0, 2.0 and 3.0mL of whole cell saponized trial vaccine, respectively. While R4

served as negative control which received 1.0mL of normal saline. The rabbits were

observed twice daily for 14 days post vaccination for any physiological and clinical

complications.

6.2.7 Vaccinal trial in experimental animals

Two species of small ruminant, sheep and goat were used as experimental

animals for testing the efficacy of indiginous saponized and commercially available

lyophilized Mmc vaccine (Plate 6.1).

6.2.7.1 Sheep grouping and vaccine inoculation

For the evaluation of immunogenic efficacy of vaccine 15 male sheep of Kari

breed about one year of age were kept at Livestock experimental farm, the University

of Agriculture, Peshawar. The animals were divided into three groups A, B and C. Each

group were housed at different premises by providing clean water, green fodder and

concentrate for a period of 15 days. All animals were dewormed with Nilzan Plus (ICI,

Pakistan) at the dose rate of 10.5mg/kg body weight. The animals were screened for

any previous Mycoplasma infection. Nasal swab were taken from all sheep cultured on

Hayflick broth for confirmation of any growth. Group A and B were inoculated with

1.0mL whole saponized Mmc vaccine and commercially available lyophilized Mmc

vaccine of VRI Lahore, respectively at subcutaneous route around thoracic area. The

booster dose of 1.0mL vaccine was given at 14th

day post vaccination (Plate 6.2). The

group C severed as negative control by injecting 1.0 mL of sterile Hayflick media.

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6.2.7.2 Goats grouping and vaccine inoculation

The indigenous vaccine was further tested for efficacy in goats of local breed.

A total of 18 animals about 6-18 months of age were kept at Arid zone small ruminant

research station Kohat. The animals were divided into three groups A, B and C,

comprising six animals each and reared in clean environment. The animals were

offered clean drinking water at labitum and feed with green fodder and concentrate. All

the groups were dewormed and examined daily for any respiratory signs for a period of

15 days. Nasal swab were taken from each animal for culturing and confirmation of

mycoplasmosis through PCR. Group A and B was divided into sub-groups, the

unchallanged group consisted of A1, A2, A3 and B1, B2, B3 that were inoculated with

1.0mL whole cell saponized vaccine and commercially available lyophilized Mmc

vaccine prepared by VRI Lahore, respectively. The booster dose of 1.0mL of both

vaccines was repeated at day 14th

post vaccination. The animals A4, A5, A6 and B4,

B5, B6 were vaccinated as per above procedure and were challenged with Mmc antigen

at the dose rate of 1x108 CFU/mL (1mL) on day 21

th post vaccination as described by

Wesonga et al. (2004). Group C severed as negative control that received 1.0mL of

normal saline.

6.2.7.3 Examination of vaccinated animals and blood sampling

After the vaccination the animals were thoroughly monitored for recording of

any clinical signs and development of lesions. The rectal temperature was recorded

daily at morning and evening and detail data of clinical examination was collected. A

blood sample of 5mL was collected in vacutainer from all experimental animals on

days 7, 14, 21, 28, 35, 42, 49, 60 and 75 post vaccinations. Collected blood samples

were allowed to stand for two hours at room temperature followed by centrifugation (Z-

300K, Hermle, GmbH, Germany) at 2500 rpm for 5 minutes to separate serum (Tuck et

al., 2009). Collected serum was taken in 1.5 mL eppendorf tube and stored at -20 °C till

further use for the detection of antibodies against Mmc through IHA as described

previously (Zahid et al., 2013; Cho et al., 1976).

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6.2.7.4 Preparation of Mycoplasma antigen

The inoculum was prepared by suspending pure and single Mycoplasma colony

taken from modified Hayflick agar media and transferred into fresh broth maintained in

sterile glass tube and adjusted the growth to 1x104 CFU/mL.

6.2.7.5 Sensitization of sheep erythrocytes (RBC)

Equal volume of washed sheep RBC was gently mixed with 0.2%

glutaraldehyde solution then incubated at 37 °C for 20 minutes. These RBCs were

washed three times with normal saline solution containing 0.1% sodium azide (Sigma-

Aldrich) and finally mixed with 0.01M phosphate buffer saline (PBS) to maintain 20%

working solution.

6.2.7.6 Indirect Haemagglutination (IHA) test

Hyperimmune sera were separately raised in sheep and goats according to

standard protocol as described by Rahman et al. (2003). IHA was performed by the

method with slight modification as described by Cho et al. (1976). Serum samples of

sheep and goats were heat inactivated at 56 °C for 30 minutes in water bath (Sakura,

Japan). Test sera of 1% of sheep and goats and control group were serially two fold

diluted in normal saline (25 µL). Sensitized treated sheep RBC 2% was mixed

separately to each serum sample in 96 well microtiteration plate. The plate was

incubated at 37 °C for one hour and read for hemagglutination. The antibody titer

against Mmc was described as reciprocal of highest dilution that showing definite

agglutination of antigen sensitized sheep RBCs.

