protein based approaches to understand and prevent contagious …235819/fulltext02.pdf · 2009. 9....

Protein based approaches to understand and prevent contagious bovine pleuropneumonia

Carl Hamsten

Royal Institute of Technology

School of Biotechnology

Stockholm 2009

Carl Hamsten 2009

ISBN 978-91-7415-417-7

TRITA-BIO Report 2009:17

ISSN 1654-2312

Royal Institute of Technology

School of Biotechnology

AlbaNova University Center

SE-106 91 Stockholm

Sweden

Printed at Universitetsservice US-AB

Drottning Kristinas väg 53B

SE-100 44 Stockholm

Sweden

iii

Carl Hamsten (2009): Protein based approaches to understand and prevent contagious

bovine pleuropneumonia. School of Biotechnology, Royal Institute of Technology (KTH),

Stockholm, Sweden

ABSTRACT

Contagious bovine pleuropneumonia (CBPP) is a severe infectious disease caused by Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) and is a vast problem in Africa. Current CBPP prevention is based on attenuated live strain vaccines, but these are limited by factors such as short-term immunity, cold-chain dependence and retained virulence. CBPP can be diagnosed using post-mortem examination, identification of the agent using culture and PCR based methods as well as serological diagnostic methods, but the latter are generally not sensitive enough and there is also demand for an inexpensive, pen side field test. The research presented in this thesis was focused on using recombinantly expressed surface proteins from M. mycoides SC to characterize humoral immune responses to CBPP. Thereby candidate proteins to be used in development of serological diagnostic methods and possibly subunit vaccines could be identified. As a first step, five putative variable surface proteins of M. mycoides SC were expressed and purified from E. coli in Paper I. These proteins were analyzed using immunoblotting techniques and results showed that one protein, MSC_0364, was variably expressed on the surface of M. mycoides SC in vitro. Paper II presents expanded efforts including cloning and expression of 64 recombinant surface proteins and an assay for high throughput analysis of protein-specific IgG, IgA and IgM titers in hundreds of sera using a bead-based screening assay. The assay was evaluated by protein-specific inhibition experiments, comparisons to Western blotting and monitoring of immune responses over time in a study with sera taken from eight animals over 293 days from a previous vaccine trial. Papers III and IV present applications using the recombinant proteins and bead-based screening assay wherein proteins for diagnostic and vaccine development were identified. In Paper III, the assay was used to screen 61 proteins using well-characterized serum samples from cattle with CBPP and healthy controls, resulting in selection of eight proteins suitable for diagnostic use. These proteins were combined and evaluated in a proof-of-concept ELISA with a discriminative power that enabled 96% correct classification of sera from CBPP-affected and CBPP-free bovines. Paper IV reports the results and protein-specific analyses of a vaccine trial using the recombinant putative variable surface proteins presented in Paper I as a subunit vaccine. The vaccine conferred no protection, but a weak vaccine response could not be excluded as the cause of failure. In an effort to identity other protein candidates to be used in a subunit vaccine, protein-specific analysis of humoral immune responses elicited by the currently approved live strain vaccine, T1/44, were investigated. Here, five proteins with high IgG titers associated to immunity were identified: LppQ, MSC_02714, MSC_0136, MSC_0079 and MSC_0431. These proteins may be important in the development of a novel subunit vaccine against CBPP.

Keywords: CBPP, Mycoplasma mycoides subsp. mycoides small colony type, humoral immune

responses, recombinant surface proteins, ELISA, subunit vaccine, suspension bead array

© Carl Hamsten 2009

v

LIST OF PUBLICATIONS

This thesis is based on the following publications, which are referred to by their Roman numerals

(I-IV).

I. C. Hamsten, J. Westberg, G. Bölske, R. Ayling, M Uhlén and A. Persson (2008). Expression and immunogenicity of six putative variable surface proteins in Mycoplasma mycoides subsp. mycoides SC. Microbiology 154 (Pt 2): 539-49

II. C. Hamsten, M. Neiman, J.M. Schwenk, M. Hamsten, J.B. March and A. Persson (2009). Recombinant surface proteomics as a tool to analyze humoral immune responses in bovines infected by Mycoplasma mycoides subsp. mycoides SC. Mol Cell Proteomics, published ahead of print, doi: 10.1074/mcp.M900009-MCP200.

III. M. Neiman, C. Hamsten, J.M. Schwenk, G. Bölske and A. Persson (2009). Multiplex screening of surface proteins from Mycoplasma mycoides subsp. mycoides SC for an antigen cocktail ELISA. Clin Vaccine Immunol, published ahead of print, doi: 10.1128/CVI.00223-09

IV. C. Hamsten, G. Tjipura-Zaire, L. McAuliffe, O. J. B. Hübschle, M. Scacchia, R. D. Ayling and A. Persson (2009). Protein-specific analysis of humoral immune responses in a CBPP vaccine trial. Manuscript.

All publications are reprinted with permission from their copyright holders.

vi

RELATED PUBLICATIONS

Not included in the thesis

G. Tjipura-Zaire, L. McAuliffe, C. Churchward, R.A.J. Nicholas, M.D. Fielder, O.J.B. Hübschle¸M. Scacchia, R. Gonçalves, A. Persson, C. Hamsten and R. D. Ayling (2009). Comparison of T1/44 Vaccine and Two Novel Vaccines to Protect Against an Experimental Infection of Contagious Bovine Pleuropneumonia and Comparative Pathogenicity of Planktonic and Biofilm Grown Mycoplasma mycoides subsp. mycoides Small Colony Cells in Cattle. Manuscript

vii

To my family

ix

TABLE OF CONTENTS

INTRODUCTION .................................................................................................................1

1 CONTAGIOUS BOVINE PLEUROPNEUMONIA..........................................................................2 1.1 Clinical signs.......................................................................................................................................2 1.2 Pathology ............................................................................................................................................3

1.3 Occurrence...........................................................................................................................................5

2 MYCOPLASMA MYCOIDES SUBSP. MYCOIDES SC ....................................................................7

– A MOLLICUTE CAUSING CBPP ............................................................................................7 2.1 Mollicutes - taxonomy and phylogeny ...................................................................................................7 2.2 Consequences of a small genome – host specificity..................................................................................9 2.3 Using mycoplasmas to find a minimal gene set for cellular life .............................................................10

3 PATHOGENICITY FACTORS OF M. MYCOIDES SC .................................................................12 3.1 General pathogenicity factors..............................................................................................................12 3.2 Lipoproteins......................................................................................................................................13

3.3 Variable surface proteins...................................................................................................................14

4 PAST AND PRESENT PREVENTION OF CBPP .......................................................................17 4.1 Attenuated live strain vaccines ...........................................................................................................17

4.2 Chemotherapy ...................................................................................................................................18

5 DIAGNOSIS OF CBPP .......................................................................................................19 5.1 Clinical, pathological and culture based methods.................................................................................19

5.2 PCR-based molecular identification....................................................................................................19 5.3 Serological diagnosis of CBPP – currently approved methods ..............................................................20 5.4 Newly developed serological diagnostic methods ...................................................................................23

6 RESEARCH ON CBPP-SPECIFIC IMMUNE RESPONSES ..........................................................25 6.1 Is interferon- production associated to CBPP recovery?......................................................................25

6.2 Can M. mycoides SC modulate the immune response?........................................................................28

7 DEVELOPMENTS TOWARDS NON-LIVE STRAIN VACCINES ...................................................30 7.1 ISCOM vaccines...............................................................................................................................30 7.2 Other vaccine formulations.................................................................................................................31 7.3 A DNA vaccine for CBPP? ............................................................................................................31

PRESENT INVESTIGATION............................................................................................32

8 AIMS...............................................................................................................................32

9 INVESTIGATING PUTATIVE VARIABLE SURFACE PROTEINS (PAPER I)...................................33 9.1 Rationale ..........................................................................................................................................33 9.2 Results..............................................................................................................................................33

9.3 Conclusions.......................................................................................................................................35

10 CREATING A SURFACE PROTEIN ARRAY ASSAY (PAPER II)................................................36 10.1 Rationale ........................................................................................................................................36

10.2 Results............................................................................................................................................36 10.3 Conclusions.....................................................................................................................................41

11 SELECTION OF ANTIGENS FOR A NOVEL SEROLOGICAL DIAGNOSTIC ASSAY (PAPER III) .....42 11.1 Rationale ........................................................................................................................................42 11.2 Results............................................................................................................................................42 11.3 Conclusions.....................................................................................................................................46

x

12 ANALYSIS OF A CBPP VACCINE TRIAL (PAPER IV)..........................................................48 12.1 Rationale........................................................................................................................................ 48

12.2 Results ........................................................................................................................................... 49 12.3 Conclusions .................................................................................................................................... 54

13 CONCLUDING REMARKS AND FUTURE PERSPECTIVES .......................................................56

ACKNOWLEDGEMENTS.................................................................................................59

LIST OF ABBREVIATIONS..............................................................................................62

REFERENCES ....................................................................................................................63

CARL HAMSTEN 2009

1

INTRODUCTION

Dear Readers,

This thesis is a summary of the work I have been doing for the last couple of years, which

involves various efforts to identify proteins with potential to be used in diagnosis and prevention

of contagious bovine pleuropneumonia (CBPP). It is a bacterial disease in cattle caused by

Mycoplasma mycoides subsp. mycoides small colony type. The disease is not relevant in a purely

Swedish perspective, as the last recorded outbreak occurred in 1856 (1), but the last European

outbreaks occurred during the 1990’s and CBPP is currently a problem in many African countries.

