pathogenesis, molecular characterization, chemotherapy and vaccine...
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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
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)
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 Materials and methods ......................................................................... 144
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 Materials and methods ......................................................................... 173
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
5.2 Antimicrobial activity of commercially available agents against local
isolates of Mycoplasma capricolum subsp. capripneumoniae using agar
disc diffusion assay. ......................................................................................... 149
5.3 Antimicrobial activity of commercially available agents against local
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
5.5 Anti-mycoplasmal activity of methanolic extract of Azadirachta indica,
Calotropis procera & Artemisia herba-alba using agar well diffusion
assay against Mycoplasma capricolum subsp. capripneumoniae. ................... 156
5.6 Anti-mycoplasmal activity of methanolic extract of Azadirachta indica,
Calotropis procera & Artemisia herba-alba using agar well diffusion
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
5.2 Average MICs value of different antimicrobial agents against the local
isolates of Mccp. ............................................................................................... 153
5.3 Average MICs value of different antimicrobial agents against the local
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
5.5 Anti-mycoplasmal activity of methanolic extract of A. indica, C. procera
and A.herba-alba against the local isolates of Mccp ....................................... 159
5.6 Anti-mycoplasmal activity of methanolic extract of A. indica, C. procera
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
5.2 MIC of different antimicrobial agents using broth micro dilution method
against Mycoplasma capricolum subsp. capripneumoniae in PPLO broth. ... 151
5.3 MIC of different antimicrobial agents using broth micro dilution method
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
PATHOGENESIS, MOLECULAR CHARACTERIZATION,
CHEMOTHERAPY AND VACCINE DEVELOPMENT FOR
MYCOPLASMOSIS IN SMALL RUMINANTS
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
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
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
3.4.1 Biochemical tests
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
3.7.2 Biochemical tests
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
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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
***Highly significant, NS= Non significant
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.
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Table 3.13 PCR result for confirmation of M. putrefaciens and proportional
difference using Z- test analysis for different climatic zones
Pairs Prop. Difference Z- value P- value
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***
***Highly significant, NS= Non significant
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
Pairs Prop. Difference Z- value P- value
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
***Highly significant, NS= Non significant
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
multiple alignment. The phylogenetic tree was constructed by neighbor-
joining algorithm using the software MEGA version 7.2.
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
multiple alignment. The phylogenetic tree was constructed by neighbor-
joining algorithm using the software MEGA version 7.2.
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
constructed tree indicated that the local isolated field strain is different from the strains
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.
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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 Materials and Methods
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).
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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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6. Ethyl alcohol 50% 1 min
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
13. Ethyl alcohol 100% 1 min
12. Ethyl alcohol 100% 1 min
vi. Counter staining
11. Eosin (Annexure-11) 1 min
vii. Dehydration
10. Ethyl alcohol 75% 1 min
9. Ethyl alcohol 100% 1 min
20. Ethyl alcohol 100% 1 min
19. Ethyl alcohol 100% 1 min
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.
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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.
111
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).
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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
3 Pneumonia 97 53.88
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
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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
3 Pneumonia 40 67.77
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).
121
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).
122
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).
123
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).
124
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.
127
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
130
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
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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.
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5.2 Materials and Methods
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
147
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.
Antimicrobial Concentration
(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.
Antimicrobial Concentration
(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
Table 5.5 Anti-mycoplasmal activity of methanolic extract of A. indica, C. procera
and A. herba-alba using agar well diffusion assay against Mccp.
Plant species
Concentrations
(mg/mL)
Diameter zone of inhibition (mm)
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
Table 5.6 Anti-mycoplasmal activity of methanolic extract of A. indica, C. procera
and A. herba-alba using agar well diffusion assay against M.
putrefaciens.
Plant species
Concentration
(mg/mL)
Diameter zone of inhibition (mm)
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
Fig 5.5 Anti-mycoplasmal activity of methanolic extracts of A. indica, C. procera
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
Fig 5.6 Anti-mycoplasmal activity of methanolic extracts of A. indica, C. procera
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;
163
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,
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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.
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6.2 Materials and Methods
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
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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).
Method of preparation
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.
Method of preparation
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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Method of preparation
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.
184
185
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
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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
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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.
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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
constructed tree indicated that the local isolated field strain is different from the strains
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
Name of chemical Concentration/Volume
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%)
Name of chemical Concentration/Volume
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)
Name of chemical Concentration/Volume
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)
Name of chemical Concentration/Volume
NaCl 0.5g
Distilled water 25mL
Methanol 75mL
Concentrated HCl 0.5mL
Annex-10 Ammonia alcohol (Scharlau, Barcelona, Spain)
Name of chemical Concentration/Volume
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)
Name of chemical Concentration/Volume
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
Distilled Water 1000 mL
Polysorbate 80 (optional) 5.0 Ml
Annex-14 Composition of TSB Soybean-casein digest media (Merck, Germany)
Chemical name Quantity/ volume
Pancreatic Digest of Casein 17.0 g
Papain Digest of Soybean Meal 3.0 g
Glucose monohydrate/anhydrous 2.5 g/2.3 g
Sodium chloride 5.0 g
Dipotassium hydrogen phosphate, K2HPO4 2.5 g
Polysorbate 80 5.0 mL
Distilled Water 1000 mL
239
Annex-15 Mannitol Salt Agar (MSA) media (Oxoid, England)
Chemical name Quantity/ volume
Sodium chloride 40.0 g
Mannitol 10.0 g
Beef extract 1.0 g
Phenol red 0.025 g
Agar 15.0 g
Distilled Water 1000 mL
Annex-16 Sabourad dextrose agar media (Oxoid, England)
Chemical name Quantity/ volume
Dextrose (Glucose) 40.0 g
Peptone 10.0 g
Agar 15 g
Distilled Water 1000 mL