SEROLOGICAL AND MOLECULAR STUDIES OF MYCOPLASMA … PHD THESI… · Figure 1: Phylogenic tree of...

i

SEROLOGICAL AND MOLECULAR STUDIES OF MYCOPLASMA MYCOIDES

MYCOIDES SMALL COLONY IN NORTHERN NIGERIA

By

DR. NWANKPA NICK DOUGLAS PG/PHD/03/34614

DEPARTMENT OF VETERINARY PATHOLOGY AND MICROBIOLOGY

UNIVERSITY OF NIGERIA, NSUKKA

FEBRUARY, 2008

ii

SEROLOGICAL AND MOLECULAR STUDIES OF MYCOPLASMA MYCOIDES

MYCOIDES SMALL COLONY IN NORTHERN NIGERIA

By

DR. NWANKPA NICK DOUGLAS PG/PHD/03/34614

A THESIS SUBMITTED TO THE DEPARTMENT OF VETERINARY PATHOLOGY AND MICROBIOLOGY, FACULTY OF VETERINARY MEDICINE IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF THE DEGREE OF DOCTOR OF PHILOSOPHY IN VETERINARY MICROBIOLOGY UNIVERSITY OF NIGERIA, NSUKKA

FEBRUARY, 2008

iii

CCEERRTTIIFFIICCAATTIIOONN

We certify that Dr. Nwankpa, Nick Douglas carried out this research work in the

Department of Veterinary Pathology and Microbiology at the University of Nigeria

Nsukka.

The work presented herein is original and has not been previously reported anywhere

else.

__________________________

Professor, S. I. Oboegbulem

(Supervisor)

__________________________

DATE

__________________________ ____________________________

Dr. J. I. Ihedioha Prof. G. O. Egwu

(Head of Department) (External Examiner)

__________________________ ____________________________

DATE DATE

iv

DDEEDDIICCAATTIIOONN

To the very few who live a life of service to mankind and derive pleasure in touching

positively the lives of people and bringing sunshine into their lives.

v

AACCKKNNOOWWLLEEDDGGEEMMEENNTTSS

I would like to thank the National Veterinary Research Institute Vom for financing my

PhD program, and Centre International Recherché Agronomic pour Development

(CIRAD) Montpellier, France for financing the bench work carried out in their

laboratory. I am most grateful to Dr. (Mrs.) L. H. Lombin, Chief Executive of NVRI

Vom. She was truly the driving force behind this work. Her resilience and motivation

were a great source of encouragement and inspiration to me. She is a mother, a lady and

a leader. I am grateful and truly honoured to be associated with her.

I wish to thank my supervisors Prof. S. I. Oboegbulem for accepting me as his student

and for the constant support, advice, and providing directions during the research and

writing of this thesis. Sincere thanks also go to Dr. K. F. Chah for the dedication,

constructive criticisms, and encouragement during the work.

I wish also to thank my supervisor at CIRAD Dr. Francois Thiaucourt, the head of

Bacteriology and World Reference Laboratory for Contagious Bovine Pleuropneumonia

(CBPP) and Dr. Manso-silvan Lucia. Their technical skills, ideas, determination, and

commitment made this work worthwhile. I also thank all the other members who have

also contributed (Lorenzon Sophie, Armelle Peyraud, Woubit Sallah, Marie Caroline,

and Yaya Abubakar).

I wish to express my appreciation to my superiors and colleagues in NVRI, Dr. A.A.

Makinde, Dr. J. U. Molokwu, Dr. David Shamaki, Dr. P. A. Okewole, Dr. Atanda

Olabode and others too numerous to mention.

vi

I also thank all the staff, Faculty of Veterinary Medicine, University of Nigeria, Nsukka,

Dr. S.V.O. Shoyinka, Dr. J.I. Ihedioha, and Dr. J.I. Onunkwo. I appreciate all my

friends too numerous to mention especially Dr. C. O. Nwaigwe and his Lolo for their

very wonderful hospitality at Nsukka, and Mr. Sunday Makinde, Bacterial Vaccine

Production, always there to lend a helping hand, you are truly a friend and a brother.

I truly wish to appreciate my darling wife, Veronica Rume Nwankpa for her love,

understanding, and support. I thank her particularly for accepting the PhD as a genuine

excuse for me to leave my home, sometimes at very odd periods and for the beautiful

bouncing baby girl Michelle Fatima Nenpin whose delivery coincided with the end of

the work.

Finally, I thank our heavenly father for giving me the grace to contribute to the benefit

of mankind.

vii

________________________________________________TABLE OF CONTENTS

TITLE PAGE _________________________________________________________ i

CERTIFICATION ____________________________________________________ iii

DEDICATION _______________________________________________________ iv

ACKNOWLEDGEMENTS _____________________________________________ v

TABLE OF CONTENTS _____________________ Error! Bookmark not defined.

LIST OF FIGURES _________________________ Error! Bookmark not defined.

LIST OF PLATES __________________________ Error! Bookmark not defined.

LIST OF TABLES ______________________________________________ xiii

ABSTRACT___________________________________________________xiv

CHAPTER ONE ___________________________ Error! Bookmark not defined.

INTRODUCTION ______________________________________________________ 1

1.1 Background of the Study _________________________________________ 1

1.2 Statement of the Problem _________________________________________ 2

1.3 Aims and Objectives ____________________________________________ 3

1.4 Relevance and Justification ______________________________________ 4

CHAPTER TWO _______________________________________________________ 5

LITERATURE REVIEW ________________________________________________ 5

2.1. The Mycoplasmas ______________________________________________ 5

2.1.1 Phylogeny and Genome characteristics ___________________________________ 8

2.2 Contagious Bovine Pleuropneumonia (CBPP) ______________________ 13

2.2.1 Historical Perspective ___________________________________________________ 13

2.2.1.1. CBPP in Africa _____________________________________________________ 15

CBPP in Tanzania _______________________________________________ 18

CBPP in Botswana _______________________________________________ 18

CBPP in Nigeria _________________________________________________ 19

2.2.2 Transmission and spread of CBPP ______________________________________ 21

viii

2.2.4 Clinical Signs __________________________________________________________ 24

2.2.4 Pathology ______________________________________________________________ 28

2.2.5 Serology _______________________________________________________________ 28

2.2.6 Molecular diagnosis ____________________________________________________ 31

2.2.7 Economic importance ___________________________________________________ 33

2.2.8 Vaccines ________________________________________________________________ 34

2.3 Mycoplasma mycoides cluster ____________________________________ 37

2.3.1 Taxonomy and Phylogeny of the Mycoides cluster ________________________ 38

2.3.2 Pathogenicity of members of the M. mycoides cluster _____________________ 40

2.3.2.1 Capsular polysaccharide ______________________________________________ 40

2.3.2.2 Hydrogen peroxide ___________________________________________________ 41

2.3.2.3 Variable surface protein _______________________________________________ 41

2.4 Molecular methods for the identification and characterization of

Mycoplasmas ________________________________________________ 42

CHAPTER THREE ___________________________________________________ 50

SEROLOGICAL STUDIES _____________________________________________ 50

3.1 Experiment 1: Identification of MmmSC in twelve States of Northern Nigeria

by competitive Enzyme Linked Immuno-Sorbent Assay (c-ELISA). ____ 50

3.1.1 Introduction ___________________________________________________________ 50

3.1.2 Materials and Methods _______________________________________________ 51

3.1.2.1 Competitive Enzyme Linked Immuno-Sorbent Assay (c-ELISA) ____ 54

3.1.3 Results _________________________________________________________________ 54

3.1.4 Discussions ____________________________________________________________ 56

CHAPTER FOUR _____________________________________________________ 60

ISOLATION AND IDENTIFICATION OF MMSC in _______________________ 61

northern nigeria ______________________________________________________ 61

4.1 Experiment 2 : Isolation of Mycoplasma mycoides mycoides ______Small

Colony _____________________________________________________ 61

4.1.1 Introduction ___________________________________________________________ 61

ix

4.1.2 Materials and Methods ________________________________________________ 61

3.2 .1 Culture and isolation _________________________________________________ 62

4. 1.3 Results ________________________________________________________________ 62

4 .1.4 Discussions ___________________________________________________________ 64

4.2 Experiment 3: Identification and confirmation of Mycoplasma mycoides

mycoides Small Colony isolates by specific Polymerase Chain Reaction

(PCR). _____________________________________________________ 67

4.2.1 Introduction ___________________________________________________________ 67

4.2 .2 Materials and Methods ________________________________________________ 68

Extraction of DNA ___________________________________________________ 68

Diagnostic PCR for the detection of MmmSC ________________________ 68

Agarose (2%) gel electrophoresis _____________________________________ 69

4.2.3 Results _________________________________________________________________ 69

4.2.4 Discussions _____________________________________________________________ 75

4.3 Experiment 4: Identification of isolates by MmmSC Specific QPCR ____ 75

4.3.1 Introduction ___________________________________________________________ 75


4.3.3 Results _________________________________________________________________ 78

4.3.4 Discussions _____________________________________________________________ 81

CHAPTER FIVE_____________________________________________________82

5.0 MOLECULAR CHARACTERIZATION OF MMSC ISOLATES ____________ 82

5.1 Experiment 5: Multi-Locus Variable Analysis (MLVA) Variable Number

Tandem Repeats (VNTR) MmmSC TR-34 PCR ____________________ 82

5.1.1 Introduction ___________________________________________________________ 82

Agarose (4%) Gel preparation and migration _________________________ 83

Polyacrylamide (10% ) Gel Electrophoresis __________________________ 84

5.1.3 Results ________________________________________________________________ 85

10% Polyacrylamide Gel electrophoresis ____________________________ 89

5.1.4 Discussions ____________________________________________________________ 92

x

5.2 Experiment 6: Multi-Locus Sequence Analysis (MLSA) on Loc-PG1-0001

and Loc-PG-0103 ____________________________________________ 92

5.2.1 Introduction ___________________________________________________________ 92


PCR for Loc-PG1-0001and Loc-PG1-0103 ___________________________ 96

5.2.3 Results ________________________________________________________________ 97

Allelic profile analysis _______________________________________________ 97

Alleles defined in non-coding sequences Loc-PG1-0001 _______________ 98

Alleles defined in genes of unknown function: Lipoproteins (Lpp) and

Conserved hypothetical proteins (Chp) Loc-PG1-0103 ________________ 98

CHAPTER SIX ______________________________________________________ 109

GENERAL DISCUSSIONS ___________________________________________ 111

6.1 Competitive Enzyme Linked Immuno-sorbent Assay for the estimation of

CBPP prevalence ___________________________________________ 111

6.2 Isolation and Identification of Mycoplasma mycoides mycoides Small

Colony from Northern Nigeria. _________________________________ 113

6.3 Multi-Locus Variable Analysis (MLVA) of Variable Number Tandem Repeats

(VNTR) 34 and Multilocus Sequence Analysis MLSA ______________ 114

6.4 Multi-locus Sequence Analysis (MLSA) on Loc-PG1-0001 and Loc-PG1-0103

115

CONCLUSIONS _____________________________________________________ 123

MAJOR CONSTRAINTS OF THE STUDY_______________________________125 RECOMMENDATIONS______________________________________________126 REFERENCES______________________________________________________128 APPENDIX

I. Preparation of broth and agar medium base for Mycoplasma cultures _________ 156

II. Preparation of supplement for mycoplasma culture medium ________________ 158

III. Preparation of supplement for mycoplasma culture medium _______________ 158

IV. Preparation of broth and agar medium base for Mycoplasma cultures ________ 158

xi

V. Preparation of supplement for mycoplasma culture medium ________________ 158

xii

LIST OF FIGURES

Figure 1: Phylogenic tree of Mollicutes constructed based on the 16SrDNA sequence 11

Figure 2: Map of Nigeria showing the 12 States . _____________________________ 53

Figure 3: Rate of CBPP infection in Bulls and Cows based on the total number sampled

____________________________________________________________________ 57

Figure 4: Graph showing average PI, average OD and infection rates for samples

collected from the various states. _________________________________________ 59

Figure 5: Specific QPCR for the identification of MmmSC _____________________ 79

Figure 6: Temperature dissociation curve for the isolates ______________________ 80

Figure 7: Geographical distribution of the different MmmSC TR 34 types _________ 91

Figure 8: Distribution of alleles of Loc-PG1-0001 in Northern Nigeria __________ 103

Figure 9: Distribution of strains based on the number of repeats for Loc-PG1-0103 in

Northern Nigeria _____________________________________________________ 105

Figure 10: Overall distribution of alleles for combined MLVN TR, MLSA on loc-PG-

0001 and 0103 in Northern Nigeria. ______________________________________ 108

Figure 11: Overall distribution of alleles for combined MLVN TR, MLSA on loc-PG-

0001, and 0103 in Northern Nigeria ________________ Error! Bookmark not defined.

xiii

LIST OF PLATES

PLATE 1 : Electron Micrograph of thin sectioned Mycoplasma. __________________ 7

PLATE 2: Colonies of Mycoplasma mycoides mycoides SC isolated from the specimens

____________________________________________________________________ 66

PLATE 3: MmmSC diagnostic PCR MSC1: 275 bp product from all clones of the

samples ______________________________________________________________ 70

PLATE 4: M. mycoides mycoides Small Colony (048 Bovine, Birnin Kebbi) Isolate __ 73

PLATE 5: (044 Ovine, Fadan kaje) Nasal swab collected from ovine adult in contact

with a CBPP infected herd in Fadan Kaje, Kaduna State _______________________ 74

PLATE 6: MLVA VNTR MmmSC TR-34 PCR: 4% Agarose gel electrophoresis ___ 87

PLATE 7: MLVA VNTR MmmSC TR-34 PCR: 4% Agarose gel electrophoresis ___ 88

PLATE 8: MLVA VNTR MmmSC TR-34 PCR: 10% Poly Acrylamide Gel

Electrophoresis _______________________________________________________ 89

PLATE 9: MLVA VNTR MmmSC TR-34 PCR: 10% Poly Acrylamide Gel

Electrophoresis _______________________________________________________ 90

PLATE 10: Analysis of PCR products on 1 % agarose gel electrophoresis prior to

sequencing __________________________________________________________ 100

PLATE 11: Alignment of sequences on locus Loc-PG1-0001 ___________________ 101

PLATE 12: Alignment of sequences on locus Loc-PG1-0001 ___________________ 102

PLATE 13: alignment of the sequences on locus PG1-0103.There is 4 alleles on this

locus _______________________________________________________________ 104

LLIISSTT OOFF TTAABBLLEESS

xiv

Table 1: Estimated prevalence of CBPP by c-ELISA in 12 Northern States of Nigeria 55

Table 2: Culture and identification of isolates from specimens __________________ 63

Table 3: Isolates identified by Specific PCR for the diagnosis of MmmSC. _________ 71

Table 4: TR 34 Differentiates the Mycoplasma isolates into 5 alleles _____________ 86

Table 5: Overall distribution of alleles for combined MLVN TR, MLSA on loc-PG- 0001

and 0103 in Northern Nigeria.MmmSC strains. _____________________________ 107

Table 6: Polymorphisms between the allelic profiles of Vaccine strains and isolates

from Northern Nigeria. __________________________ Error! Bookmark not defined.

xv

ABSTRACT

Serological and Molecular studies of Mycoplasma mycoides mycoides Small Colony

(MmmSC) was undertaken in Northern Nigeria. The study was aimed at ascertaining

the presence of MmmSC in Northern Nigeria by serological and cultural techniques

including characterization of isolates using current Molecular tools. This study

established links between isolates and the distribution of Contagious Bovine

Pleuropneumonia (CBPP), a disease, caused by this organism in some Northern parts of

Nigeria. A total of 2026 bovine sera samples from twelve states of Northern Nigeria

were screened by c-ELISA technique and 213 tested positive for CBPP. The prevalence

of infection ranged from 2.1 % to 32.5 %, with an overall prevalence of 10.8 %. A total

of 287 tissue samples were also collected from CBPP outbreaks in 5 Northern States

and processed for isolation and identification of Mycoplasma organisms. Among these

is a pleural fluid sample which was collected from a two week old lamb. Thirteen

MmmSC isolates were recovered from the outbreaks with an isolate representing one

outbreak. The isolates were confirmed by conventional and Real Time PCRs and then

characterized by Multilocus Variable-Number Tandem Repeat Analysis (MLVA) and

Multilocus Sequence Analysis (MLSA). All Polymerase Chain Reaction (PCR)

amplifications were performed according to specific protocols. PCR products were

controlled by electrophoretic separation in 1 % agarose gels. Tandem Repeat (TR) 34

PCR products were analyzed on 4 % agarose and 10% polyacrylamide gels. The

relevant loci were sequenced with the corresponding primers. The sequences were

assembled and aligned with the software Vector NTI SuiteTM (InfoMax, 2001). Seven

profiles were defined for the thirteen isolates giving more precision in the definition of

the origin of the strains. The variations observed within the M. mycoides subsp.

mycoides isolates characterized indicates that the problems of differentiation associated

with homogeneity of the Mycoides cluster group can be avoided and the variations also

cast some doubts over the protective ability of a single strain for CBPP. A new allele

was described in this work and assigned a no. 7. Presently, the isolates in this study are

the only MmmSC isolates in Nigeria to be identified and characterized to molecular

level. The study also indicated that the isolates recovered from sheep are identical to

those of bovine, making sheep a potential source of infection for bovine. The tools used

in this study may be useful in the characterization of other isolates of MmmSC from

other parts of Nigeria.

1

CCHHAAPPTTEERR OONNEE

IINNTTRROODDUUCCTTIIOONN

1.1 Background of the Study

Contagious Bovine Pleuropneumonia (CBPP) is a highly contagious disease primarily

of cattle caused by Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC)

bovine biotype (Osiyemi, 1981; Provost et al., 1987; Terlaak, 1992; Taylor et al.,

1992a). On account of its transmissibility and economic impacts, CBPP is now

recognized as a priority trans-boundary disease and has thus been categorized as the

only bacterial disease in the OIE list A diseases (Litamoi et al., 2004). The disease is

considered to be the most economically important cattle disease in Africa causing

greater losses in cattle than any other disease including Rinderpest (OIE, 2003).

Presently the disease is wide spread in Nigeria and the recent wave of civil and religious

unrest which swept most parts of the country only helped to spread the disease by the

unrestricted movement of nomads across state boundaries which made accurate

monitoring difficult (Nwankpa et al., 2003). Several authors have documented the

outbreaks, prevalence, and the economic importance of the disease in Nigeria (Egwu et

al., 1996, Osiyemi, 1981, Nwanta and Umoh 1992). The disease was present in most

sub-Saharan countries and had not only reinfected countries like Uganda and Kenya but

has also infected countries like Tanzania (1990) , Botswana (1995) and Rwanda (1995)

which had been CBPP free (Nicholas and Bashiruddin 1995).

Control of CBPP in Nigeria was probably achieved by 1965 following 10years of mass

vaccination, well organized disease reporting, efficient laboratory diagnosis, effective

quarantine, and strict control of cattle movement. This did not last long as the disease

re-emerged a few years later perhaps from chronic carriers (Lungers) through one or all

2

of the neighbouring countries of Niger, Chad and Cameroon (Nwanta and Umoh, 1992).

In spite of an eradication campaign launched in 1970, outbreaks rose rapidly from 1986

onwards to a peak in 1989 where over 10,000 cattle were affected (Nwanta and Umoh,

1992). Alarmed by the situation, the meeting of the FAO/OIE/OUA IBAR Consultative

Group on Contagious Bovine Pleuropneumonia was convened for the first time in over

25years in June 1998, to discuss the deteriorating situation of the disease in Africa

(FAO, 2000). The meeting focused on the wealth of new data accumulating on the

causative organism of CBPP, Mycoplasma mycoides subspecies mycoides small-colony

type (MmmSC). Disease status, current knowledge, surveillance and control strategies,

as well as research directions were extensively discussed (FAO, 2001). The meeting

concluded that, “CBPP was the most important threat to the cattle industry in Africa.”

Although deterioration of Veterinary services could not be demonstrated, it was further

observed that the inadequacy of veterinary services, epidemiological knowledge, the

inadequacy of control systems and regional coordination, coupled with civil unrest

contributed to the endemicity of CBPP in the African continent.

1.2 Statement of the Problem

Contagious Bovine Pleuropneumonia (CBPP) is a contagious disease primarily of cattle.

The disease is prevalent in Africa where it is responsible for high economic losses and

is a limiting factor in cattle production. While relatively accurate data can be gathered

on which areas are infected, what is less clear is the prevalence of infection, as reported

cases are usually inaccurate and subjective (Nicholas et al., 2000). The establishment of

the true prevalence rates of CBPP in infected countries is a crucial prerequisite to

mounting a successful disease control programme, and a precursor to national efforts

(FAO, 2004). The increasing importance of the disease emphasizes the need for an

3

accurate data on the prevalence of the infection if prompt and effective control measures

are to be instituted. Presently, in Nigeria, the extent and pattern of CBPP prevalence is

largely unknown (Aliyu, et al., 2000).

Although M. mycoides SC has been isolated from goats and sheep (Brandão, 1995;

Srivastava et al., 2000; Kusiluka et al., 2000), the role of small ruminants in the

epidemiology of CBPP has not been demonstrated and no reservoir either in wild

animals has been established (Bell et al., 1990; Masiga et al., 1996). There is therefore,

the need to determine if small ruminants are involved in any way in the epidemiology of

the disease in Nigeria. There is also a need to determine the type of strains of MmmSC

involved in the various out breaks in Nigeria. It has been shown that cattle affected with

CBPP in Europe since 1980 have shown less severe clinical signs and lower mortality

than affected animals in Africa (Nicholas et al., 1996). Studies of experimental

infections in cattle, with a representative strain isolated from European outbreaks of

CBPP have indicated that the strain was less pathogenic than a typical African strain of

M. mycoides subsp. Mycoides SC (Abdo et al., 1998). It thus raises the question of the

source of current outbreaks in Africa. Are the current outbreaks due to a resurgence of

dormant African strains from the eradication campaigns of the early 60’s, or is it

possible that Africa is witnessing an import of the European strains causing the current

wave of outbreaks.

1.3 Aims and Objectives

The aims and objectives of this research are to:

1. Determine the prevalence of Mycoplasma mycoides mycoides Small Colony in

Northern Nigeria by serological and cultural methods.

4

2. Confirm the presence of the aetiological agent of CBPP (Mycoplasma mycoides

mycoides Small Colony) using conventional and Real-Time PCR

3. Molecularly characterize the isolates of Mycoplasma mycoides mycoides Small

Colony, the agent of CBPP in Nigeria, using Variable Number Tandem Repeat 34

(VNTR 34) and Multi-locus sequence Analysis (MLSA).

4. Determine the role if any of small ruminants in the epidemiology of Contagious

Bovine Pleuropneumonia in Nigeria.

1.4 Relevance and Justification

1. Various reports have indicated that the disease is endemic in Nigeria hence the

need for better concerted efforts in control strategies.

2. Field experiences have shown that the current vaccines are not protective enough

and vaccinations campaigns not very efficient.

3. Outbreaks of CBPP have indicated differences in the pathogenicity of the agents

involved thereby indicating a likelihood of several circulating strains of MmmSC

in the field.

4. Presently, current tools for the diagnosis of CBPP have a lot of limitations and

drawback, hence the need for better and efficient ones.