6.2.8 Data analysis

Data was arranged in Microsoft excel sheet and geometric mean (GMT)

value was calculated for antibodies titers.

Polynomial two degree quadratic equation was used to find out relation

between the antibodies titer and the days.

Indepedenet samples t-test was applied to compare the significant difference

between the averages GMT of two selected vaccines.

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6.3 Results

6.3.1 Viable counts and protein concentration of inactivated stock culture

The Mycoplasma mycoides subsp. capri (Mmc) viable count estimated 1x108

CFU/mL from stock culture was obtained having estimated protein contents 0.2g/mL.

Thrice washed cells of Mycoplasma in 1% PBS were successfully inactivated with

3mg/mL autoclaved saponin for eight hours at 37 °C. The inactivated cell was cultured

on blood agar and modified Hayflick agar for any contamination and Mycoplasma

growth, respectively. No culture was observed on agar plates after 96 hours post

incubation that confirmed successful inactivation of Mycoplasma.

6.3.2 Sterility testing

The whole cell of Mmc saponized vaccine was further investigated for sterility.

The vaccine inoculated in sensitive media including tryptose soya broth (TSB), Fluid

thioglycollate medium (FTM), SBCDM agar and Sabourad dextrose agar. No bacterial

and fungal growth was observed after 48-72 hours post incubation at 37 °C in all the

mentioned media. The vaccine thus declared sterile and safe for in-vivo use.

6.3.3 Safety of whole cell saponized vaccine

For the safety evaluation the whole cell saponized Mmc vaccine was inoculated

into four rabbits (RI, R2, R3 and R4). All rabbits were observed twice daily for any

physiological and clinical complications like elevation in body temperature, nasal

discharge, cough, salivation, restlessness, behavior change, GIT disturbances and any

palpable swelling at the site if inoculation. After 14 days observation no adverse

clinical complication and lesions were recorded among the vaccinated and control

rabbits and vaccine declared safe for in-vivo use.

6.3.4 Estimation of antibodies titer through IHA

Serum sample from vaccinal and control groups of sheep were collected on day

0, 7, 14, 21, 28, 35, 42, 49, 56, 60 and 75 post vaccinations. The data was analyzed by

geometric mean for the calculation of IHA antibodies titer raised in sheep and goats

against whole cell saponized Mmc vaccine and commercially available lyophilized

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Mmc vaccine. The serum samples of group A, which received saponized vaccine

showed average antibodies 27.86 at day 21th

post vaccination. The antibodies titer rose

to GMT of 128, 147 and 147 on day 28, 35 and 42 respectively. GMT titer was

maintained 147 at day 35 and 42 and then decreased to 48 on day 49. The lowest GMT

titer was recorded 4.59 at day 75 post vaccination (Fig 6.1).

Fig. 6.1 Average GMT value of whole cell saponised Mmc vaccine antibodies

titer in sheep.

Fig. 6.2 Average GMT value of lyophilized Mmc vaccine VRI, Lahore

antibodies titer in sheep.

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The group B, which received Mmc vaccine of VRI Lahore showed average

antibodies GMT titer of 24.3, 84.4 and 128 at day 21, 28 and 35 post vaccination. The

titer was decreased to GMT 111.4 and 32 at day 42 and 49, respectively. The lowest

GMT value was recorded 3.5 at day 75 (Fig 6.2). The result revealed that both vaccines

produced protective antibodies in the blood of experimental sheep. However, the group

A vaccinated with whole cell culture saponized vaccine showed higher GMT value of

147 at day 35 and 42 post vaccination. The quadratic relation (R2) revealed that

saponised and lyophilized vaccine dependent 57.4% and 55% on days, respectively

(Fig 6.3).

Fig. 6.3 Comparative GMT value of whole cell saponized vaccine and

lyophilized Mmc vaccine of VRI in sheep. Independent sample T test was applied to compare average GMT of both vaccine (t

value=0.51, df=18, P=0.617), non-significant difference (P>0.05) was found between

two vaccine.

In the 2nd

vaccinal trial the immunogenic potential of two vaccines was further

evaluated in goats. In group A that received saponized Mmc vaccine the antibodies

GMT value of unchallenged animals (A1, A2, A3) was 101.59 on day 21 post

vaccination. The maximum antibodies titer of 176 was recorded on day 28 and then

gradually decreased to GMT 160, 112 and 64 on day 35, 42 and 49, respectively. The

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lowest GMT 8 was recorded on day 75. In group A of challenged animals (A4, A5, A6)

showed maximum antibodies GMT titer 224 on day 28 and maintained up to day 35

post vaccination respectively. The GMT value was gradually decreased to 176 and 128

at day 42 and 49 post vaccination. The lowest GMT was recorded as 16.8 on day 75

post vaccination. The results revealed that maximum antibodies GMT was recorded in

challenged animals as compared to unchallenged animals on day 28 and was

maintained up to day 35. The quadratic relation (R2) indicated that antibodies titer

produced by saponised vaccine in challenged and unchallenged was dependent 61.17%

and 63.5% on days, respectively (Fig 6.4).