Since it affects livestock, CBPP has both economical and nutritional consequences. In the

introduction I will give an overview of the disease, its causing agent, available options for

prevention and diagnosis as well as current research. This will hopefully assist in putting my

research, presented in the Present investigation section, into the larger context of finding efficient

means of controlling CBPP.

Enjoy!

Carl Hamsten

PROTEIN BASED APPROACHES TO UNDERSTAND AND PREVENT CONTAGIOUS BOVINE PLEUROPNEUMONIA

2

1 Contagious bovine pleuropneumonia

Contagious bovine pleuropneumonia (CBPP) is severe respiratory disease affecting cattle and

buffalo, caused by Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC, see

chapters 2-3). The main signs of CBPP are respiratory distress and coughing, evident on

stimulation of resting animals (2). The disease is transmitted by direct contact with an infected

animal through inhalation of infective droplets or contact with contaminated fodder and fomites.

Although the causing agent has been isolated from goats and sheep (3), the role of small

ruminants in the epidemiology of CBPP has not been established (4). M. mycoides SC can be

present in respiratory aerosol droplets, urine and foetal fluids (2, 5, 6). Airborne transmission up

to 200 m is believed to be possible, even though the causing agent is sensitive to drying. In this

context, shared night accommodations and water holes among nomadic herds is a reason for the

widely spread CBPP in Africa. The incubation period for naturally infected animals is a matter of

dispute and the reported range encompass three weeks to six months, where about a month is

seen as most common (4, 7). Cattle of all ages are affected by CBPP, although calves up to six

months often develop lameness from swollen limb joints rather than the pulmonary disease (4).

The mortality rate of CBPP in Africa varies between 10-70% (7), although 50% is rarely exceeded

(6). Mortality rates in recent European outbreaks were almost zero, even though pathological

lesions were identical as in African outbreaks, which could be explained by different strains of the

causing agent, different cattle breeds or differences in animal health status (8-11).

1.1 Clinical signs

The clinical manifestations of CBPP in cattle range from hyperacute through acute, subacute and

chronic forms. The hyperacute form is most often seen at the start of an outbreak and involves up

to ten percent of the infected animals. Death is sudden, within a week of respiratory signs or

without prior signs at all. The early stages of acute CBPP are indistinguishable from any severe

pneumonia with pleuritis (2) and acute CBPP affects approximately 20 percent of infected

animals. Clinical signs start with fever, dullness, anorexia, irregular rumination and only slight

respiratory distress. Respiratory symptoms including laboured and painful breathing and severe

cough that increase in severity as the disease progress and lung lesions develop. An affected

animal often present a typical stance with arched back, extended neck and forelegs spread apart in

an effort to ease breathing. A nasal discharge and frothy saliva around the mouth is often seen.

Subacute CBPP is the most common form (40-50%) and is a less severe form of the acute disease

CARL HAMSTEN 2009

3

with only slight respiratory symptoms and intermittent fever. This form commonly progresses

into chronic CBPP, which is a natural evolution of both acute and subacute CBPP. The chronic

form, which can also be the initial stage of disease, is characterized by an apparently healthy state

of the animal even though chronic lung lesions are present. These “silent” carriers of CBPP are

infectious and thought to be an important factor in spreading the disease among cattle herds. It is

estimated that up to 25% of affected cattle become chronic carriers (6).

1.2 Pathology

The pathogenesis of CBPP is not completely understood and the potential role of induced auto-

immune responses or hypersensitivity reactions is still to be determined. (See chapter 3 for further

details on pathogenicity factors). Except for young calves, pathological lesions are generally

confined to the chest cavity. Usually one lung and pleura are affected and a large volume of

pleural exudate containing clots of fibrin is common. Large fibrin “omelettes” make the lung

adhere to the chest wall and at these sites, the pleura is thickened and opaque. The interlobular

septa of affected lung regions are also thickened due to adsorption of the exudates and “frame”

the lung lobules, which vary in colors of red, grey and yellow due to different stages of

inflammatory lesions. This gives a characteristic ”marbled” appearance when dissecting the lung.

Pleural exudate is rarely seen in chronic cases of CBPP, but adhesions between lung lobes and to

the chest wall are more common. Necrotic lesions in the lung are surrounded by capsules of

fibrous connective tissue forming structures called sequestra. Sizes of sequestra can vary from one

to 30 cm in diameter, the capsule can be up to one cm thick and sequestra of different sizes can

be found simultaneously in one lung. The inner parts of the sequestra often retain the (lung)

structure of the acute lesion but the necrotic material may liquefy or become caseous over time.

Fibrous scars can replace small sequestra over time, but large sequestra may persist for years. A

sequestrum is believed to be a source of infection if it is ruptured or drained by a bronchus, but

no evidence of this has been published. The pathological description of CBPP has been adapted

from (2, 4, 6, 7, 12) as well as own observations during a CBPP vaccine trial.


4

Figure 1.1 Typical lesion of CBPP, seen at post-mortem examinations. The top image shows a lung adhering to the chest wall, the middle image shows a marbelled lung with thickened intralobular septa and the bottom image shows a encapsulated sequestrum with a necrotic core.

CARL HAMSTEN 2009

5

1.3 Occurrence

CBPP is a disease notifiable to the World Organization for Animal Health (OIE) since it has a

major impact on livestock production and a potential for rapid spread across national borders. As

a result, CBPP-infected countries are excluded from international trade. At present, the disease

causes vast problems in Africa with severe socio-economical consequences (13, 14). During the

time period 2006-2008, OIE reported outbreaks in 21 African countries as detailed in figure 1

(15).

Figure 1.2 CBPP occurrence in African countries during 2006-2008 (15).

The history of CBPP has been reviewed on several occasions and clinical descriptions of CBPP-

like disease have been traced as far back as 1550 AD (4, 7, 16, 17). The disease spread in Europe

during the 18th and 19th centuries as a consequence of wars and trade. CBPP was introduced in

USA, Asia and Australia via cattle imported from Europe in the 19th and the beginning of the 20th

centuries. Cattle from the Netherlands introduced CBPP in South Africa in 1854 and spread the

disease to neighbouring countries. CBPP could also have been introduced into East Africa by


6

cattle imported from India by British expeditionary forces in the 19th century (4). The disease was

eradicated from most parts of Western Europe in the beginning of the 20th century but has re-

emerged in every decade since (8). Several outbreaks of CBPP occurred in France, Portugal, Italy

and Spain during the 1980s and 1990s but Europe has been CBPP-free since 1999 (1). The last

outbreaks of CBPP in USA and Australia were recorded in 1892 and 1967 respectively (1) and the

countries have been disease-free since. Disease-monitoring in the Middle-East and Asia have not

been as thorough (7) but CBPP was reported in Quatar, Burma, United Arab Emirates, Kuwait

and Bangladesh during the 1990s (1) and Saudi Arabia in 2005 (15).

CARL HAMSTEN 2009

7

2 Mycoplasma mycoides subsp. mycoides SC

– a mollicute causing CBPP

The causative organism of CBPP was isolated 111 years ago by the French scientists Nocard and

Roux (18). It was classified as a mycoplasma in 1956 (19) and given its current name Mycoplasma

mycoides subspecies mycoides small colony type in 1978 (20). Before details regarding pathogenicity

factors are given in chapter 3, this chapter will give an overview of Mollicutes – the class of

bacteria within the phylum Firmicutes that M. mycoides SC belongs to – to give a broader perspective

of mycoplasmas.

2.1 Mollicutes - taxonomy and phylogeny

Taxonomically, M. mycoides SC belongs to the class Mollicutes, order Mycoplasmatales, family

Mycoplasmataceae and genus Mycoplasma (21). Mollicutes (mollis; soft, cutis; skin, in Latin) are separated

from all other bacteria by their small size and lack of a cell wall (22). Different genera within

Mollicutes are summarized in table 2.1, but the trivial name mycoplasmas often refer to all species

therein. In a pure human perspective, Mycoplasma pneumoniae is the most important species, causing

respiratory tract diseases such as tracheobronchitis, pharygitis and pneumonia (23). Other proven

human pathogens are Mycoplasma genitalium, Mycoplasma hominis and Ureaplasma species (24). M.

genitalium can cause nongonococcal urethritis (NGU); Ureaplasma spp. can cause NGU,

spontaneous abortions or premature births; and M. hominis can cause post-partum fevers and

systemic infections in immunocompromised individuals (23).

The phytoplasmas have an undefined taxonomic status, as they are currently not culturable.

Phytoplasmas are plant pathogens that spread via insect vectors and are responsible for hundreds

of diseases, many in economically important plants, causing morphological changes such as

witches broom, proliferation, flower alterations, stunting and general decline (25). Focusing on

bovines, there are several mycoplasmas except M. mycoides SC that cause disease. Several species

are associated with respiratory disease, mastitis and arthritis, the most important (common) being

Mycoplasma bovis (26).


8

Genus No. of species Habitat Mycoplasma 107 Humans, animals Ureaplasma 7 Humans, animals Entomoplasma 6 Insects, plants Mesoplasma 12 Insects, plants Spiroplasma 34 Insects, plants Acholeplasma 14 Animals, some plants, insects Anaeroplasma 4 Bovine/ovine rumen

Asteroleplasma 1 Bovine/ovine rumen

Phytoplasma* (6) Plants, insects

Table 2.1 Genera within the class Molli cu t es . *Phytoplasmas are currently unculturable and hence their taxonomic status is not defined but several Candidatus Phytoplasma species have been published. Table adapted from (22, 27).