5

CHAPTER TWO

LLIITTEERRAATTUURREE RREEVVIIEEWW

2.1. The Mycoplasmas

Mycoplasma is a trivial name for a group of microorganisms which belong to the class

Mollicutes (Maniloff 1992). Mollicutes evolved from AT-rich, gram-positive bacteria

to become the smallest self replicating organisms known to date. During their

degenerative evolution, their genomes considerably reduced in size and many genes,

common to most bacteria, were lost. Most characteristically, mollicutes (mollis= soft,

cutis= skin) lost the genes involved in the synthesis of a cell wall. The presence of a cell

membrane as the only boundary implies an intrinsic resistance to antimicrobial agents

that inhibit cell wall synthesis, sensitivity to osmotic shock and an ability to pass filters

typically used to sterilise solutions (Stakenborg et al., 2005). Moreover, because of their

small genomes, mollicutes have limited biosynthetic capabilities and occur as obligate

parasites in a wide diversity of plant and animal hosts. Thus far, tremendous efforts

have led to the description of more than 200 species, and still, this number likely

represents only a minor fraction of the mollicutes present in nature (Stakenborg et al.,

2005). Of the eight genera currently described within the class of Mollicutes, the genus

Mycoplasma is by far the most studied (Stakenborg et al.,, 2005).

Incidentally, the first mollicute to be isolated and described was Mycoplasma mycoides

subsp. mycoides small colony (SC) in 1898, but it took another few decades before

other animal Mycoplasmas were found (Bové, 1999). For instance, the porcine pathogen

M. hyopneumoniae was only demonstrated in 1965 (Mare and Switzer, 1965). The first

human Mycoplasma, M. pneumoniae, was described in 1937 (Asai et al., 1993).

6

Mycoplasmas are wide spread in nature as parasites of humans, mammals, reptiles, fish,

arthropods, and plants. It has been indicated that the extanct Mycoplasmas are the

surviving descendants of exceedingly primitive bacteria that existed before the

development of a peptidoglycan based cell wall (Razin, 1992). The class mollicutes

consists of nine genera, Ureaplasma, Entomoplasma, Mesoplasma, Spiroplasma,

Acholeplasma, Anaeroplasmas, Asteroleplasma, and Phytoplasma. In general,

Mollicutes are the smallest and simplest self-replicating organisms, being made of a

plasma membrane, ribosomes, and a circular double-stranded DNA molecule (Razin

1997b). They are characterized by their small genome size and are thought to have

undergone reductive evolution, losing many genes possessed by more complex bacteria

in the process (Plate. 1). They lack many genes, including those for cell wall synthesis

and for the production of all 20 amino acids, as well as genes encoding enzymes of the

citric acid cycle and the majority of all other biosynthetic genes (Razin et al., 1998).

They can survive with a reduced genome since they are able to acquire these products

from their host in vivo and as such they are considered the model organisms for the

study of essential functions in living cells. It could be argued that Mycoplasmas are

close to the concept of "ideal parasites," usually living in harmony with their host.

(Nicholas, 2004). Despite their small genome size, Mycoplasmas cause a wide range of

disease in both humans and animals. The primary habitats of human and animal

Mycoplasmas are the mucous surfaces of the respiratory and urinogenital tracts, the

eyes, alimentary canal, mammary glands and joints. They are associated with

pneumonia, arthritis and reproductive disorders. Infections with pathogenic

Mycoplasmas are rarely of the fulminant type but, rather, follow a chronic course.

7

0.5 µm

PLATE 1 : Electron Micrograph of thin sectioned Mycoplasma.Cells are bounded by a single membrane showing in section the characteristic trilaminar shape. The cytoplasm contains thin threads representing sectioned chromosome and dark granules representing ribosomes (Adapted from Shmuel Razin, 1996 Courtesy of RM Cole, Bethesda, Maryland).

However, very little is known about their pathogenicity factors or mechanisms of

persistence in the host. There are only a few characterized virulence factors of

Mycoplasmas; these include, in certain

hydrogen peroxide (Miles

scavenge arginine from host cells (Sasaki

to explain how Mycoplasmas

their paucity of virulence factors and lack of cell wall. Presently, about 190

Mycoplasma species have been described under the class

2.1.1 Phylogeny and Genome characteristics

Early attempts to classify the mollicutes by immunological and molecular approaches

provided useful taxonomic data but

studies on the phylogenetic relationships of the mollicutes have concentrated on the

highly conserved Mycoplasma

constraints, has changed much less than the bulk of the genome (

widely agreed that the mollicutes that have already been characterized and

taxonomically defined constitute only a part, apparently a minor one, of the mollicutes

living in nature. Therefore,

hosts is the willingness of a

isolate and taxonomically characterize

al., 1998). There is a consensus among bacterial taxonomists

sequences of bacterial genomes will form the basis for phylogeny and, ultimately,

taxonomy. However, as long as complete genomic sequences are available for a few

bacteria only, current bacterial taxonomy, including that of mollicutes (

Committee on Systematic Bacteriology

8



; these include, in certain Mycoplasma species, the production of

e (Miles and Taylor,, 1991), the carbohydrate capsule, the ability to

scavenge arginine from host cells (Sasaki et al., 2002) and T-cell mitogens. It is difficult

Mycoplasmas manage to cause such severe and chronic infection given

ity of virulence factors and lack of cell wall. Presently, about 190

have been described under the class mollicutes (Rottem 2003)

Phylogeny and Genome characteristics

Early attempts to classify the mollicutes by immunological and molecular approaches

provided useful taxonomic data but with little information on the trends. More recently,


Mycoplasma ribosomal ®RNA which, because of very tight structural

constraints, has changed much less than the bulk of the genome (Nicholas, 200



Therefore, the main factor for adding an animal or plant to the list of

hosts is the willingness of a Mycoplasmologist to invest the effort and funds required to

isolate and taxonomically characterize new Mycoplasmas from different host (

). There is a consensus among bacterial taxonomists that the complete



bacteria only, current bacterial taxonomy, including that of mollicutes (

Committee on Systematic Bacteriology Subcommittee on the Taxonomy of



species, the production of

1991), the carbohydrate capsule, the ability to

cell mitogens. It is difficult

manage to cause such severe and chronic infection given

ity of virulence factors and lack of cell wall. Presently, about 190

(Rottem 2003).

Early attempts to classify the mollicutes by immunological and molecular approaches,

trends. More recently,


because of very tight structural

Nicholas, 2004). It is



plant to the list of

to invest the effort and funds required to

host (Razin et

that the complete



bacteria only, current bacterial taxonomy, including that of mollicutes (International

Subcommittee on the Taxonomy of

9

Mollicutes, 1995), relies on the combination of phenotypic characteristics and

phylogenetic data based on partial genomic sequences, particularly those from the

conserved rRNA genes (Razin et al., 1998). According to 16S rRNA sequences

(Weisburg et al., 1989) the mollicutes are divided into five phylogenetic units (clades).

At the moment, the genus Mycoplasma encompasses a pneumoniae group, while other

groups are spiroplasma, or mycoides group, hominis and an anaeroplasma with only one

species. The haemobartonella and Eperythrozoon species were recently and correctly

placed within the genus Mycoplasma (Neimark et al., 2001), but also currently enlisted

Mycoplasma species should be reclassified to other genera. The current problems are

probably best exemplified by the taxonomic position of the M. mycoides cluster.

Unequivocal evidence places this cluster more closely to spiroplasmas than to

Mycoplasmas (Gasparich et al., 2004). However, owing to practical and legislative

complications, taxonomic changes have not been realized and one species of the M.

mycoides cluster, referred to as Mycoplasma Bovine Group 7 (MBG 7) and closely

related to M.capricolum sp., but still has no official name. Some species of veterinary

medical importance are found within the phylogenetic spiroplasma group in the so-

called Mycoplasma mycoides cluster, encircled in blue (Figure 1). An important feature

of mollicute phylogeny proposed by Woese (Woese, 1987) is the rapid pace of their

evolution, in line with the marked genotypic and phenotypic variability characterizing

the mollicutes as a group. The great weight given to 16S rDNA sequences in

Mycoplasma phylogeny, taxonomy, and species identification (Patterson et al., 1996;

Razin, S. 1992) led the Mollicutes Taxonomy Committee (International Committee on

Systematic Bacteriology, 1997) to recommend the inclusion of the 16S rDNA sequence

in any description of a new Mycoplasma species (Fig. 1). Although the 16S rRNA

sequence has proved to be very effective tools in the phylogeny and taxonomy of

10

mollicutes, it was thought that additional phylogenetic markers are desirable to support

the conclusions based on the 16S rRNA data.

11

Figure 1: Phylogenic tree of Mollicutes constructed based on the 16SrDNA sequence The six species of the M. mycoides cluster are encircled in blue. Among which M. mycoides subsp

mycoides LC spotted in red is the species of interest in the present study (Adapted from MolliGen Web site http://cbi.labri.fr/outils/molligen/).

100

100

100

100

100

100

100 100

100

100

100

100

100

100

100

70

90

83

52

87

43

99

86

98 50

51

50

0.05

Sp

irop

lasm

a

Pn

eum

on

iae

Ho

min

is

Ph

yto

pla

sma

M. haemofelis

M. sp bovine group 7 M. capricolum subsp. capripneumoniae

M. capricolum subsp. capricolum

M. mycoides subsp. mycoids SC

M. mycoides subsp. mycoides LC

Mesoplasma florum Spiroplasma citri

M. mycoides subsp. capri

Spiroplasma kunkelii

U. urealyticum/parvum

M. penetrans

M. gallisepticum M. pneumoniae

M. genitalium

M. mobile

M. arthritidis

M. hominis

M. orale

M. hyopneumoniae

M. pulmonis

M. synoviae

M. fermentans

M. bovis

M. agalactiae

Acholeplasma laidlawii

Aster Yellow phytoplasma

Onion Yellow phytoplasma

Stolbur phytoplasma Western X phytoplasma

Flavescence Dorée phytoplasma

12

In fact, such markers have already been applied, including the conserved ribosomal

protein genes (Gundersen et al., 1996), the elongation factor EF-Tu (tuf) gene (Kamla et

al., 1996), the heat shock protein gene hsp70 (Falah and Gupta, 1997), and the

16S/23SrRNA intergenic sequences (Smart et al., 1996). Use of these markers has

supplemented and complemented the 16S rRNA comparative data. A priori, wobble in

the genetic code permits more variations in protein gene sequences, even of highly

conserved genes, than is possible in rRNA sequences. Thus, even though these

ribosomal protein-encoding genes are quite conserved, they vary considerably in size

and primary sequence more than the 16S rRNA genes do (Razin et al., 1998). The

reassignment, in most mollicutes of Uridine-Guanine-Adenine (UGA) from a stop

codon to a tryptophan codon, a feature found in mitochondria is the apparent outcome

of codon reassignment under strong A+T pressure. Not all mollicutes do share this

property, the phylogenetically early acholeplasmas and phytoplasma use conventional

Uridine-Guanine-Guanine (UGG) codon for tryptophan retaining UGA as a stop codon

(Razin et al., 1998).

There is now solid genetic support for the hypothesis that Mycoplasmas have evolved as

a branch of gram-positive bacteria by a process of reductive evolution (Woubit et al.,

2007). During this process, the Mycoplasmas lost considerable portions of their

ancestral chromosomes but retained the genes essential for life. Thus, the Mycoplasma l

genomes carry a high percentage of conserved genes, greatly facilitating gene

annotation. The significant genome compaction that occurred in Mycoplasmas was

made possible by adopting a parasitic mode of life (Razin, 1989). The recent sequencing

of several Mycoplasma genomes (Bork, et al., 1995; Fraser et al., 1995; Himmelreich et

al., 1996) has provided some information on Mycoplasma l genes homologous to cell

13

division genes of walled-covered bacteria. The comparative genomics data reveal the

lack in Mycoplasmas of a significant number of genes belonging to this category,

findings which may be relevant in the consideration of the relative importance of the

different genes in the prokaryotic cell division process.

Considerable advances were also made toward a better understanding of Mycoplasma

pathogenesis. Most impressive are the findings concerning the interaction of

Mycoplasmas with the immune system, macrophage activation, cytokine induction,

Mycoplasma cell components acting as super antigens, and autoimmune manifestations.

Evasion of the host immune system by antigenic variation of Mycoplasma l surface

components, as well as molecular definition of Mycoplasma l adhesins, has also gained

much attention recently (Razin et al., 1998). The most important finding, perhaps, is

that of the ftsZ gene in Mycoplasmas which indicates that ftsZ gene is a highly

conserved and ubiquitous gene (found also in archeons and chloroplasts), fulfilling a

key role in prokaryote cell division. In eubacteria, the ftsZ protein is a polymer-forming,

GTP-hydrolyzing protein with tubulin-like elements; it is localized to the site of

septation and forms a constricting ring (the Z ring) between the dividing cells (Bork et

al., 1995; Fraser et al., 1995; Himmelreich et al., 1996).

2.2 Contagious Bovine Pleuropneumonia (CBPP)

2.2.1 Historical Perspective

Contagious Bovine Pleuropneumonia (CBPP) is thought to have existed in Europe

many centuries ago and it is note worthy that eradication was achieved in most

European countries before anything was known concerning the etiological agent as it

was first identified in 1898 by Nocard and Roux (Thiaucourt, 2000b). The disease was

14

believed to have spread throughout Europe in the 18th century through uncontrolled

movements of cattle that were caused by wars, transhumance and trade (Provost et al.,

1987; Turner, 1959; Blancou, 1996). Early descriptions of the disease were found in

writings from Gallo in Italy (1550) and also from C. Testienne in France (1554).

However, it was only during the 18th century that the disease was clearly described, in

Switzerland in 1732 by J. Scheuchzer and in France in the monts d’Auvergne and

Vosges, in Italy the Piemont and in Germanic States in Bavaria and Wurtemberg

(Thiaucourt, 2000b). “The spread of CBPP throughout Europe started at the end of the

18th century and culminated in the middle of the 19th century. All European countries

became infected: Northern France 1922, Belgium 1828, Holland and Prussia 1830,

Schleswig Holstein 1841, Sweden 1847, Norway (then Swedish territory 1860), the first

cases seen in Spain in 1846 in Barcelona region and later on in 1864 in central Spain”

(Thiaucourt, 2000b) CBPP was introduced into the United States of America, Asia,

Australia and Japan in the 19th and 20th century by importation of cattle from Europe.

South Africa was infected in 1854 through importation of cattle from the Netherlands;

thereafter, the disease spread to other countries in southern Africa. In Namibia, the

disease was first recorded in 1856 and is reported to have caused large scale cattle

losses for the next 40 years. The disease spread rapidly aided by people trying to flee

with their animals from the disease, wars and the use of trekking cattle for freight. Laws

aimed at controlling the disease were promulgated in 1885 and CBPP became a

notifiable disease and has remained so since 1887 (Masiga et al., 1996). Vaccination,

adequate movement control, and good extension services greatly reduced the prevalence

of the disease and by 1904 only minor outbreaks were reported. The disease was

completely eradicated by 1944; however, reintroduction of the disease from neighboring

Angola saw a resurgence of the disease in 1983. Presently the disease is considered

15

endemic in some parts of Namibia. It is believed that East Africa was infected in the

19th century by cattle imported from India (Masiga et al., 1996). Through vigorous

control efforts involving slaughter of the affected animals, quarantine and strict control

of animal movements, CBPP was eradicated from most of Western Europe, U.S.A. and

Japan by the beginning of the 20th century (Provost et al., 1987). Some foci of the

disease remained in south-western Europe (TerLaak, 1992; Regalla et al., 1996) where a

resurgence of CBPP occurred in the early 1980s with reports in southern France on a

few occasions, between 1980 and 1984. In Italy the disease reappeared in 1990,

however vigorous eradication efforts again were successful (Nicholas et al., 2000). The

Peoples Republic of China eradicated CBPP in the 1980s while Australia eradicated

CBPP in 1973 through animal-movement control, vaccination and slaughter of affected

and in-contact animals, combined with an efficient disease-surveillance system

(Newton, 1992). Zimbabwe eradicated CBPP in 1904, South Africa in 1924 (FAO/OIE,

1995). Most countries in southern Africa eradicated CBPP by the end of 1939 but the

disease remained in war-torn Angola and Namibia, from which it spread to Botswana in

1995 (Trichard et al., 1989; Masiga et al., 1996; Amanfu et al., 1998). The current

status of CBPP in Eastern Europe and the Mediterranean region, the Middle East and

Asia is not well known and this poses a threat to Western Europe (Thiaucourt, 1999).

Sporadic outbreaks have been recognized in the Middle East most likely from

importations of African cattle (FAO, 2001).

2.2.1.1. CBPP in Africa

CBPP is presently considered to be the most economically important cattle disease in

Africa, causing greater losses in cattle than any other disease including Rinderpest. The

growing sense of impatience and frustration connected with this disease is the fact that

16

the stunning successes achieved in other parts of the world in terms of eradication,

simply have not been repeated. On the contrary CBPP has increased the size of its

territory, and threatens to increase further, in a continent with growing human

population but dwindling meat supply (FAO, 2001). Presently, CBPP is found in an

area south of the Sahara, from the Tropic of Cancer to the Tropic of Capricorn and from

the Atlantic to the Indian Ocean. Endemic infection extends throughout the pastoral

herds of much of western, central and eastern Africa, with Angola and Northern

Namibia in southern Africa. Malawi, Mozambique, South Africa, Swaziland, and

Zimbabwe are currently free (Masiga et al., 1996).

By the end of 1999, CBPP was present in at least 27 countries in equatorial, central and

southern Africa although it is difficult to be certain due to the discrepancy between

official and non-official reports (Nicholas et.al. 2000). The two main CBPP infection

foci in west and central Africa are the Inner Delta area of Niger and the Lake Chad area.

With the exception of countries like Senegal and Gambia in West Africa and Gabon and

Congo Brazzaville in central Africa whose CBPP status remains unknown, all other

countries are currently infected (FAO, 2000).

In Africa, Contagious Bovine Pleuropneumonia (CBPP) is present in at least 29

countries and the disease was said to have made its initial entry into Africa in 1854,

when an infected bull was introduced to Mossel Bay, South Africa, from the

Netherlands. Nearly one hundred and fifty years later, the disease is still enzootic in

large areas of sub-Saharan Africa (Nicholas, 2004). In the 1960s and 1970s, sustained

research on CBPP in Nigeria, Kenya, Chad and other African countries, coupled with a

massive international campaign code-named Joint Project 16 (JP 16) resulted in the

17

disappearance of clinical disease from most parts of Africa. However, because of

economic decline and poorly financed veterinary services, the disease made a

spectacular comeback in the late 1980s and early 1990s. Today, more countries are

affected by CBPP than was the case 20 years ago (FAO, 2001). Endemic infection

extends throughout the pastoral herds of much of western, central and eastern Africa,

with Angola and Northern Namibia in South Africa. CBPP continues to be a

constraining factor in African livestock production and a deterrent to investment in the

livestock sector. The disease is currently endemic in much of West Africa and in the

greater horn of Africa and Angola; it has in recent years spread into Tanzania, is

established in Northern Namibia, has made repeated incursions into Zambia; and after a

brief epidemic was eradicated from Northern Botswana (Paskin, 2000). Seasonal cattle

movements by pastoralists resulted in the spread of the disease in 1990 from Kenya,

where the disease has been endemic for some time, into the Ngorogoro crater in

Tanzania, which had been free of CBPP for 30 years. As a result of drought, failure to

report outbreaks of the disease and continued, unrestricted movement of livestock,

CBPP spread further south in 1995 causing over 14,000 cattle deaths in just six months.

Most of Central and Eastern Africa, from Uganda down to Zambia, now have cattle

infected with CBPP. Most countries in Central and Eastern Africa are infected. Angola

is still infected and the prevalence of the disease in the country is not known because of

civil conflict. Newly-infected areas in the 1990s include much of Uganda, parts of

Kenya, the Ituri Region of the Democratic Republic of Congo and most of Tanzania,

where recently the disease has spread alarmingly; Rwanda (1994), Botswana (1995,

now free), Burundi (1997) and Zambia (1997). Currently, the disease is absent in some

southern African countries, i.e. Botswana, Lesotho, South Africa, Swaziland and

Zimbabwe, and parts of Namibia and Zambia (http://www.vm.iaState.edu).

18

2.2.1.1.1 CBPP in Tanzania

CBPP was introduced into Tanzania from southern Kenya in 1916 (Hammond and

Branagan, 1965). The disease spread to Northern regions of Tanzania encompassing

Arusha, Mara, Kilimanjaro and Tanga, achieving its maximum spread in 1941.

Restriction and control of animal movements, quarantine and vaccination resulted in

eradication of CBPP from Tanzania in 1964 and the country was declared free from the

disease in 1966 (Lwebandiza, 1969). In the late 1980s, a new episode of CBPP emerged

in East Africa. Movements of animals spread the disease from Northern and eastern

Uganda to the south-western part of that country (Rweyemamu and Benkirane, 1996).

From Northern Kenya, the disease spread southward and crossed into the Ngorongoro

district of the Arusha region in Northern Tanzania in June 1990 and since then it has

spread widely, threatening the entire national cattle herd. Because of lack of a clear

disease-control policy, uncontrolled cattle movements, lack of public awareness and

commitment, ineffective legislation, attempts to control and eradicate the disease for the

last 10 years have failed (Bölske et al., 1995; Anon., 2000).

2.2.1.1.2 CBPP in Botswana

In Botswana, the disease re-emerged in early 1995 after an absence of 50 years. Border

controls which were relaxed after the end of the Namibian War of Independence

allowed the uncontrolled movement and smuggling of livestock, resulting in the

outbreak of CBPP. Despite rapid control efforts, including restriction of cattle

movement, the erection of fences & the slaughter of infected herds, the disease

continued to spread and, in 1996, 300,000 cattle in the infected region had to be

19

slaughtered. This proved effective and, in January 1997, Botswana was declared

'provisionally free' from CBPP ([email protected]).

2.2.1.1.3 CBPP in Nigeria

CBPP is currently endemic in Nigeria with pockets of outbreaks occurring in the North

of the country, where most of the cattle population is located (Osiyemi, 1981; Fayomi

and Aliyu, 1992; Ameh et al., 1998). Most cattle in Nigeria are owned by nomadic

Fulanis who move for long distances (thereby enhancing the spread of the disease).

In Nigeria, CBPP was regarded as extinct in 1965 and this was brought about by ten

years of mass vaccination, well organised disease reporting, laboratory diagnosis,

quarantine, slaughter policy, and strict control of cattle movements (Knowles, 1955 and

1960, Griffin and laing, 1966). Although achieved by 1965, control of CBPP did not

last long as the disease re-emerged a few years later perhaps from bordering countries

of Niger, Chad, and Cameroon. In spite of an eradication campaign launched in 1970,

outbreaks rose rapidly from 1986 onwards to a peak in 1989 when over 10,000 cattle

were affected (Nwanta and Umoh, 1992).