Fig. 6.4 Average GMT value of whole cell saponized Mmc vaccine antibodies

titer in goats. Independent sample T test was applied to compare average GMT of both groups (t

value= -0.72, df=18, P=0.478), non-significant difference (P>0.05) was found between

chanllenged and unchallanged vaccinated groups.

The group B that received lyophilized Mmc vaccine of VRI Lahore was also

evaluated in this study. The antibodies GMT value of vaccinated unchallenged goats

(B1, B2, B3) were 96 on day 21 post vaccination. The GMT was increased to 160 on

day 28 and maintained up to day 35 post vaccination followed by gradual decreased to

96 and 64 on day 42 and 49, respectively. The lowest GMT value was noted as 6.3 on

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day 75 post vaccination. In group B the challenged animals (B4, B5, B6) showed

antibodies GMT titer 12.7 and 80.63 on day 14 and 21, respectively. The antibodies

titer was increased to 192 on day 28 and 35, then decreased to 160 and 96 on day 42

and 49 post vaccination, respectively. The lowest GMT titer was recorded 16 on day 75

post vaccination. The results revealed that maximum antibodies GMT was recorded

after challenge dose on day 28 and was maintained up to day 35. The quadratic relation

(R2) showed that antibodies titer produced by lyophilised VRI vaccine in challenged

and unchallenged group was dependent 60.23% and 54.81% on days, respectively (Fig

6.5).

Fig. 6.5 Average GMT value of antibodies titer of lyophilized Mmc vaccine VRI

Lahore in goats. Independent sample T test was applied to compare average GMT of both groups (t

value= -0.57, df=18, P=0.574), non-significant difference (P>0.05) was found between

chanllenged and unchallanged vaccinated groups.

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6.4 Discussion

Since the discovery of new chemotherapeutic agents the fatality of diseases are

drastically decreases and the life span of human and animals population is greatly

increased. However, some emerging and reemerging diseases are still a challenge for

the researchers and the development of drug resistance creates havoc among the

scientists. The failure of chemotherapy provides an opportunity for the researchers to

adopt control strategies for combating these fatal diseases. Immunization is the possible

way to effectively control and prevent the infectious diseases (Sumithra et al., 2013).

Similarly, several infectious diseases including mycoplasmosis can be effectively

control through specie specific vaccination (Howard et al., 1987). In the recent past a

number of human and animals diseases like polio, small pox, diphtheria and rinderpest

are completely eradicated due to efficient vaccination (Ghanem et al., 2013). Still a

number of infectious diseases are responsible for the death of millions of human and

animal population due to non-availability of effective vaccine (Curtiss, 2011). The

specie specific vaccine is useful tool to encounter many infectious diseases of livestock

population (OIE, 2004). Vaccination is cheaper than treatment, and it also ensures good

protection in many endemic diseases and maintains good herd health (Nicholas et al.,

2009). In Pakistan only one specie specific vaccine is in practice to control the CCPP in

small ruminants. However inspite of mass vaccination the disease is still wide spread in

the country causes huge economic losses. The reason for the failure of vaccine is due to

existence of several other pathogenic species of Mm cluster and non-cluster (Shahzad et

al., 2016; Hira et al., 2015; Awan et al., 2009). The other possible reasons might be due

the difference in the antigenic structure of field strains with the vaccinal strain. To

address the above issue the present study was design to prepare indigenous vaccine

from the local isolate of Mmc.

Saponin has been used successfully as adjuvant in the preparation of inactivated

vaccine from the local isolates (Nicholas, 2002; Kensil et al., 1991). Saponin inactived

Mycoplasma vaccine has been used in different regions of the world with variable

immunogenic efficacy (Nicholas and Churchward, 2012). In present study Mycoplasma

mycoides subsp. capri (Mmc) was the most prevalent (13.53%) pathogenic Mycoplasma

specie isolated from naturally infected small ruminant. Therefore, this pathogen was

considered a candidate for vaccine preparation to evaluate its immunogenic potential

186

against CCPP in the study area. Saponin inactivated Mmc vaccine was prepared and

evaluated for immunogenic potential in sheep and goats. Saponin was used at the dose

rate of 3.0mg/mL and was found effective for inactivation of Mycoplasma whole cell.

The statement is supported by the findings that saponin having both property of

inactivation Mycoplasma cell and as well as vaccine adjuvant (Ahmad et al., 2013;

Kensil et al., 1991). Similarly, saponized vaccine for contagious agalactiae has been

provided good protection as compare to formalize or heat killed vaccine. It is justified

by the facts that saponin preserved the major antigenic part in untreated Mycoplasma

whole cell (Tola et al., 1999). In another experimental study saponin at the rate of

2.0mg/mL was successfully used for inactivation of the culture of M. bovis (Ahmad et

al., 2013).