Phylogenetic clustering using 16S rRNA sequencing classifies the Mollicutes into five groups,

placing M mycoides SC in the spiroplasma group (28). Within that group, it is one of nine closely

related species that form the Mycoplasma mycoides cluster, detailed in table 2.2 (29, 30). Until

recently, six of these formed the classical Mycoplasma mycoides cluster (31) where all members are

highly pathogenic and cause severe diseases in ruminants making correct identification of these

species important. The taxonomy of the Mycoplasma mycoides cluster was emended in 2009 resulting

in the combination of Mycoplasma mycoides subsp. mycoides large colony type and Mycoplasma mycoides

subsp. capri into a single subspecies and the assignment of the Mycoplasma sp Bovine group 7

strains into the separate species Mycoplasma leachii (32). Unfortunately, serological cross-reactions

between the species in the cluster are considerable and inconsistent which cause problems for

diagnosis (20, 33).

CARL HAMSTEN 2009

9

Species Main host (other) Disease M. mycoides SC Cattle (buffalo, goat sheep) CBPP

M. mycoides subsp. capri Goat, sheep (cattle) Arthritis, pleuropneumonia M. capricolum subsp. capricolum Goat, sheep (cattle) Arthritis, mastitis, pneumonia M. capricolum subsp. capripneumoniae

Goat (sheep)

Pleuropneumonia (CCPP)

M. leachii Cattle (goat, sheep) Arthritis, mastitis, calf pneumonia

M. yeatsii Goat - M. cottewii Goat - M. putrefaciens Goat Arthritis, mastitis

Table 2.2 Members of the classical (top five) and the phylogenetic (all) Mycoplasma mycoides cluster. Table adapted from (27, 30, 32, 34).

2.2 Consequences of a small genome – host specificity

Mycoplasmas are known as the smallest self-replicating organisms (22, 35) and have evolved from

other bacteria by regressive evolution, leading to reduced genomes (28, 36) that span from 580 kb

in Mycoplasma genitalium (37) to 1359 kb in Mycoplasma penetrans (38). The 1212 kb genome of M.

mycoides SC was published in 2004 (39) and currently the genomes of 12 mycoplasma species are

completely sequenced and available in GenBank, adding to a total of 20 completed genomes

within the Mollicutes class (see table 2.3 for details). Mycoplasma genomes have a characteristically

low G+C content and most mycoplasma use the universal stop codon UGA as coding for

tryptophan (which can be explained by the pressure for increased A+T content). The low G+C

content and a low number of genes involved in recombination and repair are traits shared with

intracellular bacteria (40). However, most mycoplasmas are extracellular (41, 42) but there are

some exceptions: Mycoplasma penetrans can invade and survive in host cells and Mycoplasma

fermentans, Mycoplasma pneumoniae, Mycoplasma genitalium as well as Mycoplasma gallisepticum sometimes

reside in nonphagocytic cells (43). The genomic reduction in Mollicute evolution has led to a

limited metabolic capacity (44) and thus reliance of the host to supply metabolites such as nucleic

acid precursors and amino acids. In general, mycoplasmas depend on the host for all amino acids

except for cysteine, aspartic acid and glutamic acid. In the end, the reductive evolution of Mollicutes

has led to strict host and tissue specificities (22).


10

2.3 Using mycoplasmas to find a minimal gene set for cellular life

Since Mycoplasma genitalium is the smallest organism that can be grown in culture (37, 45), it has

acquired a lot of scientific interest as a starting point in finding the minimal set of genes required

for bacterial growth (45). About 100 of the 482 protein coding genes were found to be non-

essential when disrupted individually under optimal laboratory conditions (45, 46). In another

approach, a set of 256 genes were suggested as close to the minimal set when comparing the

proteome of Mycoplasma genitalium and Haemophilus influenzae (47). However, 11 of these are missing

in the M. mycoides SC genome (39). An in silico metabolic model of Mycoplasma genitalium published

this year (48) was based on 189 genes and consisted of 262 reactions and 274 metabolites.

However, this model excluded essential genes not directly related to metabolic processes and

essential genes of unknown function (as determined in vivo by previous efforts), thus it did not

represent a full minimal set. To find the true non-redundant set of genes, the whole Mycoplasma

genitalium genome was recently synthesized (49) to allow tailored reduction of the genome by

simultaneous removal of a number of genes. This requires methods for removal of the native

genome and replacement with the synthetic one within a living cell. The successful transplantation

of a M. mycoides subsp. capri (formerly M. mycoides subsp. mycoides large colony type) genome to

Mycoplasma capricolum subsp. capricolum cells, that subsequently acquired the M. mycoides subsp. capri

phenotype, represents a step towards that goal (50).

CARL HAMSTEN 2009

11

Species Genome size (kb) GC content

(%) Completed Habitat

Mycoplasma genitalium 580 31,7 1995 (37) Human

Mycoplasma pneumoniae 816 40 1996 (51) Human

Mycoplasma pulmonis 964 26,6 2001 (52) Murine Mycoplasma penetrans 1359 25,7 2002 (38) Human

Mycoplasma gallisepticum 996 31 2003 (53) Avian

Mycoplasma mobile 777 25 2004 (54) Animal M. mycoides SC 1212 24 2004 (39) Animal

Mycoplasma hyopneumoniaea 893-920 28,5-28,6

2004 (55), 2005 (56)

Swine

Mycoplasma synoviae 799 28,5 2005 (56) Avian

Mycoplasma capricolum subsp. capricolum 1010 23,8 2006 (42) Animal Mycoplasma agalactiae 877 29,7 2007 (40) Animal Mycoplasma arthritidis 820 30,7 2008 (57) Rat

Ureaplasma parvum serovar 3 752 25,5 2000 (58) Human

Ureaplasma urealyticum serovar 10 847 25 2008b Human

Mesoplasma florum 793 27 2004c Human, insect, plant

Acholeplasma laidlawii 1497 31,9 2007d Animal, plant, insect

Onion yellows phytoplasma 861e 27,7 2004 (59) Plant

Aster yellows witches-broom phytoplasma 707e 26,9 2006 (60) Plant

Candidatus Phytoplasma austrailense 880e 27,4 2008 (61) Plant

Candidatus Phytoplasma mali 602 21,4 2008 (62) Plant

Table 2.3 Complete Molli cu t e genomes available in GenBank (accessed in March 2009). aThree strains have been sequenced, bGenBank accession no. CP001184, cGenBank accession no. AE017263, dGenBank accession no. CP000896, eExcluding plasmids.


12

3 Pathogenicity factors of M. myco ides SC

Genes encoding classical virulence factors such as adhesins, invasins or toxins were not found in

the genome sequence of M. mycoides SC (39), but potential virulence genes have been found in

some mycoplasma species such as the adhesins of Mycoplasma pneumoniae (63, 64). The lack of

classical virulence factors in most mycoplasmas could be a consequence of their minimal

genomes, but factors mediating adhesion to the host should arguably be demanded by their

parasitism (22). Nonetheless, no surface receptors mediating adhesion have been reported for M.

mycoides SC but other pathogenicity factors have been reported and were recently reviewed (35). I

will summarize the pathogenicity factors believed to be important in the following sections.

3.1 General pathogenicity factors

Many mycoplasmas cause oxidative damage to the host tissue by secretion of H2O2 (65) and M.

mycoides SC has been shown to accumulate H2O2 after oxidation of glycerol (66). The strains

responsible for the European outbreaks of CBPP in 1980-1999 are considered less virulent (10,

67) and were found to produce less H2O2 in the presence of glycerol than other strains (68, 69).

This identified the glycerol catabolism system and its product H2O2 as a potential virulence factor

(68, 70, 71). The system consists of a ATP-binding cassette (ABC) transporter, GtsABC, and a

membrane bound glycerol-phosphate oxidase (GlpO). European M. mycoides SC strains were

found to be missing a functional GtsABC component (71, 72). It has been shown that GlpO is

directly correlated to cytotoxic effects against embryonic calf nasal epithelial cells (ECaNEp cells),

since blocking it with mono-specific anti-GlpO antibodies hindered the release of H2O2 and the

subsequent cytotoxic effects (70). However, an expanded study of cytotoxic effects to ECaNEp

cells concluded that the non/low-virulent vaccine strain T1/44 was releasing high amounts of

H2O2 and was thus also highly cytotoxic (73). Hence there must be other virulence factors

contributing to M. mycoides SC pathogenicity. Nonetheless, work in finding mutation sites in the

glpO gene that are useful for future live vaccine development was recently published (74). In this

study, the binding site for the cofactor FAD was deleted resulting in GlpO devoid of glycerol-

phosphate oxidase activity. Polyclonal antibodies directed towards this mutant also bound native

GlpO, neutralizing the H2O2 production and thereby proving the use of this mutant in the

development of live attenuated vaccines (74).