In 1998, under the Technical Co-operation Project (TCP), the FAO/IAEA introduced

the competitive ELISA for the measurement of CBPP antibodies to laboratories in

Africa. This new technique is considered to be more sensitive than Complement

Fixation Test (CFT) and almost as specific for the organism as the CFT (Le Goff and

Thiaucourt, 1998; Nicholas et al., 1996). The National Veterinary Research Institute,

Vom was one of the centers chosen for the project. Prior to this, diagnosis of the disease

was mostly based on clinical signs, isolation of the organism and serology. The

20

serological techniques like the simple Plate Agglutination Test and Complement

Fixation Test which at that time was the prescribed test by the OIE (Campbell and

Turner, 1953) were then in use. In 2002 there was a meeting of Ten Research Contract

holders from 10 African Countries (Botswana, Cote d’Ivoire, Ethiopia, Ghana, Kenya,

Mali, Namibia, Tanzania, Uganda and Nigeria), and three Research Agreement Holders

(CIRAD/EMVT), France, National Veterinary Institute, Sweden, Scottish Agricultural

College, UK) and representatives of FAO and IAEA. During the meeting presentations

on the situation and diagnosis of CBPP and the diagnostic and surveillance capabilities

in these countries were given and discussed. The meeting recognized the need for an

improved diagnosis of CBPP in Africa and the need for the establishment of better

CBPP surveillance systems and recommended the confirmation under field conditions

of the performance of the monoclonal antibody based competitive ELISA for the

detection of antibodies to Mycoplasma mycoides mycoides SC in the 10 laboratories

participating in the programme (FAO/OIE, 1995).

The Pan- African Programme for the Control of Epizootics (PACE) which covers 32

countries including Nigeria came into limelight after the conclusion of the Project on

Sero-monitoring by the IAEA/FAO. In order to support the CBPP control efforts at the

national and regional levels, PACE adopted several strategies which included the

reinforcement of animal epidemiology services and control of the major diseases. The

objectives were to enhance national capacities, improve the distribution of veterinary

services and medicines within the country and eliminate the last reservoirs of

Rinderpest. Others were to verify freedom from the disease, and control major

epizootics (FAO, 2001). Under this programme, PACE was to improve abattoir services

and commission specific surveys. These were aimed at improving surveillance of

21

clinical cases, improving the national disease reporting and surveillance systems and

introduce a compatible system of data management for all the countries involved. It was

also to introduce a Data Management System at OAU/IBAR to assist strategic decision

making at the sub-regional and continental levels in Africa. Unfortunately, none of

these were effectively achieved before the programme wound up in 2002.

2.2.2 Transmission and spread of CBPP

2.2.2.1 Transmission of CBPP

The epidemiology of CBPP is influenced by many factors including the virulence of the

M. mycoides SC strains, host susceptibility and management systems (Provost et al.,

1987, TerLaak, 1992, Nicholas and Palmer, 1994, Masiga et al., 1996). Spread and

Transmission of the disease occurs through inhalation of infective droplets from

clinically sick or carrier animals. Infection can also be acquired from fodder and

fomites contaminated with infected urine and fetal fluids (Masiga and Domenech, 1995;

Windsor and Masiga, 1977b). Close proximity between infected and healthy animals

facilitates the rapid transmission of the disease within and between herds. In Africa,

cattle movements through transhumance, nomadism and trekking of trade cattle are

responsible for the maintenance and spread of the disease within and across country

borders (Provost et al., 1987; Masiga et al., 1996).

CBPP affects cattle and buffalo but reports of isolation of the causative agent in small

ruminants especially sheep and goats have been made (Brandão, 1995; Srivastava et al.,

2000; Kusiluka et al., 2000). However, the role of small ruminants in the epidemiology

of CBPP has not been demonstrated and no reservoir in wild animals has been

established, either (Bell et al., 1990; Masiga et al., 1996). Any severe pneumonia with

22

pleurisy in cattle may be indistinguishable from the early stages of CBPP.

Consequently, inability to isolate Mycoplasma mycoides mycoides Small Colony

organism from cases with typical lesions should be expected since other infectious

agents may mimic the presenting features (Regalla et al., 1996).

2.2.2.2 Spread of CBPP

Contagion occurs through direct and repeated contacts between infected and susceptible

cattle essentially through expectorations of coughing (Provost et al., 1987; Nicholas and

Bashidurin, 1995). Excretion of organisms from the respiratory tract has been shown to

occur before the onset of detectable serological responses in experimentally infected

animals. (Bashiruddin et al., 1994; Miserez et al., 1997; Frey et al., 1998). The positive

findings from the swab samples taken in Portugal show the presence of MmmSC in

nasal secretions of infected cattle in the field and thus confirm the respiratory shedding

of organisms as a mode of contagion dispersion. Involvement of chronic carriers in the

perpetuation of the infection has been suggested by several authors (Mahoney, 1954;

Martel et al., 1985; Provost et al., 1987; Egwu et al., 1996) but is still debated (Windsor

and Masiga, 1977). Risk factors for its spread include high-density confinement in night

housings and use of common grasslands and watering places (Provost et al., 1987). In

Africa, between-zone or country spread essentially is related to cattle movements

caused by trade, transhumance and social troubles (Roeder and Rweyemamu, 1995). In

this continent, up to a third of cases that recover from acute disease become potential

carriers. This figure was probably higher in Europe where there is a far more

widespread use of antimicrobials (Nicholas, 2004). The risk of infection of CBPP-

uninfected herds by carriers can also be enhanced by animal exchanges (e.g. by loaning

contracts) between farmers which are quite frequent in mixed crop–livestock systems.

23

Chronic carriers are difficult to detect and their importation in the herds might be a

major risk or between-herd spread of the disease (Laval, 2002; Lesnoff et al., 2002).

Unfortunately, longitudinal data on the within-herd spread of CBPP are rare in general

(Bygrave et al., 1968) and absent for mixed crop–livestock systems. These systems are

common in Africa (especially in the East African highlands) and characterized by small

herds managed by individual farmers (Gryseels and Anderson, 1983). MmmSC chronic

carriers might present time-delimitated infectious phases during re-activations of lesions

or break-downs of sequestra. Therefore, inapparently infected animals may freely

transmit infection to susceptible animals (Hammond and Branagan, 1965). Chronic

carriers often were suspected to generate field outbreaks and endemic situations in cattle

populations (Curasson, 1942; Mahoney, 1954; Martel et al., 1985; Provost et al., 1987;

Dedieu et al., 1996; Egwu et al., 1996), although this hypothesis remains unproven. For

example, in experimental conditions, Windsor and Masiga (1977) did not observe any

disease transmission after challenging healthy animals with chronic carriers. Those

authors concluded that carriers (if infectious) play only an occasional role in the

epidemiology of CBPP.

The virulence of the African strain of the organism has been determined to be much

more than that of its European counterparts as such the disease in Europe has low

morbidity and mortality and appears to be less severe than in Africa with a high

percentage of infected animals with chronic lesions. The lower virulence of strains of

the recent European isolates if compared to African field strains seems to be due to an

attenuation most probably caused by a distinct deletion of 8.84 kb involving disruption

of the operon gtsABC for glycerol uptake, resulting to reduced production of hydrogen

peroxide (Houshaymi et al., 1997; Vilei and Frey, 2001). This operon was shown to

24

differentiate the strains of the recent European isolates from all other M. mycoides

subsp. Mycoides SC strains (Vilei and Frey, 2001). Other suggested explanations for the

milder disease in Europe include better animal husbandry and more frequent use of

antibiotics and anti-inflammatory drugs which may favour the formation of chronic

lesions in carrier animals (Provost et al., 1987).

2.2.3 Incubation Period

The incubation period in naturally infected animals ranges from 3 to 8 weeks and may

even be longer (Masiga et al., 1996; Baker, 1998; Radostitis et al., 1999). When control

cattle were placed in contact with naturally affected cattle from a recent outbreak in

Namibia, seroconversion was seen after 6 weeks, rose rapidly in the next two weeks by

which time 40% of contacts had died (Nicholas, 2004). In a fully susceptible cattle

population, the morbidity may reach 100% and mortality 50% (Masiga et al., 1996;

Baker, 1998). In experimental or natural outbreaks, most of the CBPP seroconversions

seem to continue until 6–7 months after the initial introduction of CBPP (Hudson and

Turner, 1963; Bygrave et al., 1968; Provost et al., 1987). However, in the field survey,

seroconversions continued more than 8 months after the disease onset (Lesnoff et al.,

2004).

2.2.4 Clinical Signs

The disease is characterized clinically by severe coughing, weakness, emaciation and

sometimes by elevated temperature (Provost et al., 1987; Egwu et al., 1996). The

severity of symptoms range from hyper acute, through acute, sub acute and chronic

forms of pleuropneumonia, while calves up to six months normally develop arthritis and

show lameness from swollen, hot and painful limb joints (Persson, 2002; Egwu, et. al.,

25

1996). Animals show dullness, anorexia, irregular rumination with moderate fever and

may show signs of respiratory disease. Coughing is usually persistent and is slight or

dry. Sometimes fever goes up to 40 – 42 0C, and the animal prostrates with difficulty of

movement. As the lung lesions develop, the signs become more pronounced with

increased frequency of coughing and the animal becomes prostrate or stands with the

back arched, head extended and elbows abducted (Nicholas, 2004). Cattle of all ages

can be affected by CBPP. The clinical picture of CBPP is more suggestive of damage

due to host immune and inflammatory responses rather than to direct toxic effects by

the Mycoplasma l cell components (Razin et al., 1998). The specific reactions elicited

by invading Mycoplasmas, essential for resistance and protection against Mycoplasma

infections, have also been shown to play a role in the development of lesions and

exacerbation of Mycoplasma induced diseases, as described and reviewed previously

(Biberfeld, 1985; Cassell et al., 1985; Cole et al., 1985; Howard and Taylor, 1985). In

addition to eliciting anti-Mycoplasma l immune responses, Mycoplasmas exert a wide

range of nonspecific immuno-modulatory effects upon cells making up the immune

system. Several documentations have been made on the clinical and pathological

features of CBPP (Provost et al., 1987; Masiga et al., 1996; Regalla et al., 1996). The

major signs being associated with respiratory stress (Scundamore, 1995), especially

after exercise where a soft dry cough is evident. All ages of animals are susceptible but

young animals develop joint swelling rather than lung infection. Calves are more

resistant to CBPP than adults (Curasson, 1942; Provost et al., 1987) and are generally

kept away from the main herd which greatly limits contact between the young ones and

the adults. Typically when first introduced into a herd, CBPP is severe and mortality

relatively high. However a small proportion of cattle may die rapidly without showing

any signs other than fever (FAO, 2002). Some animals appear to be naturally resistant

26

and subclinical forms are frequent. Severe respiratory signs are the most prominent

features observed in the clinical cases, and are associated with typical lesions of

pleurisy and pneumonia (Lesnoff, 2004).

2.2.4.1 Acute infection

The acute form involves cessation of rumination, nasal discharge, a dry cough and

difficulty in breathing (Scundamore, 1995; Ross, 1993, Curasson, 1942; Martel et al.,

1985; Provost et al., 1987); a marbled pneumonia and an exudative pleurisy are the

most-obvious lesions. Recovered cattle often have necrotic lung tissue, encapsulated in

sequestra where Mycoplasmas can survive (Lesnoff, 2004). The clinical signs observed

in the acute form are much accelerated. The pathological signs are usually characteristic

with marked pleural adhesion accompanied by exudative pericarditis (Regalla et al.,

1996). Affected animals may die within a week exhibiting classical respiratory signs.

2.2.4.2 Sub acute infection

Signs in the sub acute form, may be limited to a slight cough only noticeable when the

animal is exercised. CBPP in Europe, unlike that caused in Africa where mortality rates

are typically 10-70% in epizootics, is characterized by low morbidity and low or non-

existent mortality with the majority of infected cattle showing chronic lesions; this is

characteristic of endemic disease; the sub-acute form is most common in Africa

(Regalla et al., 1996).

27

2.2.4.3 Chronic infection

However, chronic lung lesions with viable MmmSC can persist in sick and/or recovered

cattle—in particular, when they develop ‘sequestra’ (i.e. necrotic tissues surrounded by

a fibrous capsule) and these animals could persist as carriers (Curasson, 1942; Provost

et al., 1987). There is no consensus on carrier parameters. According to Bygrave et al.,

(1968), most sequestra resorb into sterile fibrotic scars within 4 months. Nevertheless,

some examples of MmmSC isolations 1 or 2 years after the infection has been reported

from such lesions (Turner, 1954; Windsor and Masiga, 1977a).

2.2.5 Diagnosis of CBPP

Diagnosis of CBPP is achieved by the demonstration of typical pathology and/or the

presence of MmmSC after postmortem examination and isolation of the causative agent.

Although MmmSC is not difficult to cultivate, primary isolation of the pathogen from

lung lesions is made difficult by the use of therapeutic agents and cultures are often

negative from sequestered lesion material (Bashiruddin, 1998; Aliyu et al., 2000).

However, confirmation of the disease is usually done in two ways: detection of the

causal organism in affected tissue, and detection of serum antibodies to the organism

(FAO, 2002). The inability of CFT to discriminate between natural and vaccinal

exposures in animals has led to a greater reliance on Post Mortem examination of lung

lesions for monitoring and surveillance of CBPP in Nigeria. Moreover, some diagnostic

correlation exists between lung lesions of affected animals and serological techniques

such as CFT and enzyme-linked immunosorbent assay (ELISA) (Nicholas et al., 1996).

28

2.2.5.1 Pathology

Lesions are confined to the lungs and thoracic cavity which includes distension of

pulmonary lobules, consolidation, and marbling with varying colours of yellow, grey

and red hepatisation, bronchopneumonia and pleurisy, typified by adhesion of parietal

and visceral surfaces (Provost et al., 1987; Scundamore, 1995), and are mostly

unilateral. In a study in Nigeria, 95% of lesions were restricted to a single lung (Egwu et

al., 1996) with the diaphragmatic lobes being more commonly affected than cranial

lobes. Adhesions to the chest with roughened pleural membranes are common.

Significant quantities of straw coloured pleural fluid can be found in acute cases which

in most cases yields pure growth of the causative organism. The interlobular septa are

often distended and lungs show the typical marbled appearance with lung lobules

showing great variations in colour from red, grey to yellow depending on the stage of

inflammation. Associated lymph nodes undergo hypertrophy. In chronic cases the

sequestra is the main lesion type and consists of necrotic material surrounded by a

fibrotic capsule ranging from 10 to 100 mm in diameter. Necrotic foci have been

reported in the kidneys of affected cattle.

2.2.5.2 Serology

Serodiagnosis plays a key role in survey and control programs to combat Contagious

Bovine Pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides

SC. At the species level, serologic relatedness has until recently overshadowed all other

features used in routine mollicute identification, but the weight given to the

determination of molecular properties in mollicute classification and identification is

steadily increasing (Razin, 1992). The function of antigenic variation in several

Mycoplasmas like M. hyorhinis, M. gallisepticum (Yogev et al., 1994), M. bovis

29

(Rosengarten et al., 1994), has been attributed to either immune evasion or, for

structural proteins, microorganism/host interactions essential for pathogenesis.

Antigenic variation, an important mechanism of infection, is due to membrane surface

proteins (Vsps) mainly lipoproteins (Wise et al., 1993). The high rate of surface

antigenic variation characterizing mollicutes may impose some limitations on the use of

monoclonal antibodies to surface antigens as tools in Mycoplasma identification

(Rosengarten, 1996); though these difficulties are usually not encountered when

polyclonal antibodies are used (International Committee on Systematic Bacteriology,

1997). Antigenic variation of M. mycoides SC of bovine origin has been demonstrated

(Costas et al., 1987; Poumarat and Solsona, 1995) with isolates from different animal

species and geographic locations (Gonc,alves et al., 1994, 1996). These studies showed

differences among strains isolated from cattle, small ruminants and water buffalo

originating from European countries, in particular Italy. In a study, European strains

form a genetic clonal lineage (Cheng et al., 1995), and antigenic differences within the

Portuguese (with one exception), Spanish and French strains which together formed one

group with nearly identical immunoprofiles were seen which are distinct from the

Italian strains (Goncalves et al., 1998). Differences in protein profiles between M.

mycoides SC strains usually reflected variations in the concentrations of individual

proteins. In a study, protein profiles of PG1 and the Australian strain V5 were found to

be similar, both lacking the 30 kDa band but, the absence of specific proteins was noted

in some cases (Goncalves et al., 1998). Previous studies by Gonc,alves et al., (1994),

indicated that the type strain PG1 of unknown origin was characterized by the absence

of 54 kDa protein. In a related study, a strong IgA reaction to the membrane lipoprotein

P72 of M. mycoides SC, in bronchial lavage samples of cattle experimentally infected,

confirmed the induction of a specific local immune response (Abdo et al., 1997).

30

Furthermore, these studies showed that other reacting antigens with IgA, such as 110,

95 and 48 kDa, are lipoproteins. Proteins partitioning into TX-114 phase has been

demonstrated to be important immunogenic surface components recognized by host

antibodies during infection (Riethman et al., 1987; Rosengarten and Wise, 1991).

The serological techniques mostly in use today are those developed in the 1950s: the

serum agglutination slide test SAST (Turner and Etheridge, 1963), the complement

fixation test CFT (Campbell and Turner, 1953; Gambles, 1956) and the detection of

circulating antigen by Agar Gel Immunodiffusion AGID (Griffin, 1965; White, 1958;

Shifrin, 1967). These serological methods have been proven useful for the detection of

outbreaks and they have had an important role in the successful CBPP eradication

campaign in Australia (Newton, 1992). These tests though quite useful have some

limitations which has made them sometimes unreliable in the field. In a CBPP-free

region, the use of classical serological techniques might be disappointing as the

percentage of false positives will be quite high and consequently, the predictive value of

a positive result will be very low (Stark et al., 1995 ). The Slide-Agglutination Blood

Test, Agar Gel Diffusion and Complement-Fixation Tests were highly sensitive for

detection of acute cases but less so for chronic cases (Campbell and Turner, 1953;

Turner, 1962; Shifrine and Gourlay, 1967). The Slide Agglutination Test on its own is

not very sensitive and it may give some false positive reactions but easy to perform in

the field, but will detect positive cases in acute outbreaks (Adler and Etheridge, 1964) .

It can be recommended for the diagnosis of acute outbreaks when immediate actions are

to be taken (Aliyu et al., 2000). Though the Complement-Fixation Test (CFT) is

commonly used as a diagnostic method in most CBPP endemic countries of Africa, its

sensitivity in detecting chronically affected animals is low (Provost et al., 1987;

31

Nicholas et al., 1996). It also has the disadvantage of being quite difficult to standardize

because of the use of antigens or fresh red blood cells of various qualities, and it

requires skilled technicians to perform the test. In the case of an acute outbreak, the

sensitivity is quite good as it detects up to 70% of positives and the rapid waning of

antibodies might be an advantage since vaccination elicits very low titers that usually

return CFT results to negativity after 3 months. Once again, this test can be used to

detect acutely infected herds. Performing mass screening is relatively rapid and easy by

testing a single dilution, thus, allowing the determination with CFT of incidence of the

disease in a country, i.e., by detecting the herds that have suffered from an outbreak

during the 3 months before the sampling. It will not permit the detection of all infected

animals and therefore, cannot be recommended as an individual testing method for

import restrictions. (Le Goff and Thiaucourt, 1998).

2.2.5.3 Molecular diagnosis

In general, species within the M. mycoides cluster share many immunological,

biochemical and genetic properties, which result in major problems for diagnostic

laboratories in the identification of field strains because of the use of classical

techniques, such as the Growth Inhibition Test, that have some limitations (Persson,

2002, Cottew et al., 1987). The Classical technique was considered for a long time as a

gold standard but, in practice, gives varying degrees of cross-reactions (Thiaucourt, et

al., 2000a). Though some degree of success has been achieved in immunological

experiments carried out to differentiate the various strains (Vilei and Abdo, 2000),

these tests and most others were found not to be as reliable as molecular based tests

such as the Polymerase Chain Reaction (PCR) (Bashiruddin et al., 1994). The advent of

molecular biology has greatly enhanced the capability to detect and identify species, to

32

classify and characterise strains and to assess the genetic diversity of populations

(Stakenborg et al., 2005). Diagnostic tests based on DNA amplification by polymerase

chain reaction (PCR) promise high sensitivity and specificity and are suited to large-

scale performance in 96-well format thermal cyclers. The traditional method for the

analysis of PCR products has been agarose gel electrophoresis which will continue to be

useful for the optimization and analysis of PCR tests with small sample numbers. Gel

electrophoresis is not suitable for large-scale routine use (Bashiruddin et al., 1998). The

use of PCR has overcome most of the problems encountered with immunological tests

and it has permitted major advances both in phylogenetic studies and in diagnostic

methods (Thiaucourt et al., 2000a). The specific identification of MmmSC strains can

now be obtained by various PCR methods but these techniques are universal and

recognize pathogenic as well as vaccine strains (Dedieu et al., 1994). The PCR

techniques for the diagnosis of MmmSC have the advantage of being fast, specific, very

sensitive and easy to perform (Bashiruddin et al., 1994; Dedieu et al., 1994; Hotzel et

al., 1996; Miserez et al., 1997). Primers specific for the M. mycoides cluster

(Knudtson and Minion, 1993) and for MmmSC (Rovid and Roth, 1997) have been

developed, and various types of PCR assays have been developed (Rovid and Roth,

1997, Thiaucourt, et al., 2000a). Numerous detection methods able to handle moderate

to large sample numbers in the 96-well format have been described (Lazar, 1995).

These methods are based on the hybridization of oligonucleotides to either capture or

detect PCR product. (Rasmussen et al., 1994). Other methods use labeled primers to

incorporate biotin into the PCR product which is then used to either capture the product

or detect the amplicon immunoenzymatically (Bashiruddin et al., 1994). Methods which

use RNA probes followed by anti-RNA: DNA to detect captured hybrids have also been

used. A method for the specific capture and enzymatic detection of PCR product and its

33

application to the detection of Mycoplasma mycoides from a variety of clinical samples

like washed lungs with hot water or impregnated filter strips have been described

(Bashiruddin et al., 1994).

PCR can also be applied directly after denaturation in boiling water, using samples such

as lung exudates, without any DNA extraction. It can even be applied to dry samples on

filter paper. The PCR can also be performed with urine or blood. The main advantage of

the PCR technique is that it can be applied on poorly conserved samples (contaminated

or without any viable Mycoplasmas as may occur following antibiotic treatment).

Molecular epidemiological studies on genomic fragments of M. mycoides subsp.

Mycoides SC have been able to differentiate phylogenetic lineages of this organism.

(Lorenzon et al., 2003). This has made it possible to trace sources of outbreaks based on

their phylogenetic lineages. There are several assays systems developed for this

purpose. In the case of CBPP, for example, specific amplifications can be obtained

(Dedieu et al., 1994) and additional sensitivity can also be achieved by nested PCR

schemes (Miserez et al., 1997). Up till now phylogenetic studies have focused on the

16S rRNA genes (Olsen and Woese, 1993).

2.2.6 Economic importance

Contagious Bovine Pleuropneumonia (CBPP), a highly contagious disease of cattle

caused by Mycoplasma mycoides mycoides (SC) type, is still considered to be the most

economically important cattle disease in Africa, causing greater losses in cattle than any

other disease after Rinderpest (OIE, 1997). Losses per annum due to this disease are

estimated to be in the region of US$ 2 billion (Masiga et al., 1999). The Joint Project 28

(JP28) eradication programme for CBPP which was carried out in the early 70s though

34

not very successful greatly reduced the incidence of CBPP in Nigeria. Even then, the

economic losses due to CBPP in Nigeria were estimated to be in excess of 3.6 million

dollars annually nearly 20 years ago (Osiyemi, 1981). CBPP spread from Kenya to the

Ngorongoro carater in Tanzania caused over 14,000 cattle deaths in just six months (nr-

[email protected] ).