A total of 15 sheep were divided in three groups A, B and C for immunogenic

evaluation of indigenous whole cell saponized Mmc vaccine and the commercially

available lyophilized Mmc vaccine prepared by VRI, Lahore, Pakistan. At day 1st post

vaccination low antibodies titer was observed with GMT value of 1.7 through IHA in

serum of all groups. The results are supported by the findings that no antibodies were

recorded in the animals after 1st day of vaccination (Manimaran et al., 2006; Rahman et

al., 2003). The reason for low antibodies titer is due to the facts that the antibodies

needs a particular time for its activation and development. However, on day 35 and 42

post vaccination maximum antibodies titer with GMT value of 47.1 were obtained in a

group vaccinated with whole cell saponized Mmc vaccine. In contrast the lyophilized

Mmc vaccine produced maximum GMT value of 128 at day 35 post vaccination. The

results revealed that maximum antibodies titer was achieved in a group vaccinated with

locally isolated field strain. The results of the present study are justified by the facts

that vaccine prepared from any local strain of pathogen give optimum results and

confer better protection against the disease (OIE, 2014). Maximum antibodies titer was

observed at time period of 6-7th

weeks post vaccination. Similar findings were reported

that maximum antibodies titer was achieved at 6-8th

weeks post vaccination with Mmc

lyophilized vaccine in goats (Manimaran et al., 2006).

The quadratic relation (R2) revealed that saponised and lyophilized vaccine

dependent 57.4% and 55% on days, respectively. The other possible factors affected

antibodies production was not ruled out. It revealed that both vaccine having

187

immunogenic potential however more antibodies titer was recorded for saponized

vaccine. No abnormal clinical signs and pathological lesions were observed in all

vaccinated group of experimental animals during the experimental trial (75 days) that

justified the safety of locally prepared whole cell saponized vaccine. The vaccinated

sheep were recorded for mild increase in body temperature (104-104.8 °C) for 12-36

hours post vaccination. The mild increased in the body temperature justified the

immunogenic response of the whole cell saponized vaccine. The findings supported by

the fact that vaccine initially act as antigen and stimulate host immune system, trigger

several cellular and biochemical mediators responsible for elevation of body

temperature.

The goat is considered the primary host of CCPP infection among the small

ruminants with severe clinical manifestation and fatal consequences. Keeping in view

the above fact and recovery of maximum isolates of Mmc from the goat in natural

outbreak a second trial of vaccine was carried out in goats. The experiment was

conducted for the evaluation of immunogenic potential of the saponized and

lyophilized Mmc vaccine. The animals of vaccinated unchallenged group A (A1, A2,

A3) revealed the maximum antibodies GMT value of 176 on day 28 post vaccination.

The GMT value was then decreased to 160 and 112 on day 35 and 42, respectively. It

was predicted from the present study that maximum antibodies titer was achieved in

goats as compare to sheep. The minimum GTM was recorded 8 on day 75 post

vaccination. To further evaluate the efficacy and protective response of the indigenous

vaccine a trial was also conducted in animals challenged with local isolated Mmc

antigen. In this trial it was revealed that maximum GMT value of 224 was achieved on

day 28 and 35 post vaccination then the decline was recorded of GMT 176 and 128 on

day 42 and 49 post vaccination, respectively. Similar findings that indicated high

antibodies were produced by saponized vaccine at 6th

week post vaccination in goats

(Manimaran et al., 2006). The dropping in antibodies titer was continued till a

minimum titer was recorded of GMT 16.8 on day 75 post vaccination.

The findings revealed that maximum antibodies production was achieved in

challenged group as compared to unchallenged animals. It is justified by the facts that

introduction of Mmc antigen provoke fastly the immune system of host that resultantly

produced high antibodies in the serum. The quadratic equation was drawn to assess the

188

relation between the vaccinal antibodies production and days. The curve revealed that

after booster dose followed by challenged antigen high level of antibodies were

achieved and then decreased gradually up to 75 days. These results justified the

prophylactic efficacy of saponized vaccine against the local infection in experimentally

inoculated animals. The challenged pathogens are not naïve to the host immune system

resultantly produced high level of antibodies in the serum of challenged animals. All

the goats were thoroughly observed for any abnormal signs twice daily for up to 75

days. No clinical signs were observed in all vaccinated, challenged and control groups.

The findings are supported by the facts that mild clinical signs were recorded in calf

after vaccination (Nicholas et al., 2004). Such small model also recommended for

experimental trial with controlled environment is useful for the evaluation and efficacy

of vaccine in small group of challenged animals (Roth and Flaming, 1990).