CARL HAMSTEN 2009

13

A non-synonymous single nucleotide polymorphism (SNP) was found in the bgl gene coding for a

6-phospho-ß-glucosidase when searching for a tool to differentiate African strains form less

virulent strain of M. mycoides SC (75). The role of this SNP in the cytotoxicity of M. mycoides SC to

bovine lung cells was later explored (76). The group of M. mycoides SC strains expressing the Bgl

isoform Val204 showed high cytotoxicity towards embryonic bovine lung cells in the presence of ß-

D-glucosides (disaccharides) and included nearly all highly virulent African strains, whereas strains

expressing the Bgl isoform Ala204 (low virulent strains, including the vaccine strains T1/44 and

T1Sr as well as the low virulent type strain PG1) showed virtually no cytotoxicity during the same

conditions (76). The observed differences in cytotoxicity were not related to H2O2 production but

seemed to be indirectly due to the 6-phospho-ß-glucosidase activity affecting M. mycoides SC

viability in environments with high ß-D-glucoside levels (76). Thus, Bgl isoform Val204 is an

indirect virulence factor as strains expressing this isoform are more prone to survive in host

environments with elevated levels of these sugars, which may result from innate immune

responses in the host lung during infection (76).

The thick galactan layer surrounding M. mycoides SC cells, often referred to as the capsule, is

another potential virulence factor. Capsular polysaccharides (CPS) increased the virulence of the

attenuated vaccine strain KH3J when added to the inoculums prior to injection in bovines (77) and

CPS has also been shown to have a pathogenic effect when injected on its own (78). The M375

strain of M. mycoides SC isolated in Botswana produced less CPS, was more sensitive to growth-

inhibiting antisera and generated a shorter bacteraemia when compared to strains producing larger

amounts of CPS (79, 80). The capsule may also interfere with phagocytosis (81). The M. mycoides

SC genome revealed redundancies in the capsule biosynthesis genes, which may reflect the

importance of the capsule as a virulence factor (39).

3.2 Lipoproteins

As mentioned above, no adhesion factors have yet been identified in M. mycoides SC, but these and

other virulence factors are expected to be found among the lipoproteins. Lipoproteins are known

to be highly antigenic membrane proteins and have been shown to be involved in host

interactions such as adhesion and stimulation of pro-inflammatory cytokine release in other

mycoplasmas (82-88). At present five immunogenic lipoproteins of M. mycoides SC have been

described in detail; LppA, LppB, LppC, LppQ and Vmm.


14

LppA (89), also known as p72, is conserved among the species of the Mycoplasma mycoides cluster

(90, 91). LppB is conserved in parts of the Mycoplasma mycoides cluster (M. mycoides subsp. capri,

formerly M. mycoides subsp. mycoides large colony type and Mycoplasma leachii, formerly M. sp bovine

group 7) but is not present in M. mycoides SC strains from recent European outbreaks (72). The

lppB gene is within the same 8.84 kb segment containing parts of the gtsABC genes that are

missing in these strains (71) (see section 3.1). The position of lppB close to the ABC transporter

genes implies the function of a glycerol-binding subunit (39), but this has not been verified. LppC

is found in all M. mycoides SC strains and similarly to LppB in M. mycoides LC and M. sp bovine

group 7 as well, even possibly in M. mycoides subsp. capri (92). LppQ (93) is known as the

predominant antigen of M. mycoides SC (94) and is the most studied of the five lipoproteins. The

N-terminal domain of LppQ, which is surface exposed (94), induces a strong and early immune

response in cattle (93) and has been used to develop an enzyme-linked immunosorbent assay

(ELISA) for diagnosis of CBPP (95) (detailed in section 5.4). LppQ is similar to proteins with

super-antigenic character (93), which can be a cause to the apparent failure of a vaccine based on

purified recombinant LppQ (96) (detailed in section 7.2). In an effort to elucidate the role of

LppQ in pathogenesis, an LppQ-deficient strain of M. mycoides SC derived from T1/44 was

recently created (97). However, no pathogenicity studies using this strain have been reported yet.

The T cell immunogenicity of LppA, LppB, LppC and LppQ was recently evaluated as previous

studies on these lipoproteins have focused on humoral immune responses (98). This study

concluded that only LppA was recognized by lymph node lymphocytes three weeks after M.

mycoides SC exposure, a response that was maintained one year after infection. The response,

relying on CD4+ T cells and interferon (IFN-y) production, may be important as part of a

protective immune response, but needs further evaluation (see chapter 6 for further details).

3.3 Variable surface proteins

The last of the previously studied lipoproteins, Vmm, is a variable surface protein (vsp) (99).

Phenotypic switching of surface proteins to alter the antigenic repertoire was reported for

mycoplasmas in 1990 (100) and have since been widely described among several mycoplasma

species (22, 101). Antigenic variation is believed to be crucial for the survival of mycoplasmas

within their host and important functions such as adhesion, hemadsorption, membrane transport

and immunomodulation have been reported for variable surface proteins (102-108).

CARL HAMSTEN 2009

15

The simplest form of variation is an ON/OFF switch in protein expression called phase variation.

Several genetic mutations involved in phase variation have been described such as promoter

mutations (42, 57, 99, 109), changes in putative transcription activators (110, 111), mutations

leading to frame shifts or truncations (105, 112-114) and DNA inversions or gene conversions

fusing the coding gene to an active promoter (115-120). Antigenic variation is a more elaborate

mechanism of variability involving changes in protein size and/or epitope composition. This is

often accomplished on a genetic level using “cassettes” of repetitive sequences that can be

interchanged by insertion, deletion or recombination to form diverse protein variants. Antigenic

variation has not been identified in M. mycoides SC, but has been described in Mycoplasma bovis,

Mycoplasma hominis, Mycoplasma gallisepticum Mycoplasma hyorhinis, Mycoplasma arthritidis and Ureaplasma

urealyticum (109, 111, 114, 121-127). Epitope masking is another mechanism of variation wherein

epitopes of a constitutively expressed protein appears to have variable surface exposure. This is

caused by phase variation and/or antigenic variation of a secondary protein that sterically blocks

these epitopes (103, 128).

Variable proteins are often subjected to both phase variation and antigenic variation and many are

part of multigene families. Multigene family members are often differentially expressed and

chromosomal rearrangements govern which gene that is transcribed (104, 117, 120, 122, 123, 129-

132). Large multigene families with up to 70 members have been described (133). All mycoplasma

multigene families consist of highly immunogenic lipoproteins (131) and the presence of such

elaborate systems in the context of a small genome further highlights the importance of surface

variability for survival within the host, although the precise functions seldom are understood (101,

104). As more full genome sequences become available, the knowledge of mycoplasma variable

gene families expands (38-40, 42, 52, 53), which will be a helpful tool in prevention of disease

caused by these organisms.

The first vsp of M. mycoides SC, Vmm, was reported in 2002 (99). It is a 16 kDa phase variable

lipoprotein, regulated by the number of TA repeats in the promoter spacer region and parts of the

Pribnow box. Vmm-like genes were identified in M. capricolum subsp. capricolum, M. capricolum

subsp. capripneumoniae, M. sp. bovine serogroup 7, and M. putrefaciens (99). The corresponding

genes in M. capricolum subsp. capricolum have since the completion of the genome sequence been

characterized and expression of these gene products (VmcA-F, variable M. capricolum subsp.

capricolum) is phase variable in analogy to vmm but repeating domains within several Vmcs indicate

size-variation as well (42). At the completion of the genome sequence for M. mycoides SC (39), five


16

additional genes encoding lipoproteins with similar promoter spacers as vmm were found (Table

3.1). In addition, homonucleotide regions of A:s were found in the putative promoter of nine

surface proteins and may govern phase variable expression of these proteins in similar fashion.

The variability of a putative glucose phosphotransferase system permease (ptsG) in M. mycoides SC

was reported in 2004 (106). The variability was discovered by colony immunostaining with the

mAb 3F3 (134) and the variability was governed by a one base substitution leading to truncation

of PtsG upstream of the 3F3 epitope (106).

ORF Promoter repeat Lipobox Comment MSC_0117 poly(TA) VVA C

MSC_0364 poly(TA) TAS C

MSC_0390 poly(TA) VVA C vmm

MSC_1005 poly(TA) VIA C

MSC_1033 poly(TA) VIA C

MSC_0809 poly(A) -

MSC_0810 poly(A) -

MSC_0812 poly(A) -

MSC_0813 poly(A) -

MSC_0815 poly(A) -

MSC_0816 poly(A) -

MSC_0817 poly(A) -

MSC_0818 poly(A) -

MSC_0847 poly(A) TVS C

MSC_0860 - - ptsG

Table 3.1 Variable surface proteins of M. mycoides SC. MSC_0390 and MSC_0860 have been experimentally verified, the remaining are putative variable surface proteins identified in the genome sequence (39, 99, 106).

CARL HAMSTEN 2009

17

4 Past and present prevention of CBPP

Since many countries have successfully eradicated CBPP, there are obviously methods available.

Slaughter of the affected animals, quarantine and strict control of animal movements as well as

crude vaccination were factors in the successful eradication efforts of the early 20th century (USA,

Japan, parts of Western Europe) (7, 17). These methods were refined and used in the eradication

of CBPP in Australia by 1973 (5). The resurgence of CBPP in southern Europe during the 1980s

and 1990s was controlled by efficient disease monitoring and slaughtering of infected herds (7, 8).

This can in part be attributed to successful European collaborative efforts on CBPP diagnosis and

control initiated during the 1990s.