2.2.7 Vaccines

In the last decade there has been a substantial re-emergence of CBPP, despite

vaccination campaigns using freeze-dried broth cultures of live attenuated Mycoplasma

mycoides subsp. mycoides small colony biotype (MmmSC) (strain T144 or T1SR).

Information from the field and studies (Masiga, et al., 1999a and 1996, Thiaucourt et

al., 2000a, Yaya et al., 1999) have indicated that the current vaccines do not effectively

protect cattle from outbreaks of disease. The frequent failure of antibiotics and other

therapeutic approaches to eradicate Mycoplasmas and abort the infectious disease

process has led to the conclusion that development of effective vaccines is the most

promising approach to control Mycoplasma infections in humans and animals (FAO,

2002). CBPP control is achieved by eliminating the whole cattle herd population, i.e.

stamping out, wherever the disease is detected. However, this may not prove realistic,

and quarantine coupled with vaccination is the most frequently used CBPP control

measure (FAO, 2002).

The history of CBPP vaccine could be traced back to 1852, when Willems established

the ways to “inoculate” infectious material in cattle in order to protect them. This

process was recommended by the fifth international veterinary congress in 1889 as an

35

auxiliary measure to reduce significantly the number of CBPP outbreaks before

stamping out policies can be put into force and achieve complete eradication

(Thiaucourt, 2000b). This knowledge was the basis for the establishment of efficient

control strategies and eventually led to the eradication of the disease from many

countries: 1888 Holland, 1895 Switzerland, 1896 Great Britain, 1900 Belgium, 1902

France, 1919 Austria, 1924 Baltic States and 1934 Poland and Soviet Union.

In Africa, where the application of the stamping out policy of eradication is not feasible

the control of CBPP has relied on preventive immunoprophylaxis using live attenuated

cultures of the causative agent. CBPP vaccination is the method that is currently in use

in most African countries employing the vaccine strains T1/44 or its streptomycin

resistant derivative T1-SR (Litamoi et al., 2004). These CBPP vaccines do not confer

long-term immunity (Rweyemamu et al., 1995). The CBPP T1-44/2 vaccine that is

currently recommended for use in endemic areas of Africa confers immunity for ≤1 year

(Tulasne et al., 1996). The low potency of the T1/SR vaccine that was used during the

initial outbreaks of CBPP in 1990s frustrated both the farmers and livestock experts

(Masiga et al., 1996; Tulsane et al., 1996). These vaccines though have been used quite

successfully in the past for the control of CBPP in Australia, Nigeria and East Africa

(Brown et al., 1965, Hudson 1968). The T144 is noted for post vaccinal reactions while

the T1 SR induces a shorter period of immunity than the T144 (Heubschle et al., 2002).

A major factor behind poor vaccine efficacy is likely to be sub-optimum bacterial titres.

Many vaccine production laboratories do not reach the O.I.E. recommendation of

delivering a vaccine at 108 viable Mycoplasmas per animal dose (which allows for

losses during lyophilisation, storage and transport (Rweyemamu et al., 1995, Litamoi

and Seck, 1999). The pH of the growth medium is an important factor which affects

36

Mycoplasma viability (Gourlay and MacLeod 1966, Miles 1983) Current vaccine

media e.g. Gourlay’s (Gourlay, 1964) and F66 (Waite and March 2001) are poorly

buffered, containing only a dibasic (Na2HPO4) phosphate salt, and exhibit a sharp drop

in pH during MmmSC growth. This is mirrored by a rapid reduction in culture viability

once the pH begins to fall. A buffer system based upon N-[2-hydroxyethyl]piperazine-

NP-[2-ethanesulfonic acid] (HEPES) has been described (Provost et al., 1970), which

exhibits a 10-fold increase in titre and markedly increased culture survival compared to

contemporary media due to maintenance of a neutral pH during the growth cycle.

Current vaccines are freeze-dried to allow for longer term storage at 320C and to reduce

the requirement for cold-chain transport in the field. Another disadvantage is that

Mycoplasmas are extremely heat labile and as a result, current freeze dried vaccines for

the prevention of CBPP must be maintained under cold storage until used in order to

preserve the viability and potency of the immunizing organisms (Litamoi et al., 2004,

Hudson, 1968). Apart from the alleged low potency, the effectiveness of the CBPP

vaccines might have been reduced by inefficient cold chain. During the vaccination

campaigns of the past, the vaccines were stored in paraffin-fuelled refrigerators (which

were unreliable because they often failed to attain optimal cooling temperature). The

low potency of the vaccine could have resulted also from the use of harmful diluents

such as chlorinated or tap water or mishandling of the reconstituted vaccine by exposing

it to light and high temperatures for a long time (Karst, 1972).

The difficulty with the use of CBPP vaccines is that two divergent issues are sometimes

confused with each other: the efficacy of the vaccine itself, and the efficient conduct of

a vaccination campaign (Thiaucourt, 2004). The first issue can be assessed in controlled

experiments provided that all the parameters are clearly identified and analyzed. The

37

second is far more difficult, and therefore also more controversial, because the

efficiency of a vaccination campaign depends not only on the intrinsic quality of the

vaccine itself but also on the strategy and logistics for implementation of the

vaccinations (Thiaucourt, 2004). With reported cases of CBPP vaccine failures in past

vaccination campaigns (Amanfu et al., 1998), the capacity of current vaccines to control

the disease with eventual eradication of the disease has often been an issue of debate.

Even though Uncontrolled cattle movement, insufficient vaccination cover, acquired

immunity and the poor health status of vaccinated cattle were identified as some of the

major reasons attributable to CBPP vaccination campaigns failures (Thiaucourt et al.,

1998) many experts believe that “clearly, the ideal CBPP vaccine has not yet been

developed” (Thiaucourt, 2004).

2.3 Mycoplasma mycoides cluster

The Mycoplasma mycoides cluster consists of six pathogenic Mycoplasma species,

subspecies or strains causing mild to severe disease in ruminant hosts, either bovine or

caprine. These are Mycoplasma mycoides subsp. mycoides Large Colony (MmmLC),

M. mycoides subsp. Mycoides small colony type (MmmSC), M. mycoides subsp. capri

(Mmc), M. capricolum subsp. capricolum (Mcc), M. capricolum subsp.

capripneumoniae (Mccp), and Mycoplasma species bovine serogroup seven (MBG 7)

(Cottew et al., 1987). Even though the causative organism for Contagious Bovine

Pleuropneumonia (CBPP) was isolated for the first time over a century ago by Nocard

and Roux in 1898, it took another 58 years before organism was definitely identified as

a Mycoplasma and called by Edward and Freundt (1956) as Mycoplasma mycoides

subspecies mycoides (M. M. mycoides) by which time it had undergone nine name

changes (Nicholas and Bashiruddin, 1995). Two variants M. M. mycoides have been

38

recognized, the small colony (SC) type which causes CBPP in cattle, and the large

colony (LC) type which produces arthritis and mastitis in goats (Cottew and Yeats,

1978). The members of the Mycoplasma mycoides cluster are closely related both

genetically and phenotypically. When using conventional serological methods for

Mycoplasma typing, cross reactive antibodies impede the identification of pathogenic

agents of the M. mycoides cluster thereby complicating interpretation (Cottew and

Yeats, 1978; Rurangirwa and Shompole, 2000). This phenomenon was observed

between MBG 7 and Mccp (Thiaucourt, 2002) and between MBG 7 and Mcc (Bolske

1988). Analysis of the 16S rRNA categorized MBG 7 with Mcc and Mccp, and the two

species MmmLC and Mmc as a single entity (Pettersson, et al., 1996). Parsimony

analysis on an alignment of 49 DNA sequences show a subdivision of the M. mycoides

cluster into two subgroups that is in accordance with results obtained by phenotypic

methods. Two lineages exist within the capricolum subgroup, one of them clustering

strains identified as M. capricolum subsp. capricolum, M. capricolum subsp.

capripneumoniae and M. sp Bovine Group 7. However M. capricolum subsp.

capripneumoniae strains can readily be identified by three specific nucleotide positions

or by sequencing the 1298 bp long fragment (Thiaucourt et al., 2000a). There is no

clear subdivision within the mycoides subgroup, supporting the idea that M. mycoides

subsp. Mycoides LC and M. mycoides subsp. capri should not be separated into two

subspecies (Thiaucourt et al., 2000a).

2.3.1 Taxonomy and Phylogeny of the Mycoides cluster

The basis for the M. mycoides cluster taxonomy was established in 1987 (Cottew et al.,

1987) with a comprehensive review of the results on the reference strains by

conventional methods such as biochemical tests, immunological reactions (Al-Aubaidi

39

et al., 1972), isoenzyme patterns (Salih et al., 1983) one and two dimensional Poly

Acrylamide Gel Electrophoresis (PAGE) patterns (Rodwell, 1982) and DNA

hybridization. However, various techniques such as growth inhibition tests had already

shown that some species exhibited a high degree of intraspecies heterogeneity.

Therefore, Cottew et al., 1987 advocated that numerous strains of each group of strains

should be investigated in order to confirm the preliminary results (Cottew et al., 1987).

Subsequent studies with larger panels of strains, by whole cell PAGE patterns (Costas et

al., 1987; Leach et al., 1989), methylation of DNA (Bergemann et al., 1990), dot

blotting with monoclonal antibodies (Thiaucourt et al., 1994) or substrate utilization

patterns (Abu-groun et al., 1994) confirmed that this intraspecies heterogeneity was

mainly confined within the mycoides and capricolum species. By contrast, MmmSC and

Mccp seemed rather homogeneous, although Restriction Fragment Length

Polymorphism (Cheng et al., 1995; Poumarat and Solsona, 1995) as well as differences

in substrate utilization patterns (Houshaymi et al., 1997) was observed within MmmSC.

Based on the classification of the 16S rRNA, M. putrefaciencs was classified within the

M. mycoides cluster (Weisburg, et al., 1989). However, sequencing of the 16S rRNA of

M. cottewi and M. yeatsii, revealed a close similarity of M. putrefaciens with these

species, with similarity of 99.7% with M. cotttewii and 98% with M. yeatsii (Pettersson

et al., 1998). But these species, should not be regarded as members of the M. mycoides

cluster on the basis of serological, biochemical features (Pettersson et al., 1996) and

based on the phylogenetic tree derived from distance analysis of five protein coding

sequences (Figure 1) (Manso-silvan et al., 2007).

Some authors have proposed a revision of the actual classification of the M. mycoides

cluster to five subspecies (Thiaucourt et al., 2000a) Accorrding to them the new

40

calssification would have the advantage of retaining most former names (Thiaucourt et

al., 2000a). However, the proposal to reclassify this cluster accordingly has not been

adopted by the Mollicutes Taxonomy Committee (International Committee on

Systematic Bacteriology, 1997), which argues that such a reclassification would create

considerable problems in diagnostic veterinary medicine.

2.3.2 Pathogenicity of members of the M. mycoides cluster

Very little is known about the factors and mechanisms that affect the pathogenicity of

M. mycoides mycoides SC. No secreted toxins have been identified, neither receptor

molecules on the bacterial surface that mediate binding to host epithelium or induce

other cellular responses in the host tissues. However certain factors have been

associated with the pathogenesis, but the precise modes of action are still elusive

(Persson, 2002).

2.3.2.1 Capsular polysaccharide

An important pathogenicity factor in MmmSC is the capsular polysaccharide (CPS),

previously known as galactan (Woubit, 2008). It is made up of the carbohydrate

galactose (90%) and to a lesser extent glucose (2-4%) and lipid (Rodwell, 1982).

Injection of purified CPS to cattle produced severe respiratory collapse and even death

(Buttery, et al., 1976; Cottew, 1979). The CPS has been found to play a significant

role in the pathogenesis of infection, binding to the host tissue surfaces and inducing

resistance to phagocytosis. It has also been associated with the formation of autoreactive

antibodies and consequently autoimmune responses. Toxic effects of MmmSC have also

been associated with the capsule (Egwu et al., 1996, Nicholas et al., 2000, Nicholas and

Bashiruddin, 1995).

41

2.3.2.2 Hydrogen peroxide

In a recent investigation cited by Woubit (2008), there was indication that glycerol

metabolism in MmmSC strains release hydrogen peroxide (H2O2) as a byproduct,

resulting in disruption of host cell integrity. Hydrogen peroxide is produced by a

membrane located enzyme L-α-glycerophosphate oxidase (GlpO) that is involved in

glycerol metabolism (Pilo et al., 2005). The initial hypothesis was based on the fact

that virulent MmmSC African strains possessed an active ATP-binding cassette (ABC)

transport system for the utilization of glycerol, which is metabolized to

dihydroxyacetone-phosphate (DHAP) releasing H2O2, while European strains lacked

part of the glycerol uptake genes due to deletion and are less virulent (Vile and Abdo,

2000).

2.3.2.3 Variable surface protein

A report by Rosengarten and Wise (1991) indicated that Mycoplasmas express surface

proteins which can undergo reversible changes to alter the antigenic repertoire in a cell

population. The gene for these variable surface protein Vmm as they were termed

encodes a lipoprotein precursor of 59 amino acids, where the mature protein was

predicted to be 36 aa and was anchored to the membrane by only the lipid moiety, as no

transmembrane region could be identified (Woubit et al., 2007). The protein was found

to undergo reversible phase variation at a frequency of 9 x 10-4 to 5 x 10-5 per cell per

generation and this variation enables the Mycoplasma organisms to escape the host

immune defense mechanism of their host (Patersson, 2002). Vmm-like genes were also

found in the other three members of the M. mycoides cluster: Mcc, Mccp, MBG 77 and

in M. putrefaciens (Persson et al., 2002). The Vmm gene in MmmSC is an example of a

single gene encoded variable surface protein (Citti et al., 2005). A recent whole genome

sequence of Mcc type strain California kidT has revealed genes encoding a diverse

42

family of variable surface proteins termed the Vmc system. The Vmc genes of Mcc

present not only an alternative surface structure but also a system that permits high-

frequency phase variable expression as well as structural variation a feature crucial for

the survival of these wall-less microorganisms in the host (Wise et al., 2006). Variable

proteins are also involved in adhesion, hemadsorption, membrane transport and

immunomodulation (Le Grand et al., 2004).

2.4 Molecular methods for the identification and characterization of Mycoplasmas

Conventional methods for the detection and identification of Mycoplasmas

systematically involve enrichment steps in selective broth followed by morphological,

biochemical and serological tests. Although well established, these techniques have

some important drawbacks. The morphological and biochemical characteristics are in

general not discriminative, while serological cross-reactions have been frequently

reported as well. Moreover, these classical techniques are often labour intensive and

hardly ever useful to differentiate strains belonging to the same species (Stakenborg et

al., 2005).

During the past decades, the importance of molecular techniques in mycoplasmology

has greatly increased. Still, no single technique seems to be perfect. The discriminatory

power, applicability, reproducibility, ease of performance, and ease of interpretation,

may vary depending on the technique used and must be evaluated for each situation

(Olive and Bean, 1999).

The development of more accurate and faster techniques has become increasingly

important and although some generally applicable tests have been described (Melin et

al., 2004), molecular biology opened a path to shorten detection times and to improve

43

identification methods. In particular PCR methods appear very promising to replace

more and more conventional methods, although further improvements are necessary

(Vaneechoutte and Van Eldere, 1997). Currently, PCRs can be applied on purified

samples, but their effectiveness for detection and simultaneous identification may be

limited when applied to biological materials due to inhibitory effect from such

materials. The importance of sample pre-processing or DNA extraction methods, which

may need case-to case optimisation to yield compatible results (Radstrom et al., 2004),

are hard to standardise and selective enrichment steps are frequently preferred.

Moreover, PCR tests are mostly very specific and, as a consequence, only valuable to

detect and/or identify one or a few species. This is evident by the current lack of

commercially available quality-controlled, low-cost, PCR kits for the detection and

identification of Mycoplasmas. With the exception of very few commercial kits for

Mycoplasmas in cell-cultures (Razin et al., 1998), laboratories must rely on in-house

improvisions and such protocols are not often reliable. Nonetheless, molecular

techniques are likely to be an increasingly important tool for the detection and

identification of fastidious organisms like the Mycoplasmas.

While PCR can result in a direct identification of an organism by the amplification of

the species-specific product, sequence analysis can be used to discriminate between

strains. This technique does not require the cultivation of the organism and since all

molecular typing techniques are based on sequences comparisons, sequence analysis

seems the best approach. This technique also has an excellent interlaboratory

reproducibility and data can be stored online (Bashiruddin et al., 1994). But, sequence

analysis of single genomic fragments also has some important drawbacks notable

amongst which is the fact that the region under investigation is very small and is hardly,

if ever, representative of the entire genome. Besides, the region under investigation

44

must be conserved enough for amplification and at the same time variable enough to

differentiate between strains, which is not always easily attainable. In addition, the

stability of the gene must be verified over in vitro passages.

Sequence analyses of specific genomic fragments have been successfully used for the

typing of some Mycoplasma spp. In case of M. genitalium, the MG309 gene sequence

was proven stable in sequential urine samples obtained from single patients for at least

five weeks and may be valuable candidates for further typing studies (Ma and Martin,

2004). Also, sequence analysis was demonstrated for a genomic fragment of 2400 bp of

M. capricolum subp. Capripneumoniae (Lorenzon et al., 2002). Nucleotide variations in

this specific fragment were used to determine the geographical distribution of different

strains. Sequence variation of parts of the haemagglutinin encoding gene vlhA of M.

synoviae was used for strain differentiation (Hong et al., 2004) and could be linked to

the length of the expressed protein and to virulence (Hong et al., 2004)

Another technique, MLST which is even more expensive, was developed to cope with

some important drawbacks related to typing studies based on sequence analysis of

single genes (Olive and Bean, 1999). Instead of analyzing one single genomic fragment,

the partial sequences of multiple, selected genes are determined. Ideally, sequence

fragments about 500 bp in length of several, widely scattered genes should be included

to obtain a good representation of the genome, but the number may vary between

different studies. Housekeeping genes are mostly selected because they are less often

subjected to horizontal transfer events and are not liable to strong or unusual selective

pressures (Yaya et al., 2008). As a consequence, they are perfectly suited to represent

the accumulation of sequence variation in the genome. Generally in MLST analysis the

number of nucleotide differences found between alleles is disregarded and sequences

45

are given different allele numbers whether they differ at a single or at many nucleotide

positions (Yaya et al., 2008). The rationale for this is that a single genetic event

resulting in a new allele can occur by a point mutation, altering only a single nucleotide

site, or by a recombinational replacement that will often result in the modification of

multiple sites (Dingle et al., 2001). This approach has been applied successfully to a

wide variety of bacterial pathogens such as Campylobacter jejuni (Dingle et al., 2001),

Staphylococcus aureus (Enright et al., 2000), and Haemophilus influenzae (Meats et al.,

2003), and several other bacterial species of global importance (Urwin and Maiden.

2003.) for which, databases for international surveillance have been set up (Feil and

Enright, 2004). This approach has multiple applications such as phylogeny (Hanage et

al., 2005), molecular structure analysis (Tourasse et al., 2006) and molecular

epidemiology. In molecular epidemiology, MLST can be used to study the evolution of

antibiotic resistant strains (Gherardi et al., 2006), the temporal trends in strain

expansion or the distribution of strains of various lineages within a population (Lacher

et al., 2006).

However, some exceptions have been reported where a number of housekeeping genes

showed too few differences to be useful for typing (Dumke et al., 2003, Manning et al.,

2003, Nallapareddy et al., 2002). For these cases, carefully selected species specific

genes may be included instead. Only few investigators reported on the potential of the

technique for Mycoplasma spp. Even though sequence variations are known to occur

within MmmSC strains as evidenced by restriction fragment length polymorphism

(Poumarat and Solsona, 1995; Thiaucourt et al., 1998), comparative studies by SDS-

PAGE have already shown that strain variability within MmmSC is much more limited

than within other Mycoplasma mycoides mycoides cluster members (Costas et al.,

1987). So in the case of MmmSC this approach was not successful, as the variability

within housekeeping genes was too limited, if present at all. This was the case of the

genes fusA, lepA and rpoB

this may indicate that MmmSC

this Mycoplasma has adapted very recently to its bovine host. From a practical point of

view in this study, just as in the works

unknown function or non coding sequences had to be select instead in order to

differentiate MmmSC strains

multilocus sequence analysis (MLSA). This typing sc

analyses, as the gene targets used are selected for typing a biotype of a given subspecies

(MmmSC) and would not be suitable for typing strains of higher taxonomic ranks, given

their high variability (Yaya

of being a portable and very robust approach.

While the availability of complete genomic sequences is expected to provide a sound

basis for establishing phylogenetic relatedness among bacterial species and

consequently to enable the construction of taxonomic entities based on phylogeny, the

way to achieve this has not yet been worked out, since it presents several key problems.

Thus, the current, somewhat arbitrary definition of the basic taxonomic entity, that of a

bacterial species, includes all strains with approximately 70% or higher DNA homology

and with 5°C or lower

systems to generate phenotypic variations (Citti and Rosengarten, 1997) may give clues

for the understanding of pathogenesis but it also sheds light on the identification

problems encountered with reactions based on the recognition of potentially

variable membrane proteins. The sequences of a putative membrane protein gene and

partial flanking open reading frames have been obtained from various strains in this

cluster, including all reference strains. Sequence analysis showed this locus is present

46


rpoB (Yaya et al., 2008). In terms of an evolutionary perspective

MmmSC genomes are extremely homogeneous, suggesting that

adapted very recently to its bovine host. From a practical point of

view in this study, just as in the works of Yaya et al., (2008), this meant that genes of


strains. This is why the strategy has alternatively been named

multilocus sequence analysis (MLSA). This typing scheme is less universal than MLST


) and would not be suitable for typing strains of higher taxonomic ranks, given

(Yaya et al., 2008). However, as MLST, MLSA has the advantage

of being a portable and very robust approach.



tly to enable the construction of taxonomic entities based on phylogeny, the




Tm (Razin, 1992). The recent elucidation of Mycoplasma


r the understanding of pathogenesis but it also sheds light on the identification

problems encountered with reactions based on the recognition of potentially

membrane proteins. The sequences of a putative membrane protein gene and

anking open reading frames have been obtained from various strains in this



. In terms of an evolutionary perspective

genomes are extremely homogeneous, suggesting that

adapted very recently to its bovine host. From a practical point of

, this meant that genes of


. This is why the strategy has alternatively been named

heme is less universal than MLST


) and would not be suitable for typing strains of higher taxonomic ranks, given

However, as MLST, MLSA has the advantage



tly to enable the construction of taxonomic entities based on phylogeny, the




Mycoplasma l


r the understanding of pathogenesis but it also sheds light on the identification

problems encountered with reactions based on the recognition of potentially hyper

membrane proteins. The sequences of a putative membrane protein gene and

anking open reading frames have been obtained from various strains in this


47

and fully conserved in all strains of M. mycoides subsp. Mycoides SC isolated from

geographically most distant places worldwide (Thiaucourt, 2000b).

A DNA probe based on randomly chosen genomic fragments was developed for the

differentiation of members of the mycoides cluster into four groups (Taylor et al.,

1992). Genomic typing with two DNA insertion elements, IS1296 and IS1634, has

provided an efficacious tool for a preliminary differentiation of M. mycoides subsp.

Mycoides SC strains (Frey et al., 1995; Cheng et al., 1995; Vilei et al., 1999; Vilei and

Frey, 2004).

A new PCR based test has also been developed for the detection of MmmSC in animal

tissues (Bashiruddin et al., 1994) thus making it possible to identify the causative agent

without necessarily isolating them from the tissues.