Similarly the group B which received lyophilized Mmc vaccine was also

evaluated in this study. The antibodies GMT value of vaccinated unchallenged goats

(B1, B2, B3) was 96 on day 21 post vaccination. The maximum GMT was recorded

160 on day 28 and maintained up to day 35 post vaccination and then gradually

decreased to GMT of 96 and 64 on day 42 and 49, respectively. The minimum titer of

6.3 was recoded on day 75 post vaccination. In group B the vaccinated challenged

animals (B4, B5, B6) showed antibodies with GMT value of 80.63 on day 21 post

vaccination. The maximum antibodies titer of challenged goats was achieved 192 on

day 28 and 35 post vaccination.

The GMT was then slightly decreased to 160 and 96 on day 42 and 59 post

vaccination, respectively. The lowest GMT titer was recorded 16 on day 75 post

vaccination. The quadratic relation (R2) revealed that antibodies production by

saponised and lyophilized vaccine dependent 61.2% and 54.8% on days respectively in

challenged animals. The other possible factors affecting the antibodies production in

experimental animals was not ruled out. The administration of Mmc antigen stimulated

the host immune mechanism due to its earlier exposure in the form of vaccine. The

memory cells that are developed in 1st exposure of the antigen give quick response and

the activated B-cells rapidly produced high level of antibodies against the recognized

antigen. The vaccinated and challenge goats were daily observed twicely for recording

any physiological alterations like respiratory distress, GIT disturbances and pyrexia.

189

The findings revealed that no abnormal signs were noted, however moderate increase in

body temperature ranging from 104.6 to 105.2 °C for 24-48 hours in the experimental

animals were recorded post vaccination. The protection in the challenged animals is

justified by the facts that antibodies are highly specific in their action and function that

combating the antigen. The increased body temperature justified the good

immunogenic response of the whole cell saponized vaccine which is essential to

stimulate various path way of complex immune mechanism. Similar observation was

also recorded that indicated mild elevation in the body temperature after vaccination

(Stipkovits et al., 2001). Nasal swab were taken from all the challenged animals at day

14th

post infection for the detection of Mmc. No Mycoplasma was detected by PCR

from vaccinated and challenged animals. It revealed that saponized Mmc vaccine

successfully combated the infection in all the challenged animals. The results are

supported by the observation of Rurangirwa et al. (1987b), who stated that saponin

inactivated vaccine confer significant protection in the challenged experimental animals

against the infection. Similarly, saponin inactived M. bovis vaccine in an experiment

study give high protection against calf pneumonia (Ahmad et al., 2013; Nicholas,

2002). The statement are further supported by the facts saponized Mm LC and M.

agalactiae vaccine were used as prophylactic measure against contagious agalactiae in

goats (De la Fe et al., 2007). In the present study IHA was found sensitive, easy to

conduct and accurate tool for the detection of antibodies in the serum of vaccinated

animals both with saponized and lyophilized Mmc vaccine. The statement is supported

by the findings that IHA has been successfully used for the monitoring and evaluation

of several bacteria and Mycoplasma species by many researches (Rehman et al., 2013;

Gagea et al., 2006; Jaffri et al., 2006; Cho et al., 1976).

6.5 Conclusions

In experimental trial of vaccination in sheep the high antibodies titer with

maximum GMT value of 147 and 128 was recorded on day 35 post vaccination

for whole cell saponized and lyophilized Mmc vaccine, respectively.

High antibodies titer with maximum GMT value of 224 was recorded on day

35th

post vaccination in experimentally inoculated challenged goats reflecting

the potency of saponised Mmc indigenous vaccine.

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Lyophilized Mmc vaccine in challenged goats produced high antibodies titer

with GMT value of 196 on day 35th

post vaccination.

The indigenous whole cell saponized vaccine showed high immunogenic

potential in sheep and goats.

Zero mortality and no clinical signs of disease in the challenged animals

justified the safety and immunogenicity of the locally developed saponized

Mmc vaccine.

The saponin was effectively used as an inactivated agent and vaccine adjuvant

in the preparation of Mmc vaccine.

6.6 Recommendation

1. Trivalent vaccine of the three local isolates needs to be developed for effective

control of the diseae.

2. Further study is needed to determine the antigenic protein of the local isolate for

the development of sub unit and recombinant vaccine.

191

VII. SUMMARY

In the first study the isolation and molecular identification of three pathogenic

Mycoplasma species were successfully conducted. The study was carried out to identify

and characterize the pathogenic member of Mycoplasma mycoides cluster and non-

cluster species in small ruminants of three different climatic regions of

KhyberPakhtunkhwa, Pakistan. A total of 1980 samples consisted of nasal discharge

(n=1500), tracheal swabs (n=300), lungs tissues (n=147) and pleural fluids (33) were

collected from animals exhibiting respiratory sings suspected for Contagious Caprine

Pleuro pneumonia (CCPP). The collected samples were taken in transport media and

grown on modified Hayflick media incubated at 37 and 5% CO2 for 7-12 day. Out of

total samples, 737 (37.22%) showed mass turbidity and whirling movement in Hayflick

broth, while 667 (33.68%) were positive for the growth of a characteristic Mycoplasma

colonies on solid media across different climatic zones. The results revealed that

significantly (P˂0.001) higher isolates of Mycoplasma were obtained from northern

(43%) followed by southern zone (34.6%) of the study area.