Even though slaughtering of infected herds still is the most effective way to eradicate CBPP (135),

these “stamping-out” regimens cannot be readily applied in Africa. Epidemiological factors such

as transhumance, nomadism and trekking of trade cattle present unique challenges that are

aggravated by problems deploying control measures due to economical factors, political instability

and lack of movement controls (4). In the end, African countries with endemic CBPP cannot

afford eradication by slaughtering of all infected herds (5, 14, 136). This was demonstrated by a

stamping-out eradication of CBPP in Botswana during 1996, which led to negative effects on

short-term economics and increased malnutrition in children (135, 137). Extensive vaccination

programs and chemotherapy are the remaining options for CBPP control in Africa and of these,

vaccination still is the preferred method (14, 138).

4.1 Attenuated live strain vaccines

The first crude vaccine against CBPP was published in 1852 by Louis Willems and consisted of

pleural exudates from diseased animals (7). Crude vaccination using pleural exudate or lung tissue,

placed under the skin at the nose or the tip of the tail, had been used long before that in Africa

and is still in use (16). This can confer some protection (17) but is discouraged as it often gives

severe adverse reactions at the insertion point. This was named the Willems’ reaction, which often

led to loss of the tail and even caused a French physician to believe he had discovered a three-

horned cattle species in 1880 (16).


18

Since then, different attenuated live M. mycoides SC strain vaccines have been used and the vaccines

currently recommended by the OIE are the live strain vaccines T1/44 and its streptomycin-

resistant derivate T1sr (139). T1/44 was semi-attenuated by 44 passages in eggs and has been in

constant use for nearly 60 years after its isolation in Tanzania (7). Although both T1/44 and T1sr

confer some level of protection, there are many limitations. Willems’ reactions are common, seen

in up to 25% of vaccinated Bos taurus (4). These gross lesions can cause loss of the tail and

occasional deaths, which are seen as accepted side effects of vaccination or even essential for

immunity (140). This indicates that T1/44 has retained significant virulence, which was confirmed

by cases of CBPP following endobronchial inoculation of the vaccine (141). Other limitations are

short-term immunity demanding yearly vaccinations, cold-chain dependence and long time for

primo-vaccinated animals to acquire immunity (7, 8, 142-144). Doubts about the efficacy of the

T1 vaccines were raised after failed campaigns in the 1990s (8) and the use of T1sr was even

discouraged (144). A vaccine trial comparing T1/44 and T1sr conducted in three African

countries revealed a protection rate of 33 to 67% after three months, depending on the challenge

strain (143). Since then, changes in the composition of the live vaccines have been suggested as

simple and inexpensive ways to improve efficacy (138), but it is apparent that novel vaccines are

needed. The status in development of novel (non live strain) vaccines is given in chapter 7.

4.2 Chemotherapy

The use of antibiotics in CBPP treatment is a controversial issue due to concerns of creating silent

carriers of the disease when only clinical signs are relieved (8, 145). Even though the practice is

forbidden in many countries, antibiotics are routinely administered (146, 147). The topic has been

discussed at recent expert meetings organized by the Food and Agriculture Organization of the

United Nations (FAO; 1998, 2003 and 2006), concluding that further studies are needed to

evaluate antibiotics as a CBPP control measure (148-150). Since mycoplasmas have no cell wall

traditional antibiotics targeting this structure such as penicillins have no function. In vitro studies of

minimal inhibitory concentrations have shown that newer antibiotics targeting protein or nucleic

acid synthesis are effective (151, 152) and recent in vivo studies have shown a reduction in CBPP

transmission after oxytetracycline (153) and danofloxacin treatment (147).

CARL HAMSTEN 2009

19

5 Diagnosis of CBPP

In this chapter, the methods available for diagnosis of CBPP will be reviewed. It will focus on

serological diagnostic methods that are currently approved by the OIE, but also give a brief

summary of recent methods that have not been approved for use as of now.

5.1 Clinical, pathological and culture based methods

Clinical diagnosis of CBPP is hard as initial signs may be slight or non-existent and are

indistinguishable from any severe pneumonia with pleuritis (2, 139). As discussed earlier, antibiotic

treatment may also relieve clinical signs (8, 145). In practice, clinical diagnosis is unreliable and

when respiratory disease occurs within a livestock population, CBPP should be investigated by

pathological, microbiological, molecular or serological diagnosis. Since the pathological lesions of

CBPP are distinctive (see chapter 1.3), abattoir surveillance of CBPP involving lung examinations

is a practical method for disease monitoring (139).

No matter which diagnosis methods are used, it is always preferential to isolate the causative

organism (as with any infectious disease). M. mycoides SC can be isolated from live animal samples

(nasal swabs bronco-alveolar lavage, blood, pleural fluid) or a post-mortem examination (lung,

pleural fluid, lymph nodes etc). The presence of M. mycoides SC varies with the stage of disease and

it may be difficult to isolate from chronic lesions, especially if antibiotic treatment is involved

(139). Hence, a negative isolation result is not conclusive (154). Unlike many other mycoplasma,

M. mycoides SC is relatively easy to grow given the right media and can subsequently be identified

by growth inhibition tests, biochemical tests, monoclonal antibodies (mAbs) and/or

immunofluorescence based methods (8, 33, 154-157). However, closely related mycoplasma

species may cross-react in these tests.

5.2 PCR-based molecular identification

Although the present work is focused on serological diagnosis, a brief description of available

nucleic acid based diagnostic methods will be given as they are equally important. These are

mainly used for final identification of M. mycoides SC when the organism has been grown and

isolated from a clinical sample.


20

Polymerase chain reaction (PCR) is a sensitive, specific, rapid and relatively easy method to

perform. The first PCR test for M. mycoides SC identification was reported in 1994 (158). It

required digestion of one of the amplicons followed by separation of the corresponding fragments

for specific identification of M. mycoides SC. It was refined to use colorimetric detection of the

amplicons in 1999, but still required separate analysis of the digested amplicon (159). A PCR

directly specific for M. mycoides SC was reported in 1994 which used restriction of the amplicon or

dot blot hybridization of the amplicon to a labelled probe to confirm and/or enhance the

specificity (160). A nested PCR using primers amplifying all members of the classical M. mycoides

cluster in the first reaction, followed by a species-specific amplification was presented in 1996

(161). Another nested PCR, targeting the p72 (lppA) gene was presented in 1997 and was shown

to have 103-105 fold higher sensitivity compared to single step PCR using the same primers or

primers from previously mentioned methods (162). Two methods based on PCR amplification of

the two 16S rRNA genes from mycoplasmas in the M. mycoides cluster were presented in 1999,

which differed in the specific detection of M. mycoides SC (163). In the PCR-laser induced

fluorescence method a M. mycoides SC-specific length difference of two nucleotides in one

amplicon was detected by gel electrophoresis in a DNA sequencer. The other method was based

on digestion of one amplicon generating a M. mycoides SC specific band. A PCR allowing specific

detection of the T1 strain vaccines, based on an unique IS1296 element in those strains, was

reported in 2000 to help distinguish CBPP outbreaks among vaccinated cattle (164).

Even though it is a quick, sensitive and specific method, PCR has problems with the high risk of

cross-contamination and carryover-contamination, especially with nested PCR (163, 165). This

requires strict separation of laboratory space used for PCR preparation and space possibly

contaminated with PCR products such as electrophoresis facilities. Nonetheless, PCR has become

the primary tool for identification of M. mycoides SC (154). Recent reports suggesting that single

PCR tests are inadequate for diagnosis of CBPP (166, 167) has led to the development of real-

time PCR assays. These have been suggested to reduce the risk of contamination and several

assays have been described (165, 168, 169), the most recent reporting a 2-3 log increase in

sensitivity compared to a conventional PCR-based detection of M. mycoides SC (165).

5.3 Serological diagnosis of CBPP – currently approved methods

In general, CBPP diagnostic systems based on serology are not sensitive enough (170) and cross-

reactions with closely related species are frequent (171). Additionally, diagnosing chronic carriers

CARL HAMSTEN 2009

21

of CBPP is problematic. As a consequence, serological diagnosis of CBPP is valid on a herd level

only since the individual animal can be in an early stage of disease prior to generation of specific

antibodies or in a late chronic stage when few animals remain seropositive (139). Despite these

shortcomings, serological diagnosis is better suited for large scale disease monitoring, especially in

Africa where PCR-based methods are not always practical (8).

The currently recommended and most widely used serological test is the complement fixation test

(CFT) developed by Campbell and Turner in 1953 (172):

The CFT test procedure is as follows (in brief):

Serum samples are diluted 1/10 and mixed with a suspension of whole cell M. mycoides SC as

antigen in a normal microplate. Complement from normal guinea-pig serum is added followed by

30 min of mixing at 37oC. Then a haemolytic system consisting of sheep red blood cells (SRBC)

and rabbit hyperimmune serum to SRBCs is added to the samples and incubated for another 30

min at 37oC. After brief centrifugation, results are read as percentage of observed complement

fixation. A positive result corresponds to 100% inhibition of haemolysis at 1/10 and results down

to 25% are doubtful. It is recommended that any observed complement fixation should be

followed by additional investigations.

In other words, if a serum sample contains antibodies to M. mycoides SC those will bind the antigen

and activate the complement, which in turn will be consumed during lysis of the M. mycoides SC

cells. When the haemolytic system is added, rabbit antibodies will bind the SRBCs but there will

be no complement left and thus no haemolysis will be observed. Although the above description

seems simple enough, the method is dependant on accurately titered amounts of antigen,

complement and hyperimmune sera as well as fresh SRBCs and good positive and negative

control sera. The method is hard to standardize due to these dependencies. Although the CFT has

been harmonized in the European union (173), it is still considered difficult to perform and

requires skilled personnel (139, 174).