The PCR–REA-based analysis of the bgl gene differentiates the African field strains of

M. mycoides subsp. Mycoides SC from the Australian strains, as well as from the type

strain PG1 whose origin is unknown. It also differentiates the T1-derived vaccine strains

from all other African strains. (Cheng et al., 1995; Vilei and Abdo,, 2000; Vilei and

Frey, 2001; Edy et al., 2004).

The use of HindIII allowed further differentiation and showed, notably, that the profiles

obtained from the vaccine strains T1/44 and KH3J were not identical (Thiaucourt et al.,

1998). The evidence of multiple copies of insertion elements present in the MmmSC

genome allowed the development of new typing tools based on Southern blot

hybridization (Cheng et al., 1995). The use of IS1296 as a probe allowed the clear

48

differentiation of recent European strains from those of African origin (Cheng et al.,

1995). The observed difference was explained later on by the identification of an 8.8

kbp deletion in the genome of most MmmSC strains of European origin (Vilei and

Abdo, 2000), resulting in a missing IS1296 band. The use of another insertion element,

IS1634, also led to different Southern blot profiles (March et al., 2000), although the

high copy number of this insertion sequence (N=60), as compared to that of IS1296

(N=28), gave rise to profiles that were difficult to analyze. In 2004 the whole genome

sequence of MmmSC reference strain PG1 was published (Vilei and Abdo,, 2000),

opening new opportunities for the development of typing tools. Analysis of the PG1

genome sequence showed that the loci and primer pairs previously selected for MLSA

were not the most adequate. Some of the primers hybridized on multiple sites, whilst

other targeted sequences that were duplicated in the PG1 genome, hampering result

interpretation.

The use of a technique based on sequencing multiple loci, designated “multilocus

sequence analysis” (MLSA), allowed shortly after the identification of 15 different

allelic profiles within a representative number of MmmSC strains (N=48) of various

origins (Lorenzon et al., 2003). Multilocus Sequence Analysis (MLSA) is very similar

to Multilocus Sequence Typing (MLST), an unambiguous procedure for characterizing

isolates of bacterial species (Enright and Spratt, 1999). MLST has been used with

various pathogenic species, such as Neisseria meningitidis, (Maiden et al., 1998; Feil et

al., 1999). For MLST, typing is based on house-keeping genes exhibiting sufficient

variations within the different strains, potentially allowing the differentiation of millions

of different strains. Previous studies have indicated that when this technique was

49

applied to MmmSC it was not successful because MmmSC strains were very closely

related and the variability within housekeeping genes was too limited, if present at all.

50

CCHHAAPPTTEERR TTHHRREEEE

SSEERROOLLOOGGIICCAALL SSTTUUDDIIEESS

3.1 Experiment 1: Identification of MmmSC in twelve States of Northern Nigeria

by competitive Enzyme Linked Immuno-Sorbent Assay (c-ELISA).

3.1.1 Introduction

Serodiagnosis plays a key role in survey and control programs to combat Contagious

Bovine Pleuropneumonia (CBPP) caused by Mycoplasma mycoides mycoides Small

Colony (Le Goff and Thiaucourt, 1998). Generally serological methods have been

proven useful for the detection of outbreaks of CBPP and they have had an important

role in successful CBPP eradication campaigns in several countries (Newton, 1992).

However, most of the serological techniques used today are still those developed in the

1950’s: the serum agglutination slide test (SAST) (Turner and Etheridge, 1963), the

Complement Fixation Test (CFT) (Campbell and Turner, 1953; Gambles, 1956), and

the detection of circulating antigen by Agar Gel Immuno-Diffusion (AGID) (Griffin,

1965; White, 1958; Shifrin, 1967). The Slide Agglutination Test is not very sensitive

and it might give some false positive reactions but it is nevertheless easy to perform

directly in the field, and it will always detect some positives in the case of acute

outbreaks (Adler and Etheridge, 1964). It can be recommended for the diagnosis of

acute outbreaks when immediate actions are to be taken. The CFT is quite difficult to

standardize because of the use of antigens or fresh red blood cells of various qualities

and it requires skilled technicians. The antibodies detected by CFT wane rapidly and the

number of positives declines dramatically when the outbreak has occurred more than 3

months before the sampling. As with SAST, a number of false positives might be found

in negative herds (Etheridge and Buttery, 1976).

51

An Indirect ELISA based on the systematic, genetic, biochemical and antigenic analysis

of surface exposed lipoproteins of M. mycoides subsp. Mycoides looked very promising

but was not specific enough (Le Goff and Lefevre, 1989). Recently, a competitive

ELISA was developed on the basis of a monoclonal antibody which specifically

recognized an uncharacterized 80 kDa antigen of M. mycoides subsp. Mycoides SC (Le

Goff and Thiaucourt, 1998). The OIE reference method for CBPP serology is the

Complement Fixation Test (CFT). This technique was used in the past for CBPP

eradication in many countries. However, it presents some disadvantages, mainly the

difficulties of antigen production, standardization and the existence of non-specific

positive results. For these reasons the CIRAD-EMVT (FAO World Reference

Laboratory for CBPP) has developed a second test, a competitive ELISA (c-ELISA)

based on a monoclonal anti-MmmSC antibody (named 117/5). This test is an alternative

to the CFT for the OIE and can be used for the official CBPP controls. For both ELISA

and CFT, it should be noted that the results obtained should be interpreted by taking

into account the results of the whole herd. The animals in the incubation stage cannot be

detected, as well as many animals in the chronic stage of the disease, since the

percentage of positive animals decreases as time progresses. So, the sampling protocol

should be adapted to this situation, and a significant number of animals to be tested

must be chosen in the suspicious herd (Institute Pourquier, c-ELISA protocol). In this

work, c-ELISA was used to estimate the prevalence of CBPP infection in the twelve

Northern States of Nigeria.

3.1.2 Materials and Methods

A total of 2026 sera samples were collected from 12 States of Northern Nigeria

indicated in Fig. 2 for analysis by c-ELISA. Sera were collected from sick or

52

apparently unhealthy animals either from suspected outbreaks in the field or through the

Pan-African programme for the Control of Epizootics (PACE) network in the Northern

parts of the country. Under the PACE programme, trained Community Animal Health

Workers (CAHW) were mandated to collect and send to Vom, sera from cattle herds

suspected to be habouring any of the transboundary diseases amongst which is CBPP.

The CAHW were trained on methods of sample collection and ensured that samples

were not collected from animals that have been vaccinated within last 3 months.

53

Figure 2: Map of Nigeria showing the 12 States .

54

3.1.2.1 Competitive Enzyme Linked Immuno-Sorbent Assay (c-ELISA)

The test was carried out according to (Le Goff and Thiaucourt, 1998). Microtitre plates

precoated with lysed Mycoplasma mycoides mycoides SC antigen were used for the test.

Non decomplemented sera (diluted 1/10) and Monoclonal Antibody (Mab) diluted in

PBS with 0.5% horse serum and 0.05% Tween 20 together with all the controls

(Positive, Negative, Monoclonal) were incubated in the plate for 1 hour at 37°C under

moderate agitation in a humid chamber. The plates were then washed twice following

which conjugate was added to all the wells (100 µl) and the plates incubated for

another1 hour at 37°C. After the incubation, the plates were washed three times and the

substrate added to all the wells (100 µl) and this time incubated for 30 minutes. The

reaction was then stopped with the addition of a Stop solution (Sulphuric acid). The

Optical Densities of the reaction was read using Multiskan EX (Labsystems) at 450 nm.

3.1.3 Results

3.1.3.1 c-ELISA

The results for the c-ELISA on isolates from Northern Nigeria are presented in Table 1.

Out of the twelve States surveyed, only two states were negative for CBPP by c-ELISA.

55

Table 1: Estimated prevalence of CBPP by c-ELISA in 12 Northern States of

Nigeria

S/N STATE NO. OF

SAMPLES

NO.

POSITIVE

*(%)

1 Adamawa 304 8 (2.63)

2 Bauchi 154 50 (32.47)

3 Borno 96 2 (2.08)

4 Gombe 18 9 (50.5)

5 Jigawa 135 18 (13.33)

6 Kaduna 313 39 (12.46)

7 Kano 215 11 (5.12)

8 Katsina 74 0 (0)

9 Plateau 591 72 (13.54)

10 Sokoto 108 4 (3.7)

11 Taraba 15 6 (40.0)

12 Kebbi 3 0 (0)

TOTAL 2026 219 (10.81)

* Figures in brackets are percentage positive.

56

3.1.3.2 Rate of CBPP infection in Bulls and Cows

This study also indicated that overall, 82 out of a total of 497 (16.5%) males were

positive for CBPP infection compared to 137 out of 1529 (9%) cows tested. There was

an association between sex and infection rate, i.e. males are more likely to be infected

by the disease (X2=21.63). Both fig.3 and Appendix II shows that although majority of

animals tested were cows, a higher proportion of positive was in the males (i.e.16.5%

positive in males; 9% in females).

57

Figure 3: Rate of CBPP infection in Bulls and Cows based on the total number sampled

0

10

20

30

40

50

60

AdamawaBauchi Borno Gombe Jigawa Kaduna Kano Katsina Plateau Sokoto Taraba Kebbi

Perc

en

tag

e p

osit

ive

States

Male

Female

58

3.1.3.3 Optical Density/Average Percentage Inhibition versus infection rate

The average Optical Density (OD) and the average Percentage Inhibition were plotted

against the infection rates in the various states. The curves for the PI and OD were quite

identical and overlapped (Fig. 4). This could be explained since the PI is the degree to

which the passage of light is inhibited and OD is the degree of resistance to the passage

of light. So it was not surprising to see that both graphs were identical. However, PI is

measured in Percentage while OD is in figures. It was also interesting to note that when

both the Average PI and OD of the the various states were compared with the infection

rates, (Fig. 4) the slopes of the graphs agree with each other indicating direct

correlation with the infection trend in those states. This means that the average PI and

OD for the various states can give an idea of the infection rate in those particular states

even if the actual percentage infection rate is not determined.

59

Figure 4: Graph showing average PI, average OD and infection rates for samples collected from the

various states.

0

20

40

60

80

100

120

Adamawa Bauchi Borno Gombe Jigawa Kaduna Kano Katsina Plateau Sokoto Taraba Kebbi

Perc

en

tag

e p

osit

ivit

y

States

AV. PI

AV. OD.

% POSITIVE

60

3.1.4 Discussions

This study indicates that CBPP is present in almost all the states sampled and confirms

the assertions by Nicholas et al., (2000) that CBPP is indeed widespread in Africa,

especially Nigeria. The fact that at least half the number of states sampled gave a

prevalence rate of over 10% indicates that this disease may be assuming endemic

proportions in Nigeria. This notion is further strengthened by the fact that even though

relatively fewer males were sampled, the rate of infection in them was quite high and it

is a known fact that most farmers keep only a few males in a herd.

The study indicated a significant difference between the rate of CBPP infection in

Males and Females (X2=21.63). The difference seen in the results was as a result of a

higher number of positives recorded in the males even though the actual number of

males sampled was much smaller compared to that of the famales where higher

number were sampled but with only few positives.

It was however interesting to note that when both the Average PI and OD of the the

various states were compared with the infection rates, the slopes of the graphs agree

with each other indicating direct correlation with the infection trend in those states. This

means that the average PI and OD for the various states can give an idea of the infection

rate in thoses particular states even if the actual percentage infection rate is not

determined. This would be particularly useful in sero-prevalence studies where the raw

data for Optical density can be used to calculate the infection rate without actually

performing the calculation for percentage inhibition and this will also eliminate the

problem associated with setting up a reliable cut-off for positive reactions.

61

CCHHAAPPTTEERR FFOOUURR

IISSOOLLAATTIIOONN AANNDD IIDDEENNTTIIFFIICCAATTIIOONN OOFF MMMMSSCC IINN

NNOORRTTHHEERRNN NNIIGGEERRIIAA

4.1 Experiment 2 : Isolation of Mycoplasma mycoides mycoides Small Colony

4.1.1 Introduction

Mycoplasmas, the smallest self-replicating life forms, are primarily characterized by

their lack of a cell wall and cholesterol containing membrane (Woubit et al., 2007).

Conventional methods for the detection and identification of Mycoplasmas

systematically involve enrichment steps in selective broth followed by morphological,

biochemical and serological tests. Although well established, these techniques have

some important drawbacks. The morphological and biochemical characteristics are in

general not discriminative, while serological cross-reaction have been frequently

reported as well (Woubit et al., 2007).


Outbreaks of CBPP in five States of Northern Nigeria were investigated. The States

were Plateau, Bauchi, Kano, Kaduna and Kebbi. A total of 287 specimens comprising

49 lung and 14 pleural fluid samples, 172 nasal and 52 ear swabs were collected from

animals suspected to be in the acute stage of CBPP infection. Pleural fluid and lung

tissues were collected in cases of mortalities, while nasal and ear swabs were collected

in live animals. Specimens were also collected from sheep in contact with suspected

bovine herds or showing respiratory distress. In all, 51 specimens were collected from

the sheep which comprised 13 lung and only one pleural fluid samples, 26 nasal and 11

ear swab samples. The only pleural fluid sample was collected from a two week old

lamb which was brought to the Veterinary Clinic in Vom and died of severe respiratory

62

illness. The mother of the lamb however did not show any signs of distress. One sample

(labeled ovine lung) from an unknown source and with an unknown year of collection

was recovered from the NVRI storage freezers and added to the samples for analysis.

Another lung sample collected from ovine species suspected of Contagious Caprine

Pleuropneumonia (CCPP), dated 1970 was also analyzed for MmmSC. The number of

samples collected was based on the number of clinically ill animals available and the

willingness of the owners to permit the collection of specimens. Details of age and sex

of the animals were not considered at the final analysis because the collection of such

data was not consistent. (See Appendix I for details of the samples collected).

3.2 .1 Culture and isolation

The lung tissues, nasal and ear swabs collected from suspected bovine and ovine species

were cultured in growth medium according to Provost et al., (1987) and Freundt,

(1983), see Appendix IV. The specimens were initially incubated in PPLO Broth for at

least 48 hours at 37 0C and 5% CO2 and subsequently subcultured on agar for 2 to 4

days.

4. 1.3 Results

4.1.3.1 Isolation of Mycoplasma mycoides mycoides SC

Eighteen out of the total number of samples cultured were considered to be positive for

Mycoplasma mycoides mycoides SC based on their colonial morphologies. In all, 12 out

of 236 bovine specimens were positive for MmmSC while 6 out of 51 ovine samples

were equally positive for MmmSC. Details of the breakdown for the various positive

specimens, the colony appearance and the animal species involved, are given inTtable

2. Pictures of some of the colonies of the isolates are presented in Plate 2.

63

Table 2: Culture and identification of isolates from specimens

Registr

ation

Numbe

r

Animal

species

Specimen

Screened Colony Appearance

Tentative

identificati

on

044 Ovine adult Nasal Swab Medium, small centre

MmmSC-

like colonies

045 Ovine lamb Pleural Fluid Medium, with or without centre ,,

046 Ovine adult Nasal Swab Round, medium, with or without centre ,,

047 Ovine adult Nasal Swab Medium, dark centre, presence of films ,,

048 Bovine adult Nasal Swab Large, dark centre ,,

049 Bovine adult Ear Swab

Clone 1: large, dark central area, darker

centre, / Clone 3: larger, clear, no centre. ,,

050 Bovine adult Nasal Swab

Clone 5: large bright with darker centre /

Clone 2: small, rough, with tiny centre ,,

051 Bovine adult Nasal Swab Medium, round, clear, dark, marked centre ,,

052 Bovine adult Lungs Round, medium, with or without centre ,,


054 Bovine adult Pleural Fluid Round, medium, with or without centre ,,

055 Bovine adult Ear Swab

Medium, irregular shape, small centre

,,


057 Bovine adult Pleural fluid Round, medium, with or without centre ,,

058 Ovine Lung? Much smaller, with or without centre ,,

059 Bovine1 year Lungs Round, medium, with or without centre ,,


061 Ovine Lungs Round, medium, with or without centre ,,

64

4.1.3.2 Isolation of Mycoplasma mycoides mycoides SC from Ovine species.

The results indicated that among Mycoplasma mycoides mycoides SC isolated from 6

suspected ovine specimens is a positive case from the pleural fluid collected from a two

week old lamb. The dam did not show any signs of respiratory illness, and even though

the lamb was diagnosed with severe pneumonia, CBPP was not suspected because of

the age of the lamb. The confirmation of this disease in these animal species emphasises

the need to carry out pathogenicity studies to determine their effect on bovine.

The results also indicated that a specimen which was collected from a sheep suspected

to be suffering from Contagious Caprine Pleuropneumonia (CCPP) at the Jos abattoir

in 1970 was indeed MmmSC.

4 .1.4 Discussions

It was interesting to note that out of 287 specimens, only 18 were positive for MmmSC

giving a percentage of 6.27%. Even though the percentage positivity was apparently

higher for ovine (11.76%) than for bovine (5.08%), two major reasons could be

attributed to this. Firstly, the number of samples collected from sheep were fewer (51)

than that of cattle (236), and secondly, in cases of suspected outbreaks of CBPP,

owners usually institute treatment even though it is not recommended, which probably

is responsible for the very low number of isolates recorded in cattle. Sheep on the other

hand are usually considered as companion animals and since most owners do not

consider them to be in danger of contracting CBPP they are usually not screened or

treated, except where they show severe respiratory signs.

It was also interesting to note that there were variations in the appearance of the

colonies of Mycoplasma mycoides mycoides isolated from the different outbreaks. Even

65

though they had the characteristic small colony appearance, their appearance and size

were difficult to harmonize. There were very obvious differences between these

colonies which by their general appearance had been classified as MmmSC (See Figure

6). Some colonies were quite large with large dark centers while in others the centre

was thick and small. As a result of these variations it was not possible to make a

definitive diagnosis of Mycoplasma mycoides mycoides Small Colony for all the

isolates. These isolates had to be confirmed by a specific pcr for the diagnosis of

MmmSC and where it was negative; a sequencing of the 16SRNA was performed.

The isolation of MmmSC from the pleural fluid of a two week old lamb calls for a

reassessment of the incubation period of the disease caused by this isolate in both

bovine and ovine species. Since the lamb apparently came down with a respiratory

illness which could be attributed to the MmmSC, there is a need to determine the extent

of involvement of these species in the epidemiology of this disease.

The only plausible explanation for the isolation of MmmSC from the specimen of a

sheep suspected of CCPP could be that the MmmSC was present at the time of the

sample collection either as a contaminant or in combination with the CCPP organism,

but because of the fastidious nature of the organism, it was lost during storage.

PLATE 2: Colonies of Mycoplasma mycoides mycoides SC isolated from the specimens

66

: Colonies of Mycoplasma mycoides mycoides SC isolated from the specimens

: Colonies of Mycoplasma mycoides mycoides SC isolated from the specimens

67

4.2 Experiment 3: Identification and confirmation of Mycoplasma mycoides

mycoides Small Colony isolates by specific Polymerase Chain Reaction (PCR).

4.2.1 Introduction

In recent years, PCR has replaced traditional diagnostic tests for the identification of

members of the Mycoides cluster (Woubit, 2008). DNA amplification techniques offer a

promising identification system by avoiding variability that hinders serological methods

(Le Grand, et al., 2004). So far a number of PCR tests have been developed for the

rapid identification of species of the M. mycoides cluster. Most of the PCR systems

developed until recently and before the flourishing of genomic sequence of

Mycoplasmas were based on CAP-21 sequence fragment encoding notably for

ribosomal proteins rpsL and rpsG, and the design of specific primers for MmmSC

(Bashiruddin, et al., 1994), M. mycoides cluster and M. putrefaciens (Hotzel, et al.,

1996; Rodriguez, et al., 1997). The other most widely used target for specific PCR tests

is the 16S rRNA gene, this gene has been used for specific PCR for the identification of

Mccp (Buscanana, et al., 1999; Bolske et al., 1996) and MmmSC (Persson, et al.,

1999). In addition to these two gene that encodes for lipoprotein, p72 gene has been

used for the design of specific primers for MmmSC (Miserez et al., 1997) and MBG 7

(Frey, et al., 1998) detection. Finally gene lppA has been used for the detection of

Mccp, Mcc, MmmLC and Mmc (Monerate, et al., 1999). A non coding intergenic

sequence between MSC _0390 and MSC _ 0391 in the genome sequence of MmmSC

strain PG 1 has also been employed in the design of specific PCR primers for MmmSC

(Dedieu, et al., 1994). A recent evaluation of the above mentioned PCR tests have

revealed all PCR tests based on lipoprotein genes were not strictly specific (Le Grand,

et al., 2004) with respect to MmmSC identification. PCR tests by Dedieu et al., (1994)

68

and Bashiruddin et al., (1994) remain reliable tests as these two have been used

extensively in some CBPP diagnostic laboratories abroad (Woubit, 2008).

4.2 .2 Materials and Methods

In this experiment, all the 18 Mycoplasma isolates were subjected to Diagnostic PCR

test for the detection of MmmSC according to protocols by Dedieu et al., (1994).

4.2.2.1 Extraction of DNA

A 3 ml of MmmSC broth culture in medium was centrifuged for 10 min at 10,000g. The

pellet was washed once in 1.5 ml PBS, centrifuged again and re-suspended in 50 µl

sterile dH2O and to which 100 µl lysis buffer (100mM Tris–HCl pH 8.5, 0.05% Tween

20, 0.24 mg/ml proteinase K) was subsequently added. This was then incubated in a

water bath at 60°C for 1 hour and Proteinase K was then inactivated at 95°C for 5 min.

1 µl of the DNA extract was finally added to the reaction as the DNA template. This

template was used for all other pcr reactions carried out on the isolates.

4.2.2.2 Diagnostic PCR for the detection of MmmSC

This test was carried out according to the work of Dedieu et al., (1994). The DNA

reaction mix consisted of Sterile dH2O, 10x Qiagen Taq buffer, (15 mM MgCl2) Roche,

dNTP mix (30 mM AT / 15 mM GC), Qiagen Taq Polymerase (5 u/µl), Primer

MSC1_Forward 5’ ATACTTCTGTTCTAGTAATATG 3’ (20 µM) and Primer

MSC_Reverse 5’ CTGATTATGATGACAGTGGTCA 3’ (20 µM). To each 49 µl

PCR master mix was added 1 µl of the DNA extract from the isolates according to the

protocol. The DNA samples were amplified with the primers listed in Table 2.

Amplifications were performed using Gene Amp PCR Systems 2720 (Perkin Elmer).

Thermal cycling consisted of an initial denaturation step at 94°C for 2 minutes,

followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing at 53°C for 30

69

seconds and extension at 72°C for 1 minute. The final extension step was maintained at

72°C for 5 minutes. The PCR products were then run on 2% agarose gel.

4.2.2.3 Agarose (2%) gel electrophoresis

A 2% Agarose gel was prepared for the separation of the PCR products according to

their sizes. Briefly, 2g agarose powder was mixed with 100ml of electrophoresis buffer,

Tris-acetate-EDTA (TAE). The gel was melted in a micro-wave oven, then poured into

a gel casting tray and allowed to set at room temperature. Individual pcr products were

then mixed with a loading dye (Bromophenol blue) at a ratio of 1 to 5 and placed in

their respective wells on the gel. The gel apparatus was then connected to electric power

source and the gel run at 130 volts for 30 minutes. Finally, the gel was removed from

the electrophoresis tank, stained in Ethidium bromide solution (0.5ug/ml) for 20

minutes and rinsed in water for 15 minutes. The DNA migration was viewed under the

transilluminator (UV light of wavelength 254 nm) and photographs of taken.