The different ages, sex and species of animals were investigated for the

prevalence of CCPP. It was observed that the disease was more prevalent in young kids

up to one year of age, comprising 33.3% in sheep and 47.6% in goat’s kids. It was also

revealed that disease was significantly (P˂0.01) high in goats (58.75%) as compared to

sheep (41.24%). Similarly, high prevalence of disease was recorded in female (39%) as

compared to male 30.33%. In the country most farmers are adapted mixed farming and

keeping sheep and goats together which increases the chances of dissemination of

disease among the two species. The high prevalence of disease in female animals may

be due to various contributing factors including lactation, gestation and estrus cycle

responsible for development of stress that in turn targeted the immune mechanism and

predispose the animal to opportunistic pathogens like Mycoplasma.

The isolates were further subjected for molecular identification through specie

specific primers for mycoides cluster and non-cluster. Out of total, 553 (27.92%) were

confirmed as Mycoplasma with species distribution of 13.53%, 5.5% and 7.97% for

Mycoplasma mycoides subsp. capri (Mmc), Mycoplasma capricolum subsp.

capripneumoniae (Mccp) and M. putrefaciens (Mp), respectively. The samples were

192

collected from different sources to explore the best site for Mycoplasma isolation. It

was revealed that highest number of isolates were confirmed from pleural fluids

(63.6%) followed by lungs tissue (58.5%) and least from tracheal swabs (21%). On

sequencing of the amplified DNA of local isolates showed maximum sequence

homology 99% of 16S-rRNA gene of Mccp with the strains of neighbour countries. The


of USA and France but closely related with the strain of neighbour countries like India

and China. Similarly, the the local isolates of Mmc showed homology with MmLC

strain of Switzerland. The local strain of Mp exhibited similaraties with the strain of

USA. The classical findings of the study was the 1st time confirmation of three

pathogenic Mycoplasma species in the study area.

In the 2nd

study, a total of 1800 animal suffering from respiratory syndrome

suspected for mycoplasmosis were investigated for recording of the clinico-

pathological picture of diseases in naturally infected sheep and goats. Similarly, 180

dead animals were examined on post mortem examination for gross and

histopathological study. Different pathogenic species of Mm cluster and non-cluster are

responsible for the disease with severe clinico-pathological outcome. The clinical

manifestation of disease revealed that respiratory signs were more prominent in

diseased animals followed by other systemic involvement. Out of total examined

animals pneumonia was recorded in 61.55% animals, followed by pyrexia (58.2%),

coughing (56.83%), watery nasal discharge (52.22%) and lacrimation (40.77%). The

other clinical findings consisted of diarrhoea (22.33%), mastitis (3.7%), nervous signs

(1.6%) and abortion (1.27%). The overall mortality was recorded 15.72% in infected

animals.

The different pathogenic species of Mycoplasma has the ability to produced

lesions in various tissue, organs and system of the host. Pathomorphological study

revealed that majority of the animals exhibited lesions in the respiratory system

followed by GIT, urinary and nervous system. The most frequent lesions were recorded

in the lungs 53.88% followed by trachea 37.7% and pleural effusion 18.33%. The

multisystemic involvement of the disease was the frequent feature in lesions

distribution comprising of nephritis 18.335%, hepatitis 17.22%, pericarditis 12.2% and

spleenitis 6.11%. In few cases synovitis 6 (3.33%) and meningitis 3 (1.66%) was noted.

193

On histopathological examination majority of lungs sections showed

emphysema, atelectasis, thickning of alveolar wall and extensive leukocytic

infilteration. Some section also showed chronic inflammatory changes consisted of

aggregation of macrophages, fibroblast and plasma cells. The multi-systemic

involvement is the common feature of the findings. The other internal organs including

liver, spleen, kidneys and intestine revealed congestion, hemorrhages and accumulation

of inflammatory cell. Few brain sections showed mild congestion and leukocytic

infilteration, howevere most of the brains were presenting normal histological detail.

The gross and microscopic lesions scoring revealed the high pathogenic nature of

infection and multisystemic involvement justified the prevalence of several pathogenic

Mycoplasma species in study area. The lesions scoring revealed that respiratory tissues

of sheep and goats were more severly infected in Mycoplasma infection. The overall

lesions scoring revealed more severe nature of disease in goat as compared to sheep.