The CFT has been proven to be specific, but lacks in sensitivity. Bellini et al evaluated the

performance of the CFT in 1998 using sera colleted during outbreaks of CBPP in the early 1990s

(170). Sera from almost 34 000 cattle in healthy herds were used to determine the specificity to

98% and nearly 600 sera from cattle in infected herds were used to determine the sensitivity to

64% (170). As a comparison, isolation of M. mycoides SC from affected animals was shown to have


22

an even lower sensitivity of 54% in the same study. Sensitivity of the CFT is lowered after the

acute phase of CBPP and the test is considered insensitive three months after infection (95, 174).

This has a potential advantage as unfavourable weakly positive CFT titers seen following

vaccination using the common live strain vaccines also revert to negativity within three months

(174). Even though the CFT is thought to only detect a small proportion of animals in early stages

of the disease or chronic carriers, it is still considered to detect almost 100% of infected herds

(139).

In an effort to improve serological diagnosis of CBPP, a competitive (cELISA) using a mAb

targeting a then unknown protein was developed in 1998 (174). It was later shown that the

antibody targets the same epitope as 3F3, the variably expressed PtsG surface protein (106).

The cELISA test procedure is as follows (in brief):

An ELISA plate is coated with lysed M. mycoides SC over night. After washing, serum samples

diluted 1/10 and mAb are added and incubated for one hour at 37oC. The plate is then washed

and a HRP-conjugated secondary antibody targeting the mAb is added and incubated for an

additional hour at 37oC. After washing, a colourimetic substrate is added and the resulting

absorbance at 405 nm is noted when a control sample reach a predetermined absorbance. The

cut-off for CBPP positivity is set to 50% competition compared to a CBPP positive control.

In other words, a CBPP positive sample will reduce binding of the mAb and thus the subsequent

colour reaction will be reduced. The cELISA offers practical advantages and ease of

standardization but has a lower sensitivity in detecting acute infections compared to the CFT

(90% of CFT) (175). On the other hand, it is thought to have a better potential to detect chronic

infections (175).

The antibody classes these methods primarily detect can in part explain the differences in CBPP

detection over time. In the initial stages of infection, immunoglobulin M (IgM) responses

dominate and are characterized by lesser affinity (in general, compared to following

immunoglobulin G (IgG) responses) but higher complement fixating ability (176). This is in

favour of the CFT, relying on complement activation, over the cELISA relying on affinity to

reduce mAb binding. But as infection progress, IgM responses switch to IgG responses that have

comparably higher affinities but less complement fixating abilities. This favours the cELISA and

responses have indeed been detected longer than CFT titers (177).

CARL HAMSTEN 2009

23

5.4 Newly developed serological diagnostic methods

An indirect ELISA based on the N-terminal half of LppQ (93) was published in 2002 (95). The

N-terminal was chosen as it is surface exposed and induces a strong and specific early immune

response to M. mycoides SC in infected cattle (93-95). A recombinant protein comprising amino

acids 22-218 was tested for cross-reactivity to other mycoplasma species by Western blot and

subsequently used to create the assay. The LppQ-ELISA was pre-validated for sensitivity and

specificity using sera from 221 cattle of ten herds from Portugal, which had already been

diagnosed for CBPP by post mortem examinations, M. mycoides SC culture and CFT titers (95).

The specificity of the LppQ-ELSA seemed similar to the cELISA (174) when compared to the

CFT (about 90% in comparison). The sensitivity was also in the same range as the cELISA, which

has a good match to the CFT (95). The LppQ-ELISA was less sensitive in the early phase of

infection but gave positive results during a longer time period post-infection compared to the

CFT (95), as seen previously with the cELISA (174). Again, this was probably due to the fact that

the ELISAs measure IgG titers and the CFT IgM titers (95, 174). The LppQ-ELISA is currently

under evaluation by the IAEA (154).

A rapid latex agglutination test (LAT), giving results in two minutes using serum or whole blood,

for screening in the field was reported in 1999 (178). Slide agglutination tests had previously been

described, but were not recommended due to false-positive reactions (see (179)). The LAT used a

specific polysaccharide antigen extracted from the M. mycoides SC capsule which was bound to

latex beads (8). The test has been evaluated on European and African sera and the sensitivity was

comparable to the CFT while the LAT was simpler and more rapid to perform (8). Another LAT,

using latex beads coated with a polyclonal antibody (pAb) specific for the capsular polysaccharide

(CPS) was reported in 2003 (179). Adverse reactions were observed to M. mycoides capri and the

diagnostic correlation to the CFT was 62% at most when testing African field sera (179). The

main discrepancy was due to reduced sensitivity of the LAT compared to the CFT, but it was

argued that a high level of correlation was unlikely since the CFT detects serological responses

and the LAT detects presence of the antigen. Both are unlikely to coexist at a high level in the

animal (179). When testing known CBPP negative sera of UK origin, only 3% false positives were

detected (179). In general, LATs allow simple, rapid and inexpensive disease monitoring at the

herd level in the field, which is very suitable for the CBPP situation in Africa. Although none of

the LATs for CBPP diagnosis have been approved by the OIE, a product based on the first

mentioned LAT is on the market.


24

A diagnostic test based on western blotting named the immunoblotting test (IBT) has also been

developed and is of diagnostic value (154). The strength of this method is the analysis of humoral

immune responses in relation to the electrophoretic profile of M. mycoides SC antigens,

overcoming problems related to non-specific binding in other assays (154, 173). The test depends

on a common immunological pattern consisting of five immunodominant antigens with apparent

molecular weights of 110 (p110), 98 (p98), 95 (p95), 62/60 (p62/60) and 48 (p48) kDa, as

described in (180, 181). A field evaluation of the IBT found the sensitivity to be 92,6% compared

to 77,5% for the CFT and the specificity was 100% with no false positives (182). The IBT is

highly specific and the most sensitive serological test so far described for CBPP (173), but it is not

suitable for mass screening and should primarily be used as a confirmatory test after other tests

and all cases in which false positive results in CFT are suspected (154).

In the last sections five serological diagnosis methods presented in the last decade have been

presented along with the golden standard, the CFT. These are summarized and compared in table

5.1. In this simplified comparison, the IBT is the best test combining the highest specificity with

high sensitivity. It is however not suitable for mass screening as it is a fairly low throughput

method. It should be used in combination with a rapid, high throughput test, which in theory

could be any of the other tests. The LATs are arguably the best pen side tests for use in the field,

but a higher sensitivity and specificity than the second LAT (pAb) is preferable. Both ELISAs are

comparable to the CFT, but do not perform better. There is still a need for a serological test

surpassing the CFT in terms of sensitivity and specificity, but in a format more convenient than

the IBT.

CFT cELISA LppQ-ELISA LAT (CPS) LAT (pAb) IBT

Antigen Whole cell Lysate Recomb. LppQ CPS pAb detect. CPS Lysate

Ig class detected IgM IgG IgG IgM, IgG, IgA - IgG

Sensitivity (%) 64 96a 79a N/Ab 62a 92,6

Specificity (%) 98 97a 97a N/Ab 62a 100

Table 5.1 A summary of approved and recently developed serological diagnostic methods for CBPP. aIn comparison to CFT (95, 174, 179), breported as comparable to CFT (173). Additional data derived from (170, 173, 182).

CARL HAMSTEN 2009

25

6 Research on CBPP-specific immune responses

The basis of immune protection against CBPP is still not completely understood. A few early

studies focused mainly on humoral responses, but the importance of cell-mediated responses was

also acknowledged, although never characterized (144, 183). The humoral immune response has

been studied in the sense of identifying immunodominant proteins (67, 72, 90-93), as described in

chapters 3.2-3. The humoral immune responses have also been monitored over time under

experimental conditions by western blotting, reporting strong IgA responses to surface exposed

lipoproteins (67). An attempt to further characterize the humoral immune response in

experimentally infected cattle was reported in 2006 (177). Humoral responses were monitored

over a year following CBPP exposure and no correlation between antibody titers and clinical signs

or lung lesions could be found. Analyses of M. mycoides SC-specific antibody isotype responses

were done at the pulmonary (local) and systemic levels for animals with acute and sub-acute to

chronic infections. The most interesting result was higher specific IgA titers, both local and

systemic, in animals with sub-acute to chronic infections. A predominance of the local over the

systemic IgA response in these animals prompted the conclusion that locally produced IgA may

play a role in protection against CBPP (177). IgM, IgG1 and IgG2 responses were also compared,

but were similar among the groups and showed no correlation to disease severity (177).

6.1 Is interferon- production associated to CBPP recovery?

Studies on the role of cell-mediated immune responses in pathogenesis and control of M. mycoides

SC infections were reported for the first time in 2005 by Dedieu et al (184). Their first conclusion

was that no superantigen-like molecules were present in M. mycoides SC, which had been seen in

other mycoplasmas (185), implying that any cell-mediated responses are induced specifically. The

main conclusion of the study was that animals recovering from CBPP had persistent CD4+ TH1-

like T cell responses producing IFN- until the end of the monitoring, in contrast to animals

developing acute CBPP. The latter animals had a decreased IFN- production associated to

progression of the disease, thus a correlation between IFN- production and resistance to M.

mycoides SC infection was concluded (184). This indicated that the outcome of M. mycoides SC

infection is dependent on the type of immune response raised, as IFN- promotes TH1 responses

and inhibits TH2 responses (23).