4.2.3 Results

Thirteen out of the eighteen Mycoplasma isolates recovered from the samples cultured

were confirmed to be MmmSC by a selective Polymerase Chain Reaction Assay for the

detection of Mycoplasma mycoides subsp. Mycoides S.C. (Dedieu, et al., 1994). The

production of a band equivalent to 275 bp and at the same distance with the PG1

positive control confirms MmmSC.

70

M 1 2 3 4 5 6 7 8 9 10 1112 13 14 - M

275bp

PLATE 3: MmmSC diagnostic PCR MSC1: 275 bp product from all clones of the samples

M: molecular weight marker, 1: 045-C1; 2: 048C1, 3: 050-C1; 4: 051-SC1; 5: 051-

C1; 6: 052-C1; 7: 053-C1; 8: 054-C1; 9: 056-C1; 10: 057-C1; 11: 058-C1; 12: 059-C1; 13: 060-C1;

14: 061-C1; +MmmSC PG1 positive control; -: water blank

71

Table 3: Isolates identified by Specific PCR for the diagnosis of MmmSC.

N° Animal Species Colony Appearance Results

048 Bovine adult Large, dark centre MmmSC

051 Bovine adult Medium, round, clear, dark, marked

centre

MmmSC

052 Bovine adult Round, medium, with or without

centre

MmmSC


centre

MmmSC


centre

MmmSC


centre

MmmSC


centre

MmmSC

059 Bovine 1 year Round, medium, with or without

centre

MmmSC


centre

MmmSC

050 Bovine adult Clone 5: large bright with darker

centre / Clone 2: small, rough, with

tiny centre

Clone 5: MmmSC /

Clone 2: M. arginini

061 Ovine Round, medium, with or without MmmSC

72

centre

045 Ovine lamb Medium, with or without centre MmmSC

058 Unknown Much smaller, with or without centre MmmSC

055 Bovine adult Medium, irregular shape, small centre Clone 1: M.

alkalescens

049 Bovine adult Clone 1: large, dark central area,

darker centre, / Clone 3: larger, clear,

no centre.

Clone 1: M. yeatsii,

Clone 3: MBG 7

047 Ovine adult Medium, dark centre, presence of

films

ND (weak

amplification by

PCR)

044 Ovine adult Medium, small centre Clone 1: M. arginini

046 Ovine adult NO GROWTH ND (no growth)

The other isolates, M. arginini, M. alkalescens, M. yeatsii, and MBG 7 were identified

by sequencing of the 16sRNA.

PLATE 4: M. mycoides mycoides Small Colony (048 Bovine, Birnin Kebbi) Isolate

from pleural fluid from a Bovine in a herd suspected of CBPP in Bogodo near Birnin Kebbi in

Kebbi State. Colonies were large with dark

73

: M. mycoides mycoides Small Colony (048 Bovine, Birnin Kebbi) Isolate was recovered


Kebbi State. Colonies were large with dark centre.

was recovered


PLATE 5: (044 Ovine, Fadan kaje) Nasal swab

infected herd in Fadan Kaje, Kaduna State.

1 of these confirmed to be M. arginini

74

: (044 Ovine, Fadan kaje) Nasal swab collected from ovine adult in contact with a CBPP

infected herd in Fadan Kaje, Kaduna State. Colonies were medium in size with small center. Clone

M. arginini.

collected from ovine adult in contact with a CBPP

Colonies were medium in size with small center. Clone

75

4.2.4 Discussions

The confirmation of thirteen isolates to be Mycoplasma mycoides mycoides Small

Colony out of the eighteen initially recovered is quite significant. Some mixed cultures

of MmmSC and M. arginini were also observed. This is not so surprising since M.

arginini is very often isolated from animal samples as a contaminant and has no

pathogenic significance. However culture 055 was identified as M. alkalescens and this

agent has a pathogenic potential. Other organisms isolated included M. yeatsii and

Mycoplasma Bovine group 7 (MBG 7). Biochemical analysis was not carried out on

these samples on account of the cross reactivity of the Mycoplasmas and unreliability of

these tests. However phenotypic identification also proved difficult as it was not easy to

differentiate some non MmmSC colonies from those of MmmSC. The situation was

made worse by situations where two clones of the same culture eventually yielded two

different organisms. The fact that several colonies of MmmSC showed different colonial

morphologies makes phenotypic method of identification unreliable. Three isolates

from sheep were among the isolates confirmed as MmmSC. This is quite significant

since these animals herd together with cattle. Further work needs to be done on these

isolates to determine if they are genetically similar to the isolates from bovine. The

confirmation of thirteen isolates out of the original eighteen also indicates that infection

is widespread in Northern Nigeria.

4.3 Experiment 4: Identification of isolates by MmmSC Specific QPCR

4.3.1 Introduction

Detection of MmmSC can be done by bacterial culture, which is relatively easy as this

Mycoplasma grows well in adequate medium (Lorenzon et al., 2008). Since 1994, the

use of specific PCR tests has made detection and identification of this organism much

76

more sensitive and reliable (Bashiruddin et al., 1994; Dedeiu et al., 1994). However,

classical PCR has a number of drawbacks, the major one being the risk of

contamination during post-PCR analysis. Additionally, classical PCR does not allow

quantification of the target DNA in the sample, and may lack sensitivity when

compared to newer methods. Real Time PCR (rtPCR) assays are less prone to

contamination risks and their use in the detection of notifiable pathogens must be

promoted. Real-time PCR assays have already been described for MmmSC (Gorton et

al., 2005; Fitzmaurice et al., 2008). In these previous studies, the targets were chosen in

conserved sequences and specificity was based on very few nucleotide differences. The

availability of commercial kits has made the technique easy to perform, efficient, and

reliable. QPCR methods are easily adapted to high throughput assays, allowing

researchers to process large numbers of samples in a short period of time. In addition,

data can be collected and analyzed using specialized software designed for the specific

instrument being used, and a personal computer. QPCR has been used for many diverse

applications, including the detection of pathogenic bacteria, identification and

quantification of microorganisms from water samples, studying gene expression levels,

and detection of single-nucleotide polymorphisms (SNP’s) in genomic sequences.


The confirmatory QPCR for the MmmSC isolates was carried out using the SYBR®

Green qPCR Master mix. The SYBR Green I dye has a high binding affinity to the

minor groove of double-stranded DNA (dsDNA). It has an excitation maximum at 497

nm and an emission maximum at 520 nm. In the unbound State the dye exhibits little

fluorescence; however, when bound to dsDNA, the fluorescence greatly increases,

making it useful for the detection of product accumulation during real-time PCR.

77

During denaturation, all DNA becomes single stranded. At this stage, SYBR Green is

free in solution and produces little fluorescence. During the annealing/extension step,

the primers hybridize to the target sequence and are extended, resulting in dsDNA to

which SYBR Green I can bind. The test was carried out according to protocols by

Lorenzon et al., (2008). The SYBR® Green QPCR Master mix is a ready-to-use

cocktail containing all components, except primers and template, for the amplification

and detection of DNA in qPCR. It is supplied in a 2X concentration and contains

sufficient reagents to perform 400, 25-µL reactions. The mix is optimized for SYBR

Green reactions and contains SYBR Green I Dye, AmpliTaq Gold® DNA polymerase,

and dNTPs with dUTP, Passive Reference, and optimized buffer components.

In this reaction, to each 23 µl QPCR master mix were added 1 µl of template DNA

extracted (from Experiment 3.3.1) and 1 µl of primers Q_MSC_0382_Forward 5’

ATGCAAGAAGTTATTAATGTTTATCATTC 3’ and Q_MSC_0382_Reverse 5’

CGTAATATATTTGTTTAACATATGGAATAA 3’. The SYBR Green master mix was

mixed gently by inversion without generating bubbles to avoid the creation of optical

errors during the sample read. Amplifications were performed using Strategen-

Mx3000P® Instruments. The default dissociation curve for SYBR Green experiments

was used. This default profile dissociation curve began with 1-minute incubation at

95°C to melt the DNA and then 30-second incubation at 55°C. This was followed by a

ramp up to 95°C with all points data collection performed during the ramp.The final

cycle consisted in an initial denaturation step at 95°C for 10 minutes, followed by 40

cycles of denaturation at 95°C for 30 seconds, annealing at 56°C for 1 minute and

extension at 72°C for 1 minute. This was followed by a ramp down at 95°C a rate of

0.2°C/sec for 1 minute and then a ramp up at 55°C a rate of 0.2°C/sec for 30 seconds.

78

The final step was maintained at 95 °C for 30 seconds. The progress of the reaction was

monitored on the computer in real time.

4.3.3 Results

All positive MmmSC cultures were also screened by Specific QPCR for the

identification of MmmSC. The production of similar curves by all the isolates and

another by the PG1 positive control confirms MmmSC .

79

Figure 5: Specific QPCR for the identification of MmmSC

80

Figure 6: Temperature dissociation curve for the isolates

81

4.3.4 Discussions

Diagnosis of MmmSC by QPCR offers lots of opportunities in Mycoplasma research

especially towards CBPP control efforts. The fact that this technique is very fast means

that several samples can be handled in unit time. Considering a vast country like

Nigeria where the number of samples from the field could be especially high, this will

be of significant advantage. The monitoring of the reaction in real time also means that

end point processes like staining with Ethidium bromide which is carcinogenic and a

possible source of contamination could be avoided. Even though the melting points of

the thirteen Nigerian Isolates of MmmSC tend to show very slight variations suggesting

the possibility of strain variation, it was not possible within the scope of this work to

differentiate the isolates using this technique. Although the equipment is expensive, it is

quite affordable considering the benefits to be derived. It is relatively easy to handle and

does not require special skills in interpretation. The reaction mix is also easy to use.

82

CCHHAAPPTTEERR FFIIVVEE

55..00 MMOOLLEECCUULLAARR CCHHAARRAACCTTEERRIIZZAATTIIOONN OOFF MMMMSSCC IISSOOLLAATTEESS

5.1 Experiment 5: Multi-Locus Variable Analysis (MLVA) Variable Number Tandem Repeats (VNTR) MmmSC TR-34 PCR

5.1.1 Introduction

In contrast to the popularity of DNA based identification methods, typing methods are

only gradually implemented in the field of Mycoplasmology (Stakenborg, 2005).

Firstly, for some species, the isolation is often merely too complex and laborious to

perform. Secondly, some species are ubiquitously present and not linked to true

outbreaks, making epidemiological episodes hard, if not impossible, to define (Hege et

al., 2002). Finally, some well documented, important species are very homogeneous

and difficult or impossible to type with techniques commonly used for other

Mycoplasmas (Cousin-Allery et al., 2000; Tenover et al., 1994). Since there are

currently numerous detailed reports about intraspecific variability, it has become

increasingly important to type strains or to define specific subgroups within a species.

This increasing interest in epidemiological data of Mycoplasmas will help to elucidate

the genomic plasticity, observed for some species, to reveal the geographical spread or

transmission patterns, or to interprete differences seen on the biological level.

Ultimately, the understanding of epidemiological behaviour may offer possibilities to

control or even to eradicate Mycoplasma related diseases.

Genetic loci or sequences containing variable numbers of tandem repeats (VNTR loci)

are frequently used for typing purposes and may prove especially valuable for

Mycoplasmas. Many medically important genes have been identified based on their

linkage to a mapped VNTR locus. Individual VNTR loci have been identified in

bacteria (Andersen et al., 1996; Frenay et al., 1994; Frothingham, 1995; Goyal et al.,

83

1994). The M. mycoides subsp. Mycoides SC genome contains an exceptionally high

fraction of repetitive sequences, the highest so far in currently sequenced bacteria

(Westberg et al., 2004). Generally, repeats are somewhat arbitrarily divided in tandem

repeated sequences (or satellites) and repeats that are dispersed around the genome.

These latter repeats are often linked to important surface antigens and owing to

occurring recombination events between the multiple copies present, they may

contribute to the similarity of known insertion sequences (IS) (Mahillon and Chandler,

1998).


The 13 isolates were characterized using Multilocus Variable-Number Tandem Repeat

Analysis (MLVA) on locus TR34 (within gene nat A). The PCR was conducted using

the primers indicated in Table.4. The conditions for the PCR were same as for

Experiment 3.3.2 and the primers Eh Ch TR34L 5’ ATTCAAATGTAACCAATCAGC

3’ Eh Ch TR34R 5’ GATTGCTTTGATTAACTTGTTG 3’ corresponding to sequences

on positions 451976 and 452235 on the PG 1 genome were used for the reaction. The

premers had melting temperatures of 56oC and 58oC respectively.

5.1.2.1 Agarose (4%) Gel preparation and migration

The preparation of the gel was same as for Experiment 3.3.3 except that in this case 4 g

(4%) of agarose was used instead. Individual pcr products were then mixed with a

loading dye (Bromophenol blue) at a ratio of 1 to 5 and placed in respective wells on the

gel. The gel apparatus was then connected to electric power source and the gel was run

at 18 volts overnight (14 hours). Finally, the gel was stained in Ethidium bromide

solution (0.5ug/ml) for 10 minutes and rinsed in water for 15 minutes. The DNA

migration was viewed under the transilluminator (UV light of wavelength 254 nm) and

84

photographs of taken. The number of repeats in the DNA isolate was estimated by

comparing the bands on the gel to those of other strains which have already been

sequenced and the number of repeats determined. These known strains were

incorporated into the test to serve as controls with number of repeats ranging from 4 to

16. These strains include; 9773 (4 repeats), 04003 (5 repeats), 83162 (6 repeats), 98029

(7 repeats), Afade (8 repeats), Rita (8 repeats), Vom (8 repeats), 9048 (9 repeats), 99042

(10 repeats), 91130 (13 repeats), and 00033 (16 repeats), all of which were got from the

World Reference laboratory for CBPP in France.

5.1.2.2 Polyacrylamide (10% ) Gel Electrophoresis

A 10% polyacrylamide gel was prepared by adding 600 µl of 10% ammonium

persulfate to 30 µl of Acrylamide/Bis, 12 µl 10X TBE, 78 µl Distilled water (Milli Q)

and finally 80 µl of Tetramethylethylenediamine (TEMED). About 10 µl of each pcr

product (50 µl product plus 5 µl blue sigma loading dye) was loaded into specific wells.

10 µl Marker VIII was used as the ladder while blanks were filled with loading dye

(1/10) diluted in water. The gel apparatus was then connected to electric power source

and the gel pre-run at 100V (30mA) for 15 minutes to stabilize the apparatus. After

loading the pcr products, there was an initial run at 100V (30 mA) for 15 minutes before

it was transferred to a cold room (40C) and set to 300V (77 mA). This was left

overnight (14 hours) before stopping the run. The gel was then stained in Ethidium

Bromide for 8 minutes and destained in distilled water for 30 minutes. The number of

repeats in the DNA isolate was also estimated by comparing the bands on the gel to

those of other strains which have already been sequenced and the number of repeats

determined.

85

5.1.3 Results

When the PCR products were migrated using 4% Agarose Gel Electrophoresis, it was

difficult to differentiate the isolates because the bands were poorly separated since they

were all between 200-500bp molecular weight (See Plate 6 and 7). However when the

products were migrated using 10% Poly-Acrylamide Gel Electrophoresis (PAGE), the

bands were clearly separated making it easy to differentiate the isolates into 5 different

alleles (See Plate 8 and 9). Thus, with the locus TR34, the 13 strains from Northern

Nigeria were differentiated into 5 alleles (See Table 4), the number of tandem repeats

varied from four to twelve. Two alleles gathered the majority of isolates with 5 strains

each (alleles with 8 and 12 repeats). The three other alleles had 1 strain each. The

results are summarized in Table 4 and Fig. 7.

86

Table 4: TR 34 Differentiates the Mycoplasma isolates into 5 alleles based on the number of repeats

ranging from 4 to 14

Serial no Source No of Repeats

059 c1 Kafanchan (Ovine) 4

052 c1 Kanam 8

053 c1 Barkin Ladi 8

056 c1 Vom/Jos 8

057 c2 Vom (Fadan kaje) 8

061 c1 Jos 8

051 c1 Bauchi 9

045 c1 Vom (Lamb) 12

048 c1 Birnin Kebbi 12

050 c1 Bauchi 12

054 c1 Sanga 12

060 c1 Kano 12

058 c1 Vom (Ovine) 14

87

1,517

500/517

1,000

200

100

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 MBase pair s

PLATE 6: MLVA VNTR MmmSC TR-34 PCR: 4% Agarose gel electrophoresis

M: 100 bp molecular weight marker, 1: 045-C1; 2: 048C1, 3: 050-C1; 4: 051-SC1; 5: 051-LC1; 6:

052-C2; 7: 053-C1; 8: 054-C1; 9: 056-C1; 10: 057-C2; 11: 058-C1; 12: 059-C1; 13: 060-C1; 14: 061-

C1; 15: M 100 bp molecular weight marker

88

M 1 2 3 4 5 6 7 8 9 10 M

1,5 17

1,0 00

500/517

200

100

Base pairs

PLATE 7: MLVA VNTR MmmSC TR-34 PCR: 4% Agarose gel electrophoresis

M: 100 bp molecular weight marker, 1: VOM; 2: 83162; 3: RITA; 4: 9048; 5: 91130 6: 9773 ; 7:

98029 ; 8: 99042 ; 9: 00033; 10: 04003; M 100 bp molecular weight marker

89

10% Polyacrylamide Gel electrophoresis

PLATE 8: MLVA VNTR MmmSC TR-34 PCR: 10% Poly Acrylamide Gel Electrophoresis

B: Blank; M: Molecular weight marker, 1: 9773; 2: 059C1, 3: 04003; 4: 83162; 5: 98029; 6: 061; 7:

Afade; 8: 053-C1; M: Molecular weight marker; 9: 056-C1; 10: 057-C2; 11:B17; 12: 052-C1; 13:

051-SC1; 14: 051-LC1; 15: Rita; 16: Vom; B: Blank

90

B M 17 18 19 20 21 22 23 M 24 25 26 B

PLATE 9: MLVA VNTR MmmSC TR-34 PCR: 10% Poly Acrylamide Gel Electrophoresis

B: Blank; M: Molecular weight marker 17: 9048; 18: 99042; 19: 045-C1; 20: 048-C1; 21:050; 22:

054-C1; 23: 060-; M: Molecular weight marker 24: 91130; 25: 058; 26: 00033; B: Blank

91

Figure 7: Geographical distribution of the different MmmSC TR 34 types

Types A (4 repeats), C (9 repeats) and E (14 repeats) are found only in one State each. While Type B (8

repeats) is found in Plateau and Kaduna States. The Type D (12 repeat) is more widely distributed as it is

found in all the five States screened

4

8

9

12

14

92

5.1.4 Discussions

This technique differentiated the isolates into five different alleles and also produced

three alleles that were unique to particular States. That means if there is an outbreak of

infection involving any of these strains, a relationship between the outbreak and the

origin of the infection could be determined. If this technique is extended to other parts

of the country, it would be possible to identify all the alleles in the different parts of the

country for the purpose of documentation and control efforts. It is also interesting to

note that the isolates were segregated into five strains even though all isolates were

circulating within the central region of the country. This means that different strains are

involved in CBPP outbreaks in those areas a feature to be noted especially in relation to

the use of a single MmmSC strain for vaccination and future vaccine development.

5.2 Experiment 6: Multi-Locus Sequence Analysis (MLSA) on Loc-PG1-0001 and Loc-PG-0103

5.2.1 Introduction

High throughput sequencing technologies and new bioinformatics tools have literarily

revolutionized modern microbiology. Phenotypic characterization of bacteria may now

seem as nineteen century tools. A technique such as iso-enzyme profiling has long ago

been replaced by multilocus sequencing of all genes. Coding sequences can then be

compared and the detection of synonymous mutations has further increased the

resolving power of the technique (Woubit et al., 2007). Molecular epidemiology tools

can aid in identifying strains of Mycoplasma mycoides mycoides Small Colony at a finer

level than simply the ‘SC biotype’ level, offering the possibility of understanding

certain trends in the spread of CBPP (Lorenzon et al., 2003). Up to 1995, MmmSC

strains were considered very homogeneous (Costas et al., 1987). Specific detection

93

methods were then developed which enabled the detection of all MmmSC strains

(Bashiruddin et al., 1994) and their differentiation from the closely related strains of the

mycoides cluster. Since that time, evidence has shown that MmmSC strains can be

differentiated at the molecular level. Restriction analysis of whole DNA makes it

possible to identify strain groups and to differentiate between any two vaccinal strains,

such as T1 and KH3J (Poumarat and Solsona, 1995; Thiaucourt et al., 1998). Southern

blotting, using insertion sequence IS1296 as a probe, enabled differentiation between

strains of European and African origin. Furthermore, different profiles were obtained in

strains of African origin (Cheng et al., 1995). A major genetic difference between

European and African strains was identified later, the genome of the European strains

lacking a segment of 8.84 kb including an IS1634, an ABC transporter and other genes

(Vilei et al., 2000). Ultimately, specific PCR reactions were designed to identify the T1

vaccinal strain taking advantage of sequence variation in IS1296 flanking regions

(Lorenzon et al., 2000). Phenotypic differences were also evidenced, such as the

presence of extra bands in SDS PAGE profiles (Goncalves et al., 1998), the inability of

European strains to oxidize glycerol (Abu-goun et al., 1994 ; Houshaymi et al., 1997) or

variability in growth rates and inhibition of growth by hyper immune sera (March et al.,

2000).

While amplification of the specie-specific PCR product can result in a direct

identification, sequence analysis can be used to discriminate between strains. The

technique does not need the cultivation of fastidious Mycoplasmas and since all

molecular typing techniques are ultimately based on differences in sequences, sequence

analysis seems the best approach. Moreover, the technique has an excellent inter-

94

laboratory reproducibility and data can be stored in online databases (Jolley et al.,

2004).

More expensive Sequenced-based methods (Olive and Bean, 1999), called MLST which

were developed to cope with some important drawbacks related to typing studies based

on sequence analysis of single genes, are becoming powerful sub typing tools in

molecular epidemiology. These methods have the advantage of being easily

standardized and automated. Instead of analyzing one single genomic fragment, the

partial sequences of multiple, selected genes are determined. Ideally, sequence

fragments about 500 bp in length of at least seven, widely scattered genes should be

included to obtain a good representation of the genome, but the number may vary

between different studies. Mostly, essential (housekeeping ) genes are chosen because

they are less often subjected to horizontal transfer events and are not liable to strong or

unusual selective pressures. As a consequence, they are perfectly suited to represent the

accumulation of sequence variation in the genome. However, some exceptions have

been reported where a number of housekeeping genes showed too few differences to be

useful for typing (Dumke et al., 2003; Manning et al., 2003, Nallapareddy et al., 2002).

For these cases, carefully selected species specific genes may be included instead. Once

the base r amino acid substitution rate of the selected genes is known, mathematical

models are available to perform profound phylogenetic analyses and to efficiently

determine the clonal structure of the population (Dingle et al., 2001; Iredell et al., 2003;

Lemee et al., 2004; Sarkar and Guttman, 2004). MLST turned out to be the method of

choice for the typing of several bacterial species of global importance (Urwin and

Maiden, 2003) and for those, databases for international surveillance have been set up

(Feil and Enright, 2004). This approach has multiple applications such as phylogeny

95

(Hanage et al., 2005), molecular structure analysis (Tourasse et al., 2006) and molecular

epidemiology. In molecular epidemiology, MLST can be used to study the evolution of

antibiotic resistant strains (Gherardi et al., 2006), or the distribution of strains of various

lineages within a population (Lacher et al., 2006).