The 3rd

study was conducted for the evaluation and effectiveness of different

commercially available antibiotics and indigenous medicinal plants extract for

antimicrobial activity against the local isolates. Five different commercially available

antimicrobial agents including tylosin, oxytetracycline, enrofloxacin, gentamycin and

ceftofer sodium and three medicinal plants including Calotropis procera, Azadirachta

indica and Artemisia herba-alba were tested in-vitro by disc diffusion assay, agar well

diffusion and broth microdilution. The results revealed that maximum zone of

inhibition 19±0.71mm was produced by enrofloxacin followed by gentamycin

11.0±0.45 mm and tylosin 6.8±0.37 mm against Mmc. The isolates showed resistance

against oxytetracycline and ceftofer sodium which produced zone of inhibition 3.0±

0.32 mm and 0± 0.00 mm, respectively.

The antimicrobial effects were further investigated by broth mico dilution

method against all the local isolates of Mycoplasma. The results revealed that

enrofloxacin exhibited strong antibacterial activity with minimum inhibitory

concentrations (MICs) values of 0.001, 0.001 and 0.01mg/mL against Mycoplasma

mycoides subsp. capri (Mmc), Mycoplasma capricolum subsp. capripneumoniae

(Mccp) and Mycoplasma putrefaciens (Mp), respectively. The gentamycin was the

second effective agent with lowest MICs value of 0.01, 0.01 and 0.1mg/mL against

194

Mmc, Mccp and Mp, respectively. Interestingly, all the isolates showed resistance

against tylosin, oxytetracycline and ceftofer sodium with high MIC values.

Among the tested methanolic plant extract A. herba-alba showed maximum

zone of inhibition 16.33±0.33, 14.00±0.44 and 15.40±0.12mm at 30mg against Mmc,

Mccp and Mp, respectively. However, it was revealed that C. procera and A. indica

were moderately effective against the three species of Mycoplasma. It was concluded

that local isolates developed resistance to the commonly used antimicrobial agent like

tylosin, oxytetracycline and ceftofer sodium. However, enrofloxacin was found the

most potent agent for the treatment of caprine mycoplasmosis. Among the tested

medicinal plants A. herba-alba was showing high anti-mycoplasmal activity to all local

isolates of Mycoplasma. It was concluded from the results that medicinal plants can use

as alternative source for the treatment of ruminant mycoplasmosis.

In study 4th

indigenous vaccinal trial was conducted to prepare a saponized

vaccine from the local isolates of Mycoplasma mycoides subsp. capri (Mmc). The PCR

confirmed local isolates of Mmc having 0.2mg/mL protein content was inactivated with

saponin at the dose rate of 3.0mg/mL. The indigenous saponized vaccine and

commercially available lyophilized Mmc vaccine were inoculated in experimental

animals for evaluation and comparison of its immunogenic potential. Two species of

small ruminants i.e., sheep and goats were used for evaluating the safety and

immunogenic potential of both vaccines.

All animals were observed twice daily for any clinical and physiological

alteration throughout the experiment and the antibodies titer was monitored by IHA for

75 days post vaccination. In sheep the maximum antibodies titer was achieved with

GMT value of 147.1 and 128 for saponized and lyophilized vaccine on day 35 post

vaccination. The antibodies titer with highest GMT value of 224 was recorded on day

28 post vaccination in a challenged group vaccinated with saponized vaccine. However,

comparatively low GMT value of 192 was observed in challenged group vaccinated

with lyophilized vaccine. No abnormal clinical signs were observed in all experimental

animals throughout the experimental trial. It was concluded that saponin was

successfully used as inactivated agent and vaccine adjuvant for the preparation of

indigenous Mmc vaccine. It was conclused from the vaccinal trials that saponized Mmc

vaccine might be successfully used to encounter the infection and found as good as the

commercially available vaccine.

195

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ANNEXURE

Annex-1

PERFORMA FOR SAMPLE COLLECTION FROM SUSPECTED ANIMALS

S/No_________ Climatic Zone _______________ District _________________

Name of Farmer/owner________________________________________________

Address____________________________________________________________

Animal type /species _________________________________________________

Breed ________________________________Sex ________________Age_______

Flock size_______________ Number of sample_____________ Month __________

Husbandry practice: ___________________________________________________

Nature of sample taken: Nasal swab_______________ Tracheal swab___________

Lungs tissue ________________ Pleural fluids _______________ Serum_________

History of disease: ____________________________________________________

____________________________________________________________________

Vaccination: Yes__________________________ No_________________________

Signs and symptoms observed: __________________________________________

___________________________________________________________________

___________________________________________________________________

History of previous medication: _________________________________________

___________________________________________________________________

Post-mortem lesions: __________________________________________________

___________________________________________________________________

Morbidity: _______________________ Mortality __________________________

Treatment advised____________________________________________________

___________________________________________________________________

235

Annex-2 Composition of modified Hayflick medium (HiMedia, India)

Autoclave portion (Autoclaved at 121°C for 15 min)