26

A short summary of TH1 and TH2 responses:

The TH1/TH2 paradigm was first described for murine T helper lymphocytes in 1986 (186) and

presently the functions of TH0, TH1, TH2 and additional TH cells have been described. The

different types of TH cells are defined by their cytokine secretion (and thus their induced

responses) and the division of TH0, TH1 and TH2 responses is central to understanding the

generation of immune responses. TH0 cells are uncommitted and mature to either TH1 or TH2

cells, that once activated stimulate their own growth and inhibit development of the other type.

TH1 cells are characterized by secretion of interleukin-2 (IL-2), IFN- and tumour necrosis factor

. These cytokines stimulate inflammatory responses and production of IgM and IgG subclasses

that bind Fc receptors on neutrophils and natural killer (NK) cells and can fix complement. TH1

responses usually occur first and reinforce local responses (inflammation, delayed-type

hypertension responses via activation of macrophages, NK cells and CD8 cytotoxic T cells). TH1

responses are important for eliminating intracellular pathogens (viruses, bacteria, parasites) and

fungi but have also been associated to cell-mediated autoimmune diseases in humans. TH2

responses occur later and act systemically. The TH2 response occurs in absence of IL-2/INF-

signals and is reinforced by IL-4. It can be stimulated at a later stage of infection when antigen

reaches the lymph nodes and promotes humoral (systemic) immune responses by inducing a

switch to specific subtypes of IgG, IgA or IgE. TH2 responses can limit inflammatory and

autoimmune diseases but exacerbate intracellular infections by inhibiting TH1 responses. Above

description adapted from (23).

However, the TH1/TH2 paradigm is a simplification of a highly complex regulatory network,

which is demonstrated in bovines where polarized cytokine profiles rarely are observed (187) and

only consistently in chronic infections (188). Instead, co-expression of IL-4 and IFN- is often

observed and other cytokines (IL-2, IL-10 etc) also lack TH1/TH2 coordination (187). As a

consequence, the ratios of IL-4 and IFN- amounts are used to determine TH1 versus TH2 type

responses in bovines (187, 189).

CARL HAMSTEN 2009

27

The results of an experimental CBPP infection focusing on cell-mediated responses in lymph

nodes were reported in 2006 (190). Similar to earlier studies, a persistent M. mycoides SC specific

response mediated by IFN- secreting CD4+ T cells was found in lymph nodes of all recovering

cattle (190). These responses were also significantly higher in completely recovering animals than

in recovering animals with lung sequestra. As recovering animals are immune from further CBPP

infections (17), these IFN- producing CD4+ T cells that homed to the lymph nodes were

postulated as memory T cells responsible for protective anamnestic responses in these animals

(190). An expanded analysis of cell-mediated responses in lymph nodes taken during this

experimental infection was reported in 2008. Here, lymphocytes were analyzed in vitro to search

for direct evidence of proliferation, cytokine production and expression of activation and memory

markers in response to M. mycoides SC (191). Proliferation of CD4+ T cells and B cells when

stimulated by heat-killed M. mycoides SC was shown in cultures from animals with chronic CBPP

and not animals with acute CBPP. B cells dominated the initial proliferation, but at the sixth day

of stimulation, CD4+ T cells dominated the cultures. Initial depletion of CD4+ T cells inhibited B

cell proliferation, proving a CD4+ T cell dependent proliferation of the B cells (191). As in

previous studies, IFN- secretion was detected in cultures from animals with chronic CBPP and

here the majority of IFN- was shown originate from CD4+ T cells, even if secretion was shown

for other subsets as well (191). Finally, ratios of IFN- and IL-4 secretion were used to determine

the TH type of response. No or little IL-4 in combination with the IFN- secretion led to a

reaffirmed TH1 bias (191). The study concludes that TH1 memory lymphocytes are involved in the

control of CBPP and propose the following putative immune mechanisms: i) Production of IFN-

which potentiates phagocytic and bacteriocidal activities in macrophages as well as stimulates

opsonization by bovine neutrophiles via its IgG2 promoting effect and ii) CD4+ T cell dependent

activation of B cells (191).

The above-mentioned series of publications all point to the importance of a cell mediated

response as measured by high IFN- secretion in animals surviving CBPP. Nonetheless, there

have been recent reports questioning the role of IFN- secreting CD4+ T cells in immunity (136,

192, 193). In 2007, Scacchia et al reported the use of Cyclosporine A (CsA) to affect the

pathogenesis of CBPP (193). CsA is a fungal metabolite with immunosuppressive properties that

inhibit the transcription of genes encoding IL-2 and the IL-2 receptor, which are essential for T

cell activation (176). In the experiment, CsA delayed the clinical, pathological, humoral and cell-

mediated responses as compared to untreated controls, supporting the thesis that an

immunopathological process is part of the CBPP pathogenesis (193). Correlations of IFN-


28

release and severity of disease were not as evident as in previous studies. An apparent correlation

between chronic lesions and IFN- release was found, but the authors concluded that it is

debatable whether the animals with extensive chronic lesions can be defined as recovering (193).

Last year, Jores et al reported the results of a study assessing IFN- release upon stimulation in

peripheral blood mononuclear cells (PBMCs) collected from experimentally infected cattle (136).

No correlations between IFN- release and presence or absence of pathological lesions were

found; hence the data did not support previous studies (136). Immunocytochemistry was also

performed to investigate the involvement of different cell populations in lung lesions and revealed

an increased prevalence of myeloid cells (monocytes, granulocytes, macrophages etc) but no

increase in potential IFN- secreting cells (lymphocytes) (136).

To conclude, the role of IFN- release and CD4+ T cells in pathogenesis or control of CBPP has

to be further investigated as above-mentioned studies prove associations and correlations only.

One possible route is via in vivo depletion of T cell subpopulations (194). Nonetheless, the results

on IFN- release and CD4+ T cells should be taken into account in development of future CBPP

vaccines.

6.2 Can M. mycoides SC modulate the immune response?

There have been a few in vitro studies investigating if M. mycoides SC cells affect bovine PBMCs or

lymph node cells. In 2005, apoptotic effects of live M. mycoides SC on PBMCs were demonstrated

(195). Here, viable M. mycoides SC cells induced strong morphological changes (reduced cell size,

increased granularity) not seen with heat-killed M. mycoides SC, leading to apoptosis in all

lymphocyte subsets (195). The same effect, although less, was triggered by mycoplasma free

culture supernatants suggesting the involvement of M. mycoides SC secreted components. This

study was followed by an investigation if M. mycoides SC cells affect the response in PBMCs and

lymph node cells triggered by Concanavalin A (ConA) (196). ConA is a polyclonal mitogen

isolated from Jack bean that induce unspecific mitosis in mainly T lymphocytes (197). The effects

of ConA on PBMCs and lymph node cells from naïve cattle, measured by blastogenesis and IFN-

production, was depressed by the presence of viable but not heat-killed M. mycoides SC cells

(196). Significant effects were seen both in B and T cell populations, with the highest impact on

CD4+ T cells (196). This was in line with a study from 1973 reporting unresponsiveness to

another mitogen, phytohemagglutinin (PHA), in PBMCs taken from experimentally infected cattle

and lower responsiveness in PBMCs from T1-vaccinated cattle compared to control or recovered

CARL HAMSTEN 2009

29

cattle (183). Suppression of PHA induced responses has also been demonstrated for Mycoplasma

bovis (198), another bovine respiratory mycoplasma, which also expresses a peptide able to inhibit

ConA-triggered proliferation of bovine lymphocytes (199). Immune cell modulation has also been

demonstrated for several other mycoplasmas including Mycoplasma pulmonis, Mycoplasmas hyorhinis,

Mycoplasma hyopneumoniae, Mycoplasma fermentans, Mycoplasma salivarum and Mycoplasma pneumoniae

(200-204). Further studies on possible immunomodulation by M. mycoides SC are needed, especially

to determine if effects seen in vitro persist in vivo. Nonetheless, immunosuppressive effects may be

a reason for the short-term protection offered by live strain vaccines and need also to be

considered in future vaccine developments.


30

7 Developments towards non-live strain vaccines

There are several disadvantages with the current live strain vaccines for CBPP, as detailed in

chapter 4.1. Using inactivated or subunit vaccines instead of live culture vaccines would solve

many of these problems. Efforts in this field during the last decade are summarized in this chapter

to give an overview of the current situation in CBPP vaccine research.