MLST, while successful for the differentiation of other organisms (Feil, et al., 2000,

Kotetishvili, et al., 2002, Nicolas, et al., 2000, Zhou, et al., 2000), was unfortunately

not so with MmmSC because the techniques are quite difficult to standardize and in the

case of MmmSC the variability within housekeeping genes was too limited, if present at

all. This was the case of the genes fusA, lep A and rpoB. Therefore it was decided in

this work to use a technique based on PCR and sequencing. This technique was based

on the identification of MmmSC polymorphic DNA sequences in which polymorphisms

were amplified and sequenced and their occurrence compared in a representative subset

of strains to build a multilocus sequence analysis (MLSA) tool (Lorezon et al., 2003).

This typing scheme is less universal than MLST analysis, as the gene targets used are

selected for typing a biotype of a given subspecies (MmmSC) and would not be suitable

for typing strains of higher taxonomic ranks, given their high variability. However, as

MLST, MLSA has the advantage of being a portable and very robust approach (Yaya et

al., 2008).


A Multilocus sequence analysis (MLSA) was performed on two loci: Loc-PG1-

0001(non coding region) and Loc-PG1-0103 (coding for a hypothetical lipoprotein).

The major factor in the choice of locus of analysis was the ability of the locus to

96

differentiate strains from West Africa (Yaya et al., 2008). In that work, Loc-PG1-0001

had six alleles with the greatest variability found among strains originating from West

Africa as such it was considered as a candidate locus for the characterization of the

MmmSC isolates from Northern Nigeria. Likewise, five alleles which corresponded to a

variable number of 5 to 9 trinucleotide repeats (AAT) coding for asparagine (N) within

a putative lipoprotein coding gene were found in locus Loc-PG1-0103, and also chosen

for the analysis.

5.2.2.1 PCR for Loc-PG1-0001and Loc-PG1-0103

The PCR reactions were performed using Gene Amp PCR Systems 2720 (Perkin Elmer)

in 50 _l reaction mix. This mix contained 2.5U of Taq polymerase (Quiagen),

corresponding amplification buffer with a final concentration of 1.5mM MgCl2, 0.4 _M

of each primer, 150 _M of dGTP and dCTP and 300_M of dATP and dTTP, and the

template. Samples were amplified with the primers Loc-PG1-0001-F 5’

AACAAAAGAGATCTTAAATCACACTTTA 3’ and Loc-PG1-0001-R 5’

CCTCTTGTTTAACTTCTAGATCAGAAT 3, for Loc-PG1-0001 with Loc-PG1-

0103-D 5’ GATGGATATAATCTATACTAGCATTTA 3’ and Loc-PG1-0103-F 5’

CCTTATATAGATAAAACTCCTCCTTA 3’ for Loc-PG1-0103. Thermal cycling

consisted in an initial denaturation step at 94°C for 5 minutes, followed by 35 cycles of

denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds and extension at

72°C for 90 seconds. The final extension step was maintained at 72°C for 7 minutes.

The PCR amplification products were analyzed by electrophoresis through 1% agarose

gels (QA-Agarose, MP Biomedicas, IllKirch, France) at 100V and visualized after

staining with Ethidium bromide on a UV transilluminator. Samples with relevant

features were sent to Cogenics (Meylan, France) for sequencing with the corresponding

97

primers. Both forward and reverse strands were sequenced. Results from COGENICS

were received as electronic files. The electrophoregrammes were examined and the

sequences were assembled and aligned with the software Vector NTI SuiteTM . The

sequences were compared to those previously obtained on 50 strains of MmmSC (Yaya

et al., 2008). If the feature of a strain corresponded to one of the strains in the work of

Yaya et al., 2008, its allele number was assigned to the strain. Otherwise, a new allele

number was given.

5.2.3 Results

Prior to sequencing, all the PCR products of the isolates for the different loci were run

on 1% agarose gel to make sure they were of the required length and uncontaminated

(pure). A 538 bp product as indicated by the gel migration indicated the right amplicon

(See Plate 10).

5.2.3.1 Allelic profile analysis

The sequences obtained from each corresponding forward and reverse primers were

assembled using Vector NTI SuiteTM (InfoMax, 2001) and the extremities

corresponding to a single strand sequence or showing aberrant features were trimmed.

The sequences obtained from different strains for each locus were aligned using

ClustalW (Vector NTI). Polymorphic sites were recorded, carefully checking the

corresponding sequence chromatograms. An allele number was assigned to each change

in the nucleotide sequence in accordance with the results of Yaya et al., (2008). At the

end, each strain was characterized by an allelic profile, corresponding to the

combination of allele numbers for each of the two selected loci (See Plate 11 and 12).

98

5.2.3.1.1 Alleles defined in non-coding sequences Loc-PG1-0001

Three alleles were identified on the locus Loc-PG1-0001 (allele 1, 4 and 7) (Plates 11

and 12). Polymorphisms were at two positions: position 1523 and position 1635 on the

PG1 genome. At the first position, Isolates 051, 058 and 059 bore A instead of T for the

10 other strains. The second polymorphism was displayed at position 1635: the Isolates

048 and 054 from Kebbi and Kaduna States respectively, had A while the other 11

strains had G. This latter mutation is a new discovery as this was not observed on 51

strains previously analyzed by Yaya et al., (2008). As a result, a new allele number (n°

7) was assigned to these two isolates from Nigeria. All three alleles determined in these

loci were found circulating within the central states of Kaduna, Bauchi and Plateau

States. Alleles 1 and 4 were shared between Kaduna, Bauchi and Plateau States while

Kano State had only allele no. 4. The new allele no. 7 was shared between Kaduna and

Kebbi States with the latter state having it as the only allele (See Fig. 8).

5.2.3.1.2 Alleles defined in genes of unknown function: Lipoproteins (Lpp) and

Conserved hypothetical proteins (Chp) Loc-PG1-0103

Five alleles were defined in locus Loc-PG1-0103 (Plate 13). They corresponded to a

variable number of six to nine trinucleotide repeats (ATT) coding for asparagine (N)

within a gene coding for a putative lipoprotein from position 122-439. The 13 strains

bore 4 different alleles. There was geographic segregation of the various alleles (Fig.

9). Even though all the alleles were found circulating in the central part of the country

with the three states of Plateau, Kaduna, and Bauchi, it was interesting to note that no

single state bore all the four alleles determined in this locus. Kebbi State had a single

strain with 9 repeats which corresponds to allele 5 (Yaya et al., 2008). Kano State also

had a single allele (2) which had six repeats. Bauchi State had two alleles (6 and 7)

which had 2 and 3 repeats respectively. Kaduna and Plateau States each have three

99

different alleles (3, 4 and 5) and (2, 3 and 4) respectively which corresponds to (7, 8 and

9) and (6, 7 and 8) repeats also respectively.

100

538 bp

1321 bp

PLATE 10: Analysis of PCR products on 1 % agarose gel electrophoresis prior to sequencing

. A 538 bp amplicon is produced for loc-PG-0001 and 1321 bp amplicon for loc-PG-0103

representing the nucleotide sequence NC_005364 of reference strain PG1

PLATE 11: Alignment of sequences on locus Loc

Strains 59, 51 and 58 had a point

genome). The numbers following Loc

PG1-0001-60 is the sequence obtained on locus Loc

last five lines contain data from the sequ

101

: Alignment of sequences on locus Loc-PG1-0001 Strains 59, 51 and 58 had a point mutation (A instead of G at position 1523 of PG1

genome). The numbers following Loc-PG1-0001 refer to the strain number (e.g.: Loc

60 is the sequence obtained on locus Loc-PG1-0001 for strain n° 60). The

last five lines contain data from the sequence type defined for each allele (al1 to al5).

mutation (A instead of G at position 1523 of PG1

0001 refer to the strain number (e.g.: Loc-

0001 for strain n° 60). The

ence type defined for each allele (al1 to al5).

PLATE 12: Alignment of sequences on locus Loc

A point mutation is observed at position 1635 where A is found instead of G for two

strains (n° 054 and 048).

102

: Alignment of sequences on locus Loc-PG1-0001 A point mutation is observed at position 1635 where A is found instead of G for two

A point mutation is observed at position 1635 where A is found instead of G for two

103

Figure 8: Distribution of alleles of Loc-PG1-0001 in Northern Nigeria

1

4

7

104

PLATE 13: alignment of the sequences on locus PG1-0103.There is 4 alleles on this locus

The number of repeats varied from 6 (strains 58, 45, 50, 60), 7(strains 059, 056,

053), 8 (strains 052, 057,

061) to 9 (strains 48, 54, 51)

105

.

Figure 9: Distribution of strains based on the number of repeats for Loc-PG1-0103 in Northern

Nigeria

6

7

8

9

106

5.2.3.2 Combined MLSA and MLVNTR

Each of the 13 strains were characterised by an allelic profile and at the end, 7 different

allelic profiles were identified for the combined MLSA and MLVNTR techniques

(Table 5). The genetic events underlying the description of the various alleles for

MLSA are described in Table 6. Analysis allowed the identification of 7 main groups of

strains, ranging from one to three members per group. Six strains were included the

highest two groups, followed by two strains each in the next two groups and finally

three groups each containing a strain. The geographic positioning of the various groups

and allelic profiles is shown in Figures 10 and 11. The largest number of allelic profiles

per strain recorded in this work is 2 and this is found in all the groups except groups B

which has three members with one allelic profile each.

Also in this study, the allelic profiles of the vaccine strains determined by Yaya et

al.,(2008) was compared with that of the isolates from Northern Nigeria. Even though

five different vaccine strains were compared, only two of the vaccines have been widely

used in Africa and only one (T1SR, a T1-44 variant) is currently being used in Nigeria.

Polymorphisms were established between these vaccine strains and the field isolates

(See table 6).

107

Table 5: Overall distribution of alleles for combined MLVN TR, MLSA on loc-PG- 0001 and 0103

in Northern Nigeria.MmmSC strains.

The Profiles recorded will be entered into on-line Data Bank for future reference of all

Strains

Town

TR34:

number of

repeats

Loc-PG1-

0001

PG1-0103:

number of

(ATT)

repeats

Loc-PG1-

0103 -

Alleles Profiles

06045-C1 Vom 12 4 6 2 A

6050-C5 Bauchi 12 4 6 2 A

6060-C1 Kano 12 4 6 2 A

6052-C1 Kanam 8 4 8 4 B

6057-C2 Fadan Kaje 8 4 8 4 B

6061-C1 Jos 8 4 8 4 B

6058-C1 Vom 14 1 6 2 C

6059-C1 Kafanchan 4 1 7 3 D

6051-C1-LC1 Bauchi 9 1 9 5 E

6054C1 Sanga 12 7 9 5 F

6048-C1 Birnin Kebbi 12 7 9 5 F

6056-C1 Vom/Jos 8 4 7 3 G

6053-C1 B/Ladi 8 4 7 3 G

Global results

5 alleles 3 alleles

4 types 4 alleles

7

profiles

108

PLATE 14 : Geographical distribution of alleles for the combined MLVN

TR, MLSA on loc-PG- 0001 and 0103in Northern Nigeria

Figure 10: Overall distribution of alleles for combined MLVN TR, MLSA on loc-PG- 0001 and

0103 in Northern Nigeria.

A

B

C

D

E

F

G

109

Figure 11: Overall distribution of alleles for combined MLVN TR, MLSA on loc-PG-0001, and

0103 in Northern Nigeria

0

5

10

15

20

25

30

Po

lym

orp

his

m s

en

sit

ivit

y

Strains

PG1-0103: number of (ATT) repeats

Loc-PG1-0001

TR34: number of repeats

110

Table 6: Polymorphisms between the allelic profiles of Vaccine strains and isolates from Northern

Nigeria. Nucleotides identical to the consensus sequence are left blank. Deletions are indicated by] [. The bottom line represents the consensus sequence from the reference strain PG 1.

Polymorphisms of allelic profiles for vaccine strains were obtained from data by Yaya et al., 2008.

Loc-PG1-

Profiles Type strains Loc-PG1-0001 0103

1500 1518 1523 1524 1527 1528 1529 1530 1531 1635 122439

B05 V5 6ATT B06 Asmara 6ATT B07 T1SR 8ATT E E01 DK32 T G G01 Lederle T T ][ ][ ][ ][ ][ 4ATT I I01 KH3J 5ATT Vom 06045-C1 6ATT

Bauchi 6050-C5 6ATT

Kano 6060-C1 6ATT

Vom 6058-C1 A 6ATT

Kafanchan 6059-C1 A 7ATT

Vom/Jos 6056-C1 7ATT

B/Ladi 6053-C1 7ATT

Kanam 6052-C1 8ATT

Fadan Kaje 6057-C2 8ATT

Jos 6061-C1 8ATT

Bauchi 6051-C1-LC1 A 9ATT

Sanga 6054C1 A 9ATT

Birnin Kebbi 6048-C1 A 9ATT

PG1 Consensus C A G A T A G T A G 5ATT

111

CCHHAAPPTTEERR SSIIXX

GGEENNEERRAALL DDIISSCCUUSSSSIIOONNSS

6.1 Competitive Enzyme Linked Immuno-sorbent Assay for the estimation of

CBPP prevalence

The detection of outbreaks is a prerequisite for the success of CBPP control policies (Le

Goff and Thiaucourt, 1998). For effective control/or eradication of CBPP, its diagnosis

in live animals and identification of causal agent in tissues are necessary (Aliyu et al.,

2003). The establishment of the true prevalence rates of CBPP in infected countries is a

crucial prerequisite to mounting a successful disease control programme, and a

precursor to national efforts. CBPP is difficult to diagnose because many affected cattle

present no clinical signs as well as the inability of routinely available serological tests to

detect specific antibodies in most of the affected animals (Aliyu et al., 2003; Nicholas

and Bashirrudin, 1995; Regella et al., 1996). The virulence and immunological

mechanisms involved during M. mycoides mycoides SC infection, or following

vaccination against CBPP, are not fully elucidated. Consequently, there is no in vitro

test at present which can be used to assess accurately the immune status of bovines with

respect to CBPP (Tulsane et al., 1996). The inability of serological tests to discriminate

between natural and vaccinal exposures in animals has led to a greater reliance on Post

Mortem examination of lung lesions for monitoring and surveillance of CBPP (Aliyu et

al., 2000). However, in a comparison of c-ELISA with Complement Fixation Test

(CFT) on animals infected more than three months previously, c-ELISA able to detect

more positive cases than CFT (Le Goff and Thiaucourt, 1998) as such this test was

considered a more promising tool for the detection of CBPP. In this study, c-ELISA was

used to estimate the prevalence of CBPP in Twelve states of Northern Nigeria.

Commercial kits for ELISA have been described for the serological detection of CBPP

112

and are used today in favour of CFT because of its sensitivity and ease for large scale

testing (Nicholas, 2004). Ten out of the twelve States screened in this study, tested

positive for CBPP by c-ELISA which strongly suggests that the disease has attained

endemic proportions. This is quite contrary to the number of outbreaks reflected by

official figures for yearly outbreaks of CBPP in the country. An overall estimated

prevalence of 10.8% out of 2026 samples is quite alarming. Even though some States

like Gombe and Taraba recorded very high prevalence (50% and 40%) due to the very

low number of samples 18 and 15 respectively, States like Bauchi (154), Plateau (591)

and Jigawa (135) States with high number of samples also recorded fairly high

prevalence of infection (32.5%), (13.5%) and (13.3%) respectively. It must however be

Stated that only apparently unhealthy animals were targeted for sampling in this study,

and even then it should be noted that results obtained by c-ELISA should be interpreted

on a herd basis and not on the accuracy of individual samples. This is because c-ELISA

may not account for those animals in the incubation stage of the disease and results of

animals vaccinated less than three months prior to test may give a false outcome. In this

case however adequate vaccination history was collected and only herds not vaccinated

within three months prior to the test were considered. The two States which recorded

negative for CBPP could probably be due to the reasons given above or the fact that the

sample number for those areas was too small. In any case the positive results recorded

established the presence of CBPP in line with the objective for this work. This work

should also serve as a baseline for CBPP documentation and motivation for the

authorities to take the issue of disease outbreak documentation more seriously because

it is only by doing this that awareness about the impact of the disease in the country will

be created and international support for efforts at control will be enhanced.

113

6.2 Isolation and Identification of Mycoplasma mycoides mycoides Small Colony

from Northern Nigeria.

Thirteen (13) isolates out of the 18 recovered from this study were confirmed to be

MmmSC by conventional PCR. However phenotypic identification proved difficult as it

was almost impossible to differentiate some non MmmSC colonies from those of

MmmSC. The isolation of two different sub-species from an apparently pure

Mycoplasma culture only worsened the situation. Results from this study shows that

phenotypic method of identification for Mycoplasma should be applied only as a

preliminary step in the identification of MmmSC isolates.

The thirteen (13) isolates were also confirmed by Real Time or QPCR. Specific

identification of microorganisms by QPCR has been described for several organisms

(Lorenzon et al., 2002). However a Specific Real-Time PCR assays for the detection

and quantification of Mycoplasma mycoides subsp. mycoides SC and Mycoplasma

capricolum subsp. capripneumoniae has just be developed and accepted for publication

(Lorenzon et al., 2008). The confirmation of the 13 isolates was based on this protocol

and this is the first application of this technique for the diagnostic identification of

MmmSC isolates in Nigeria. The technique is superior to the conventional PCR because

it is more sensitive and avoids the processing of PCR products at the end of the reaction,

which is a major source of contamination and hazards for laboratories. The assay is easy

to perform, fast and robust. The equipment though expensive is affordable to most

laboratories in Nigeria.

114

6.3 Multi-Locus Variable Analysis (MLVA) of Variable Number Tandem Repeats

(VNTR) 34 and Multilocus Sequence Analysis MLSA

The causative organism of CBPP was isolated for the first time over a hundred years

ago by Nocard et al., (1898). It took another 58 years before the organism was

identified as a Mycoplasma by Edward and Freundt (1956), by which time it had

undergone nine name changes. Then 28 years ago (Nicholas and Bashiruddin, 1995),

two morphological variants of Mycoplasma mycoides mycoides were recognized.

Today, it does not only seem likely, but that further changes in nomenclature are

imminent because of new-found genetic relationships within the Small Colony variants.

The high degree of serological and DNA-relatedness between cluster members has

hitherto contributed to the difficulty of specific identification, although the recent

availability of molecular tests has helped considerably in this respect. The development

of new molecular and genetic tools has made it possible to show that there are genetic

differences between strains of MmmSC from Northern Nigeria. The ability to establish

these differences and to trace Mycoplasma mycoides subsp. mycoides SC

epidemiologically remains of great importance because of the importance of CBPP

(Litamoi et al., 2004). Not only can isolates be reliably identified today, but the

development of new techniques has aided considerably in the specific identification of

particular biotypes within a single species (March et al., 2000). The molecular

epidemiology of Mycoplasma mycoides mycoides Small Colony has advanced rapidly in

recent years, allowing an accurate characterization of strains at a molecular level,

involving both nucleic acid and protein-based approaches. In the present study, a high

level of discrimination was achieved when 13 MmmSC isolates from Northern Nigeria

were characterized into 5 alleles by the analysis of the VNTR 34 locus. MLVA was able

to discern genetic similarities and differences among these isolates. Typing by MLVA

115

can be achieved with basic equipment. The data obtained can easily be compared to

published genotypes, either at a local level or by querying data via a website created for

that purpose, http://bacterial-genotyping.igmors.u-psud.fr/. The potential Internet-based

querying tools will help make MLVA, like MLST, a good candidate for a robust and

geographically widespread control program.

Characterization of the isolates by the MLSA technique on locus Loc-PG1-0001

identified three alleles while that on locus Loc-PG1-0103 identified 4 alleles.

Combinations of MLVNTR and MLSA techniques was able to differentiate the isolates

from Northern Nigeria into 7 different profiles and amongst these were very unique

strains found only in specific locations. Presently four of the seven strains can be found

in one State each which makes it easy to relate the source of infection in case of an

outbreak involving these species. A comparison of the various molecular techniques

carried out indicated a positive correlation between them with the MLSA on Loc-PG1-

0103 being more segregative than the other two (Table 5). There was a positive

correlation between PG1-0103 and PG1-0001 using regression analysis at P<0.05.

However there was no significant correlation between Loc- PG1-0103 and TR 34.

MLSA on PG1-0103 therefore was determined to be more sensitive, followed by PG1-

0001 and then TR 34 was the least sensitive at 95% CI.

6.4 Multi-locus Sequence Analysis (MLSA) on Loc-PG1-0001 and Loc-PG1-0103

Multi-locus Sequence Analysis, a molecular approach for characterizing bacterial

species based on the principles of Multi-Locus Sequence Typing (MLST) was used in

characterizing the Isolates from Northern Nigeria. The MmmSC isolates were

characterized by MLSA based on whole genome sequence comparisons with the PG1

116

genome. Analysis of PG1 sequences indicated that primers designed on a previous

MLSA scheme (Lorenzon et al., 2003) hybridised at multiple sites or targeted

sequences that were duplicated in the PG1 genome. However the new MLSA scheme

designed by Yaya et al., (2008) seemed more robust than previous schemes and was

used for this work. In this study, three and four allelic profiles were determined for Loc-

PG1-0001 and Loc-PG1-0103 respectively for the thirteen isolates from Northern

Nigeria. Some strains share the same allelic profiles while others have profiles that were

unique. Similarly, some States have similar strains while others either have different

strains in addition or have entirely unique strains in their domain. The geographic

location of strains sharing the same allelic profile agreed with known epidemiological

data (Yaya et al., 2008). The presence of same allelic profiles in different strains from

different States of the country can be explained by a ‘clonal expansion’ of the initial

strain of the organism. Putting these two tests together, it is interesting to note the

cocktail of Mycoplasma mycoides mycoides Small Colony strains in the Northern part

of the country, which is in agreement with the transhumance and trade routes followed

by cattle herds that are raised in the north and sent to the meat markets in large cities in

the South, contrary to expectations that only a single strain is likely to be identified

since all the isolates were clustered mostly around the central part of the country.

The two major routes for cattle trade in Nigeria between the North and South are the

Lokoja and the Makurdi roads. These roads at one point go through Kaduna and Plateau

States respectively. With the Eastern axis of the road coming from Borno and the

surrounding States passing through Plateau to the South and equally, the Western axis

coming from Sokoto and the surrounding States passing through Kaduna State to the

South. Even though each road is located at least two hundred kilometres from the other,

117

these two states (Kaduna and Plateau) act as a funnel draining the livestock from the

North to the South. It is not surprising then to find these states with almost all the strains

determined for Northern Nigeria in this study.

However for these results to be meaningful, the study has to be extended to other parts

of the country so that data on the geographical distribution of all the strains would be

obtained. This would be useful in tracing future outbreaks of CBPP infection or

designing control strategies for such outbreaks. For example, an outbreak involving

profiles C, D and E would be traced easily since these strains are unique. The

information will also be useful in the selection of an appropriate candidate for future

vaccine development. Even though the results from this work may not be conclusive in

itself, it has laid the framework for relating the sources of particular outbreaks of CBPP

in Northern Nigeria.