Name of chemical Concentration/Volume

PPLO broth (Sigma, Aldrich, Germany) 21 g

Deionized water 700 mL

Agar (Sigma, Aldrich, Germany) 1% (w/v)

Membrane filtration portion (Filtered through 0.45 mµ)

Horse serum (inactivated at 560C for 30min) (Sigma,

Aldrich, Germany)

200 mL

Fresh yeast extracts (25%) (Sigma, Aldrich, Germany) 100 mL

Glucose graded (50%) (Sigma, Aldrich, Germany) 4 mL

Sodium pyrovate (25%) (Sigma, Aldrich, Germany) 8 mL

Thallium acetate (10%) (Sigma, Aldrich, Germany) 4 mL

Ampicillin (Glaxo welcome, Karachi, Pakistan)) 250 mg

Phenol red (1%) (Sigma, Aldrich, Germany) 4 mL

pH 7.8

Annex-3 Phosphate buffered saline

Salt Concentration (mmol/L) Concentration (g/L)

NaCl 137 8.00

KCl 2.7 0.20

Na2HPO4 10 1.44

KH2PO4 1.76 0.24

Ph 7.4 7.4

236

Annex-4 PCR Mixture

COMPONENT STOCK CONC. FINAL CONC. VOLUME

10X Taq Buffer 10X 1X 2.5 µl

dNTPs Mix 2.5 mM each dNTP 0.2 mM each dNTP 2.5 µl

MgCl2 25 Mm 1.5 mM 2.5 µl

Taq Polymerase 5 U/µl 2.5 U/µl 0.3 µl

Primer, Forward 100 µmoles/µl 4 µmoles 1 µl

Primer, Reverse 100 µmoles/µl 4 µmoles 1 µl

DNA N/A N/A 5 µl

DNAase- Free Deionized

Water

N/A N/A 10.2 µl

Total Master Mix Volume 25 µl

Annex-5 PCR condition

35 cycles

94oC 94

oC 72

oC 72

oC

3 min. 30 Sec.

45 Sec. 12min.

56oC 4

oC

30 Sec. ∞

Annex-6 TBE buffer


Tris acetate (pH 7.5) (Sigma, Aldrich, Germany) 0.4 mM

EDTA (pH 8.0) (Sigma, Aldrich, Germany) 20 mM

237

Annex-7 Composition of neutral buffered formalin (10%)


Formalin 37% (Scharlau, Barcelona, Spain) 100 mL

Sodium Acid Phosphate (Scharlau, Barcelona, Spain) 4.0 g

Anhydrous Sodium Phosphate (Scharlau, Barcelona, Spain) 6.5 g

Distilled Water 900 mL

Annex-8 Hematoxyline stain (Scharlau, Barcelona, Spain)


Hematoxyline (dark crystals) 10 g.

Water 70-80 °C 500 Ml

Alum (potassium alum) 20 g.

Thymol (crystals) 1 g

Annex-9 Acid Alcohol (Scharlau, Barcelona, Spain)


NaCl 0.5g

Distilled water 25mL

Methanol 75mL

Concentrated HCl 0.5mL

Annex-10 Ammonia alcohol (Scharlau, Barcelona, Spain)


Lauryl sulfate detergent salt 0.1-6% w

Bisulfite 5-15% w

Ethanol 20-40% w

Ammonia 2-10% w

Annex-11 Eosin stain (Scharlau, Barcelona, Spain)


Eosin crystals 10 g.

Distilled Water 70-80 °C 1000 mL

238

Annex-12 Vaccine production media

Name of chemical/ solution Concentration/Volume

Horse serum 20%

Seed culture of Mmc 20%

Hayflick broth 60%

Annex-13 Composition of Fluid thioglycollate media (Oxoid, England)

Chemical name Quantity/ volume

Pancreatic Digest of Casein 15.0 g

Yeast Extract (water-soluble) 5.0 g

Glucose monohydrate/anhydrous 5.5 g/5.0 g

Sodium chloride 2.5 g

L-Cystine 0.5 g

Sodium thioglycollate 0.5 g

0.1% Resazurin Sodium Solution (freshly prepared) 1.0Ml

Granulated Agar (moisture not more than 15%) 0.75 g


Polysorbate 80 (optional) 5.0 Ml

Annex-14 Composition of TSB Soybean-casein digest media (Merck, Germany)


Pancreatic Digest of Casein 17.0 g

Papain Digest of Soybean Meal 3.0 g

Glucose monohydrate/anhydrous 2.5 g/2.3 g


Dipotassium hydrogen phosphate, K2HPO4 2.5 g

Polysorbate 80 5.0 mL


239

Annex-15 Mannitol Salt Agar (MSA) media (Oxoid, England)



Mannitol 10.0 g

Beef extract 1.0 g

Phenol red 0.025 g

Agar 15.0 g


Annex-16 Sabourad dextrose agar media (Oxoid, England)


Dextrose (Glucose) 40.0 g

Peptone 10.0 g

Agar 15 g


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