Finally, the determination of a new allele which was not described in the previous work

of Yaya et al., (2008) is significant in this study. It would be necessary to extend this

study to other parts of the country in order to determine if there are other alleles of this

organism in other parts or if strains of this particular allele are distributed in other parts

of the country. It is necessary also to determine through gene expression models, the

possible roles of these polymorphisms in the pathogenicity or immunogenicity of these

strains.

118

6.5 MLSA allelic profile comparison of Vaccine strains and isolates from

Northern Nigeria.

Contagious Bovine Pleuropneumonia (CBPP) vaccines have often been the subject of

debate, possibly more so than any other bacterial vaccine. Observations from the field

(Masiga et al., 1999) and experimental studies (Thiaucourt et al., 2000a, Yaya et al.,

1999) have indicated that the current vaccines do not effectively protect cattle from

outbreaks of disease (March et al., 2002). These debates became more pronounced

when the T1SR strain of vaccine did not seem to induce sufficient protection after the

reintroduction of CBPP into Botswana (Amanfu et al., 1998) and East Africa (Masiga et

al., 1996). Presently, CBPP vaccines are used only in Africa and parts of Asia (Egwu et

al., 1996). These vaccines are live preparations produced from one of two strains, KH3J

or T144, each of which has a streptomycin resistant variant and are used in a lyophilized

form. The OIE recommended vaccine is the naturally mild T1-44 strain which has

been passaged in eggs 44 times and was isolated in Tanzania some 40 years ago (Egwu

et al., 1996). The KH3J strain which has been widely used in Western and Central

Africa (Rweyemamu et al., 1995) was isolated from the Juba region of Sudan and

passaged 88 times in embryonated eggs from which broth culture vaccines have been

prepared. The T1-44 freeze-dried vaccine is currently being used in Nigeria for CBPP

control but despite vaccination campaigns the disease continues to occur with increased

frequency (Aliyu et al., 2000).

In this study, the allelic profiles of the vaccine strains determined by Yaya et al.,(2008)

was compared with that of the isolates from Northern Nigeria. Even though five

different vaccine strains were compared, only two of the vaccines have been widely

used in Africa and only one (T1SR, a T1-44 variant) is currently being used in Nigeria.

Polymorphisms were established between these vaccines and the field isolates. The

119

genetic events underlying these polymorphisms are given in Table 6. Except for strain

058 (Vom), 059 (Kafanchan) and 051 (Bauchi) had an A instead of a G at the 1523

position of the consensus sequence while 054 (Sanga) and 048 (Birnin Kebbi) also had

an A instead of a G at position 1635. All other strains from Northern Nigeria were

identical to the vaccine strains at Loc-PG1-0001. However, greatest variability was seen

at Loc-PG1-0103 where only 052 (Kanam), 057 (Fadan Kaje) and 061 (Jos) showed

identical polymorphisms (8 repeats) with the T1SR vaccine. All other vaccine strains

had different numbers of ATT repeats (V5 and Asmara- 5 repeats and T1SR- 8 repeats).

The implications of these variations with respect to pathogenicity and immunogenicity

of the strains cannot be immediately ascertained. The fact that these isolates in some

respect appear to be different from the vaccine strains is a case for concern. This result

though, is not particularly surprising since recent finding from studies comparing T1-44

and KH3J indicated variations in immunogenicity and also experimental infections in

the past have shown virulence differences amongst African strains (Provost et al.,

1987). But what is not clear is whether these genetic differences are in themselves

responsible for serological variations witnessed in the immune responses to these

vaccines and also whether these classical vaccines can indeed protect against all these

different strains. Further work is required to determine the implications of these

variations with respect to immunogenicity and vaccine capability. It is worth

emphasizing that ‘Efficient control of CBPP in Africa, and indeed Nigeria, will require

both efficient vaccines as well as efficient diagnostic strategies’.

120

6.6 Isolation of Mycoplasma mycoides mycoides Small Colony from sheep

In this study, MmmSC was isolated in pure cultures from pleural fluid in a two week old

lamb which died of respiratory distress (045) and from two other ovine samples (058

and 061). Several reports of MmmSC isolation from small ruminants have been made

worldwide (Hudson et al., 1967; Brandao, 1995; Thiaucourt; 2000, Dujardin-Beaumetz,

1906; Yaya et al., 2000). Even though these reports tend to suggest that the role of these

ruminants in the transmission of the disease is insignificant (Mullins et al., 2000), some

authors believed that these reports should be investigated further (Egwu et al., 1996). In

Nigeria, Okoh and Ocholi isolated Mycoplasma mycoides mycoides Small Colony from

an outbreak of disease is sheep (Egwu et al., 1996). Though it was not clear from that

report whether the SC variant was confirmed or whether the isolates were pathogenic in

cattle. In the present study, some MmmSC isolates recovered from sheep were

characterized and compared with those isolated from cattle. Among these isolates was

one isolated from a two week old lamb which came to the Veterinary Clinic with a

history of respiratory distress. The farmer noticed the condition about a week after birth

and by the second week the situation had worsened with profuse mucus secretions from

the nostrils and mouth followed by difficulty in standing. The dam did not however

show any respiratory symptoms. The lamb died two weeks after birth and apart from

bilateral congestion of the lungs, there were no other visible lesions at post mortem.

MmmSC was isolated in pure culture from the pleural fluid. The isolate was confirmed

by both conventional and Real Time PCRs. Molecular characterization studies indicated

that this isolate is the same as other MmmSC isolates recovered from outbreaks of

Contagious Bovine Pleuropneumonia in cattle. Likewise results of sequence analysis for

the other strains isolated from sheep in this study indicated that only one of the strains

has a unique allelic profile (058 profile C) from those isolated from bovine (045 profile

121

A, and 061 profile B). This is particularly disturbing because it suggests that there is no

difference between isolates from sheep and those from bovine, indicating that the sheep

is a potential source of infection for bovine. This finding is significant as it implicates

sheep in the epidemiology of the disease. However, pathogenicity studies have not been

carried out in cattle to determine if the isolates are indeed pathogenic for cattle. There is

thus a need to carry out experimental infection of sheep using same isolates to

determine the risk factors associated with the transmission of infection to bovine and to

re-appraise the role of sheep in the transmission of this infection. The fact that small

ruminant species may serve as a reservoir, should be an incentive to widen the scope of

Mycoplasma isolation and identification from this host, which is known to harbour

Mycoplasma in the ear canal in the absence of pathological signs.

6.7 Mycoplasma mycoides mycoides Small Colony Isolate and serum bank

The isolates from this study are presently the only MmmSC isolates in Nigeria to be

identified and characterized to molecular level. These isolates presently form the

largest reserve of MmmSC isolates for any single country in the World Reference

Laboratory for CBPP in CIRAD, Montpellier, France and will also form the very first

batch of characterized MmmSC isolates at the Isolate bank of the Regional Reference

Laboratory for CBPP, National Veterinary Research Institute, Vom. Plateau State,

Nigeria. The sera screened in this study will also be grouped according to status and

kept in the Serum bank also of the Regional Laboratory. These would make these

isolates and sera available for future pathogenicity or immunological studies involving

this organism.

122

Finally, results from the current study would be included in a web tool dedicated to

molecular epidemiology within an EU-funded project called EPIZONE. This tool

should facilitate the dissemination of typing methods for pathogens, allow any

laboratory to compare in-house data with large databank sets and, in turn, generate

phylogenetic trees or actualized maps, provided that the geographic coordinates of all

isolates are known. Such a tool would naturally be linked to the websites of the OIE

(http://www.oie.int/wahid-prod/) and FAO.

123

CCOONNCCLLUUSSIIOONNSS

1. The presence of CBPP in Nigeria was further ascertained in this study by both

Serological and Molecular approaches.

2. A high level of discrimination was achieved using the MLVA on MmmSC

isolates. The 13 MmmSC isolates were characterized into 5 alleles using this

analysis. The Potential clinical applications of MLVNTR typing include

epidemiological investigations, identification of outbreak-associated strains and

recognition of laboratory cross-contamination. Even though the clinical

significance of M. mycoides mycoides Small Colony identified by MLVNTR

typing in this study is not yet known. VNTR typing by PCR has several

advantages. It is a rapid and reproducible method. It can be performed on

Mycoplasma killed by heat or alcohol, reducing biohazards. Results are usually

in electronic form thereby simplifying the comparison of large numbers of

strains.

3. The combination of MLVNTR and MLSA brought more precision to the

definition of the isolates in this study, with the other two loci, Loc-PG1-0001

and Loc-PG1-0103 having 3 and 4 alleles each respectively. Eventually, seven

profiles were defined for the thirteen isolates giving more precision in the

definition of the origin of the strains. This is at great variance with MmmSC

strains from southern Africa where all have the same profile, i.e. only one strain

(Yaya et al., 2008). But this is not surprising since unlike Nigeria, the region is

protected from other infected countries by a cordon of countries free of CBPP.

4. The number of strains recorded in this study (seven) in Northern Nigeria is so

far the largest for any single country in the world and the study also identified a

new allele which was not previously described.

124

5. The molecular tools used in this work if extended to other parts of Nigeria will

lead to faster and easier identification and characterization of Mycoplasma

mycoides mycoides Small and the epidemiological knowledge collected will help

to elucidate the prevalence and geographical spreading of this organism over

time or the extent and mode of its transmission during outbreaks.

6. The isolation of MmmSC in pure culture from a two week old lamb and other

adult ovine species is of major significance as it implicates ovine in the

epidemiology of CBPP in Nigeria. This agrees with the views of several authors

like Egwu et al., 1996 who suggested that small ruminants may be implicated in

the epidemiology of Contagious Bovine Pleuropneumonia.

7. As a result of this study, Nigeria now has the largest reserve of MmmSC isolates

(13) in the World Reference Laboratory for CBPP in CIRAD, France.

8. The extension of this study to other parts of the country and the determination of

the geographical distribution of the various profiles on the map will make it

possible to relate the origin of any CBPP outbreak within the country. It is even

possible to trace the source of infection especially for the unique profiles like C,

D, and E. This may be particularly useful for the country especially in

eradication programs requiring tools to trace the origin of remaining or re-

emerging CBPP foci and in control strategies especially the stamping out policy

where the source of infection is of prime importance.

9. The results obtained in this study, is the first its kind on livestock diseases in the

country to be included in a web tool dedicated to molecular epidemiology within

an EU-funded project called EPIZONE. This tool will facilitate the

dissemination of typing methods for pathogens, allowing laboratories to

compare in-house data with large databank sets and, in turn, generate

125

phylogenetic trees or actualized maps, provided that just as in this study, the

geographic coordinates of all isolates are known. Such a tool would naturally be

linked to the websites of the OIE (http://www.oie.int/wahid-prod/) and FAO

(http://www.fao.org/ag/againfo/programmes/en/empres/disease_cbpp.asp),

which provide updated information on new outbreaks for notifiable diseases.

10. Finally, over 100 years since the cause of CBPP was discovered, it is still not

possible to say with certainty how the M. mycoides mycoides Small Colony

causes disease. The use of serological tests with greater sensitivity may succeed

in identifying infected animals that are negative by c-ELISA and reduce the

burden of applying serological and abattoir survey in the determination of the

actual prevalence of CBPP. The use of Molecular tools in diagnosis is beginning

to elucidate the anatomy and behaviour of the CBPP agent even though very

little is known about the factors that play key role in the pathogenicity of the

organism and the implications of the genetic events underlying the

polymorphisms in the those strains. It is hoped that a better understanding of the

expression systems associated with these polymorphisms will enable the

formulation and production of improved vaccines to replace the semi-virulent

strains in use today. To re-echo the words of Egwu et al., 1996, ‘the persistence

of this disease in Nigeria is of great economic importance and the reports of

sporadic outbreaks in some areas in this country despite the various control

programmes in use emphasize the need…not only for control measures’ but also

for intensified commitment by all stake holders and research towards control.

126

MMAAJJOORR CCOONNSSTTRRAAIINNTTSS OOFF TTHHEE SSTTUUDDYY

a) Lack of Facilities and Reagents: Reagents and equipment were a major

requirement for this work. ELISA kits are very difficult to acquire. Presently, the

only vendor for this item is CIRAD, Montpellier, with difficulties in

administrative procedures, payments and shipment due to language barriers. As

a result of high cost of equipments, facilities like the Real Time PCR machines

are currently unavailable in the country.

b) Lack of capacity and skills in molecular biology: The various techniques applied

in this study requires capacity and skills in molecular biology especially

genomics and bioinformatics. The training I acquired from CIRAD on MLSA

was a major boost to this study.

c) Lack of reliable information from the field: Accurate information in the field

during investigation was a major problem. Most farmers would prefer to hide

outbreaks and treat their animals with antibiotics because of the lack of

compensation in the case of condemnation. Sometimes, information on the use

of vaccine before outbreak, antibiotics, and the exact period or duration of the

outbreak are very unreliable.

d) Insufficient finance: Lack of funds limited the scope of this work to Northern

Nigeria and to suspected outbreaks only. It would be interesting to find out the

situation of CBPP in apparently healthy animals, abattoir slaughter and animals

in other parts of the country.

127

RREECCOOMMMMEENNDDAATTIIOONNSS

I. There is a need to extend this study to other parts of the country to determine the

disease status in other areas and to compare isolates from such areas. The

average OD/PI of the different areas using c-ELISA may possibly give an idea

of the disease trend in such areas. Such extensions could also include abattoir

surveillance and the surveillance of apparently healthy animals. Serological

tools though not very reliable may serve as guides to instituting or supporting

control strategies.

II. This study has also indicated that the vaccines currently used for the control of

CBPP in Nigeria may not be genetically identical to the field isolates. The

significance of this finding should be investigated. In the future, the molecular

techniques used in this study may be applied to identify particular alleles with

potential to serve as possible candidates in the production of molecular vaccines

against CBPP. Though it must be pointed out that the various alleles defined in

this study are ‘strictly molecular epidemiological markers since no correlation

was established between the variations in sequences and pathogenicity.

III. The molecular tools used in this work if extended to other parts of Nigeria will

lead to faster and easier identification and characterization of Mycoplasma

mycoides mycoides Small Colony within the country and will help to determine

the geographical spread of this organism and possibly mode of CBPP

transmission during outbreaks.

IV. The isolation of MmmSC in pure culture from a two week old lamb and other

adult ovine species is of major significance as it implicates ovine in the

epidemiology of CBPP in Nigeria. Further work should be carried out to

determine the specific role played by these animals in the epidemiology of the

128

disease in Nigeria. Pathogenicity studies should be carried out in both Cattle

and Sheep to determine the virulence of this particular isolate to these species.

V. The combination of MLVNTR and MLSA increased the sensitivity of

characterization of the isolates in this study. Other loci, especially none coding

genes could be included in the MLSA. Likewise other Tandem Repeats in the

Small Colony genome should be investigated for polymorphisms.

VI. Finally, despite the use of vaccines and eradication campaigns in Nigeria, the

rate of CBPP is on the rise. It is obvious that the current vaccines are not the

magic bullet we anticipated. There is thus a need for the Government, to put

channel more efforts and resources, especially finance towards capacity building

in CBPP research and the development of next generation CBPP vaccines.

129

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AAPPPPEENNDDIIXX II

TISSUE SAMPLES COLLECTED FROM NORTHERN NIGERIA FOR MMSC ISOLATION

BOVINE SAMPLES

S/N STATES LUNGS PLEURAL FLUID

NASAL SWAB

EAR SWAB

1 PLATEAU 22 8 85 20 2 BAUCHI 7 5 23 4

3 KADUNA 9 - 28 15

4 KANO 5 - 19 5

5 KEBBI 6 1 17 8

TOTAL 49 14 172 52

OVINE SAMPLES

S/N STATES LUNGS PLEURAL FLUID

NASAL SWAB

EAR SWAB

1 PLATEAU 8 1 7 7

2 BAUCHI 1 - 2 2

3 KADUNA 2 - 11 2 4 KANO - - 4 -

5 KEBBI - - 2 -

TOTAL 13 1 26 11

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AAPPPPEENNDDIIXX IIII

Estimated number of bulls and cows infected in outbreaks of CBPP in Northern

States of Nigeria

S/N STATE TOTAL

NO. OF

ANIMALS

NO. OF

MALES

TOTAL

NO.

POSITIVE

MALES

POSITIVE

FEMALES

POSITIVE

1 Adamawa *304/**257 68 8 2 6

2 Bauchi 154 61 50 24 26

3 Borno 96 17 2 0 2

4 Gombe 18 3 9 1 8

5 Jigawa 135 52 18 3 15

6 Kaduna 313 81 39 17 22

7 Kano 215 56 11 4 7

8 Katsina 74 N/A 0 N/A N/A

9 Plateau 591 117 72 27 50

10 Sokoto 108 38 4 0 4

11 Taraba 15 4 6 4 2

12 Kebbi 3 0 0

TOTAL 2026/1979 497 219 82 137

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AAPPPPEENNDDIIXX IIIIII

Estimated infection rates of of CBPP bulls and cows in Northern States of Nigeria

NO STATE TOTAL %

INFECTION

BULLS %

INFECTION

COWS %

INFECTION

1 Adamawa 2.63 0.66 1.97

2 Bauchi 32.47 15.59 16.88

3 Borno 2.08 0 2.08

4 Gombe 50.5 5.61 44.89

5 Jigawa 13.33 2.22 11.11

6 Kaduna 12.46 5.43 7.03

7 Kano 5.12 1.86 3.26

8 Katsina 0 0 0

9 Plateau 13.54 4.75 8.79

10 Sokoto 3.7 0 3.7

11 Taraba 40.0 26.67 13.33

12 Kebbi 0 0 0

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AAPPPPEENNDDIIXX IIVV

Preparation of broth and agar medium base for Mycoplasma cultures

Principle

The procedure consists in the preparation and sterilisation of a growth medium base, whether liquid (broth) or solid (agar), which will provide a complete culture medium after addition of the appropriate supplement.

This protocol is adapted from:

Provost, A. et al. (1987). Contagious bovine pleuropneumonia. Rev. Sci. Tech. Off int. Epiz. 6, (3), 625-679.

Freundt, E. A. (1983). Culture media for classic mycoplasmas. In Methods in

Mycoplasmology (I) 405-410. Edited by S. Razin and J. G. Tully: Academic Press.

III. Related documents

- Operating manuals for all materials used (Autoclave) - Operating procedure “Quality control of mycoplasma growth media” - Registration sheet

IV. Materials and methods

a) Materials

- Weighing scales - Glassware (a 3 L beaker, a 2 L bottle, twenty 100 ml bottles, graduated

cylinders) - Magnetic stirrer with heater - Autoclave

b) Reagents

- PPLO broth Difco (without crystal violet) - Milli-Q water - Agar noble Difco - Autoclaving control tape

c) Specific safety measures

- Risk of burning whilst manipulating hot products

d) Operating procedure

PPLO agar base (1.4 litres for 20 bottles)

1- Measure 1.4 litres of milli-Q water and transfer to a beaker with a capacity of 3 litres

2- Add a magnet and agitate on a magnetic stirrer, heating until it starts boiling

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3- Weigh 20 g of agar noble Difco and add to the boiling water

4- Continue heating and agitation until the agar is completely molten (around 10-20 minutes)

5- Add 42 g of PPLO and let dissolve

6- When the mix acquires a translucent appearance, distribute 70 ml of the preparation (still liquid) into 100 ml bottles. Measure the volumes using a 100 ml cylinder and wear protective gloves to prevent skin burns.

7- Close the bottles, stick an autoclaving control tape on the lid and write on it the lot number with a permanent marker.

8- Fill in the registration sheet for production of growth media, indicating the reagents used for future reference.

9- Autoclave at 121°C for 20 minutes.

PPLO broth base (1.4 litres for 20 bottles)

1- Measure 1.4 litres of milli-Q water and transfer to a beaker with a capacity of 3 litres

2- Add 42 g of PPLO and let dissolve

3- Distribute 70 ml of the solution into 100 ml bottles

4- Close the bottles, stick an autoclaving control tape on the lid and write on it the lot number with a permanent marker

5- Fill in the registration sheet for production of growth media, indicating the reagents used for future reference

6- Autoclave at 121°C for 20 minutes

7- The quality of a lot of medium base must be tested at least once whenever a new lot of any of its ingredients is used

e) Storage

Media base may be conserved for up to 1 year at +4°C.

f) Comments - remarks

This protocol permits to obtain twenty bottles of base medium, which will provide, after supplementation, a final volume of 2 litres of ready-to-use culture medium.

Note that 30 ml of supplement are added to 70 ml of base, providing a final volume of 100 ml supplemented medium. For preparation of agar plates, melted base is cooled down to 60°C, supplemented as above and 25 ml doses are immediately added to 9 cm diameter Petri dishes.

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AAPPPPEENNDDIIXX VVII

Preparation of supplement for mycoplasma culture medium

I. Aim and field of application

This protocol aims to describe the procedure for preparation of the supplement, which is a component of the growth medium used for the isolation of mycoplasmas.

II. Principle

The procedure consists in the preparation of a mixture of all the reagents that need to be added to the “medium base” in order to produce a complete culture medium.

This protocol is adapted from:

Provost, A. et al. (1987). Contagious bovine pleuropneumonia. Rev. Sci. Tech. Off int. Epiz. 6, (3), 625-679.

Freundt, E. A. (1983). Culture media for classic mycoplasmas. In Methods in

Mycoplasmology (I) 405-410. Edited by S. Razin and J. G. Tully: Academic Press.

III. Related documents

- Operating manuals for all materials used - Operating procedure “Preparation of fresh yeast extract” - Operating procedure “Quality control of mycoplasma growth media” - Registration sheet

V. Materials and methods

a) Materials

- Water bath - Weighing scales - Glassware (a 2 L bottle, graduated cylinders) - 0.22 µm filter units - Horizontal flux hood - Sterile 50 ml “Falcon” tubes

b) Reagents

- Horse serum tested for mycoplasma culture - Fresh yeast extract tested for mycoplasma culture - Milli-Q water - D-glucose - Sodium pyruvate - Ampicillin

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c) Specific safety measures

- No identified risks

d) Operating procedure

1- The previous day, defrost two bottles of 500 ml horse serum and one bottle of 250 ml fresh yeast extract

2- De-complement the serum by incubating for 1 hour at 56°C

3- Prepare a solution of 250 ml milli-Q water, 10 g glucose, 20 g pyruvate and 2 g ampicillin.

4- Under a horizontal flux hood: Filter this solution through a 0.22 µm filter unit positioned over a 2 L bottle

5- Then filter the fresh yeast extract (250 ml) and finally the horse serum (1 L)

6- Mix well and distribute 30 ml doses into 50 ml “Falcon” tubes, annotating the lot number

7- Fill in the registration sheet for production of growth media, indicating the reagents used for future reference

9- Take one tube to test the sterility (compulsory) and the efficiency (whenever required)

10- The quality of a lot of supplement must be tested at least once whenever a new lot

of any of its ingredients is used

e) Storage

The supplement may be conserved for up to 1 year at -20°C.

f) Comments – remarks

This protocol permits to obtain fifty tubes of supplement, which will provide, after supplementation, a final volume of 5 litres of ready-to-use culture medium.

Note that each 30ml of supplement is added to 70ml medium base, producing a final volume of 100ml supplemented medium.

However, for isolation of fastidious species such as M. capricolum subsp. capripneumoniae, additional horse serum may be added (i. e.: 10 ml horse serum are added to the 100 ml supplemented medium).

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