7.1 ISCOM vaccines

A vaccine based on immunostimulating complexes (ISCOMs) was first evaluated in mice and

cattle in 1997 using detergent-solubilized M. mycoides SC cells to create a subunit vaccine with a

high number of antigens (205). A strong primary antibody response was generated in mice

yielding high IgG2a levels, up-regulated by IL-2 and IFN revealing that TH1 type responses were

induced, which was in line with previous ISCOM vaccine studies (205). In cattle, strong primary

and long lasting secondary antibody responses in similar magnitudes to naturally infected animals

were detected as well as an induced T cell response (205). The result of a field trial with this

vaccine, examining the outcome of infection in vaccinated and non-vaccinated animals was

published in 2003 (192). After vaccination, antibodies to M. mycoides SC were detected by ELISA

but the CFT test was negative. Following infection, animals vaccinated with the ISCOM vaccine

had reduced clinical symptoms and mortality compared to unvaccinated controls, but

histopathological scores were equally high in both groups (192). Based on these results, the

vaccine was not considered to induce a protective response. No difference in the formation of

sequesters was evident between the groups and immune reactions in all animals pointed to a TH1

type response, thus the likely involvement of TH1 responses in the pathogenesis of CBPP was

concluded (192). The results of a vaccine trial using a ISCOM formulation of recombinant LppQ

(93) was published in 2004 (96). The vaccine did not offer any protection to CBPP challenge in

the small group of animals used and even appeared to exacerbate the pathological lesions as

compared to unvaccinated control animals (93). Even though these trials with ISCOM

formulations have been unsuccessful, ISCOMs have been used in other veterinary vaccines and

are now included in commercial vaccines (206). The failures can be due to the M. mycoides SC

components used in these formulations and using ISCOMs is still a viable approach for mucosal

delivery of a CBPP subunit vaccine, as recently suggested (207).

CARL HAMSTEN 2009

31

7.2 Other vaccine formulations

A whole-cell M. mycoides SC vaccine inactivated with saponin was evaluated concurrently with the

LppQ ISCOM vaccine, as previously tried inactivated vaccines had been successful (96). Saponin

inactivation had previously been successful for a Mycoplasma bovis vaccine (26) but as with the

LppQ vaccine, the saponin inactivated vaccine conferred no protection and pathological lesions

appeared exacerbated compared to untreated controls (96). Another subunit vaccine based on the

capsular polysaccharide (CPS) component of M. mycoides SC was tested in a mouse model in 2002

(208). CPS is a poor immunogen and hence it was tested with different conjugations whereof only

an ovalbumin conjugation generated a significant antibody response to CPS (208). Mice given a

whole-cell disrupted vaccine were immune against challenge with live M. mycoides SC and exhibited

a reduced degree of mycoplasmaemia compared to untreated controls, which was unfortunately

not seen with the CPS-ovalbumin conjugate (208). However, results from the mycoplasmaemia

mouse model are not automatically comparable to clinical disease in cattle (140).

7.3 A DNA vaccine for CBPP?

In the context of CBPP vaccine development, a novel approach for testing DNA vaccines by

bacteriophage library screening was reported in 2006 (209). In this study, a whole genome

library of M. mycoides SC was cloned in to a bacteriophage ZAP express vector containing both

bacterial (Plac) and eukaryotic (PCMV) promoters upstream of the insert, plated on E. coli cells,

blotted and probed with convalescent-phase antiserum to identify vaccine candidates (209). Two

clones expressing a total of three different proteins were identified and the vaccine potential was

evaluated using the whole bacteriophages for immunization of mice, followed by challenge via M.

mycoides SC induced mycoplasmaemia (209). Significant responses to M. mycoides SC were observed

following vaccination with both clones and a reduced mycoplasmaemia was observed for one

clone (expressing the MSC_0397 protein). Thus, a protective effect was seen following

bacteriophage mediated DNA vaccination of MSC_0397 in mice (209). However, studies in

cattle are needed to confirm these results and have not been published yet. Even so, this method

offers potential for the development of a DNA vaccine that has the advantages of being cheap,

stable and easy to produce (140, 209, 210) but might be more controversial than traditional

vaccines.


32

PRESENT INVESTIGATION

8 Aims

The overall aim of the work presented in this thesis was to find novel protein markers to be used

in development of improved diagnostic methods and vaccines for the control and elimination of

contagious bovine pleuropneumonia. These markers were to be selected from recombinantly

expressed surface proteins from the causative organism M. mycoides SC. The strategy has the

potential to create more specific diagnostic tools and safer vaccines in comparison to live strain

vaccine strategies. To fulfil this goal, the initial aim was to create methods for expression and

analysis of the recombinant surface proteins. A second aim was to clone and express a large set of

proteins and then suitable candidate proteins were to be selected by measuring humoral immune

responses to the corresponding native proteins in sera from CBPP diseased cattle and controls.

This required the development of a suitable analysis method as a third aim. A final aim was to

select and perform initial evaluation of candidate proteins in diagnostic and vaccine applications.

In the following chapters the work included in the thesis will be presented. Each chapter will

summarize the rationale and results of a paper included in the thesis. Paper I describes a pilot

study on six putative variable surface proteins of M. mycoides SC, expressed in E. coli and studied

using immunoblotting methods. In Paper II, a method for the monitoring of IgG, IgA and IgM

titers to 64 recombinant surface proteins using a bead based suspension array assay is presented.

This assay was used to select proteins to be used in a diagnostic ELISA, presented in Paper III.

The assay was also used to evaluate a CBPP vaccine candidate based on the proteins from Paper I,

presented in Paper IV.

CARL HAMSTEN 2009

33

9 Investigating putative variable surface proteins (Paper I)

9.1 Rationale

Many mycoplasmas express variable surface proteins, as described in chapter 3.3. Only two had

been characterized in M. mycoides SC prior to this study, Vmm and PtsG (99, 106), but 14 putative

proteins with similar promoter structure to Vmm had been identified in the genome sequence

((39), table 3.1). The primary aims of this study were to investigate selected Vmm-type proteins,

and Vmm itself in reference, to detect whether humoral immune responses had been raised

against these in vivo, if they were expressed in vitro and if the in vitro expression was variable. A

second aim of this study was to develop a scheme for production of recombinant M. mycoides SC

proteins to prove the feasibility of the larger study presented in Paper II.

9.2 Results

Five of the 14 Vmm-type proteins, selected by low similarity to each other, and Vmm itself were

investigated in this study. In silico analysis identified a signal peptide for each protein (SignalP,

(211, 212)), no transmembrane regions (TMHMM, (213, 214)) and predicted all but one to be

lipoproteins (LipoP, (215)). Recombinants were designed on the full-length proteins, only

excluding the signal peptide, to get the best representation of the native protein and were fused to

an N-terminal His6ABP tag. The hexahistidine tag enabled IMAC purification as previously

described (216) while the ABP (217) was used to enhance solubility and for its

immunopotentiating properties (218, 219). A commercial kit was used to change the tryptophan

coding TGA codons in mycoplasma to TGG and all six proteins were successfully expressed in E.

coli with yields of 0.5-4.9 mg protein from 100 ml culture.

In order to analyse whether the corresponding native Vmm-type proteins were expressed in M.

Mycoides SC during infection, the recombinant proteins were subjected to dot and Western blotting

against 15 bovine sera from four CBPP outbreaks, as well as five sera from healthy Swedish cattle

and fetal bovine serum as negative controls. All sera from CBPP-affected cattle contained

antibodies binding some or all of the Vmm-type proteins (or their homologues), generating

different blot patterns for the individual animals. However, it was hard to quantify the IgG titers


34

to the different proteins using these blotting methods thus only relative titers within each blot

were estimated visually.

With the conclusion that the Vmm-type proteins or their homologues were expressed in natural

infections and a humoral immune response was raised against them, protein expression in vitro was

also examined. Colony blots of M. mycoides SC strains PG1 and M223/90 (220) were analysed

using affinity purified antibodies raised to the five recombinant Vmm-type proteins (221) and the

5G1 mAb (99, 134) against Vmm as a control. In these experiments expression of MSC_0364, as

detected by antibody A346, was concluded to be variable (see Figure 9.1). The positive control

experiment using mAb 5G1 to detect the variable expression of Vmm was successful, while the

other antibodies/proteins gave inconclusive or negative results.

Figure 9.1 Colony blotting of M. mycoides SC strain M223/90 showing using antibody A364 to visualize variable expression of surface protein MSC_0364 within and between colonies. The black colour is specific antibody staining and indicates expression of MSC_0364 and the grey colour is Ponceau S staining of all proteins, showing all colonies.

Western blots of M. mycoides SC lysates detected with the polyclonal antibodies were used as a

complement to colony blotting, supporting the results for MSC_0364 but were also inconclusive

or negative for the remaining proteins. Hence, only in vitro expression of proteins MSC_0364 and

Vmm was conclusively detected. However, expression of all six corresponding genes was detected

CARL HAMSTEN 2009

35

on an mRNA level by RT PCR using total RNA extracted from a PG1 culture. This discrepancy

could be due to differences in sensitivity between the assays. Nonetheless, in vitro expression of

MSC_0117, MSC_0816, MSC_0847 and MSC_1033 is still uncertain.

9.3 Conclusions

This study presented a scheme for expression of recombinant M. mycoides SC proteins in E. coli

with sufficient yields of protein from 100 ml culture to perform blotting experiments as well as

generate and purify polyclonal antibodies. Furthermore, the recombinant proteins could be used

to detect humoral immune responses to the corresponding native proteins generated in CBPP–

affected cattle. The five putative Vmm-type proteins or their homologues were shown to be

expressed in vivo during M. mycoides SC infection and variable expression in vitro was demonstrated

for MSC_0364. More importantly to us, these results proved the feasibility of expressing a larger

selection of M. mycoides SC surface proteins that could be used for proteins-specific analysis of

humoral immune responses in cattle. It was also apparent that a method enabling higher

throughput and resolution in the analysis of protein-specific humoral responses was needed.

These conclusions were addressed in Paper II. Since in vitro expression could only be confirmed

for the protein MSC_0364, which was variable, these proteins were interesting from a vaccine

perspective. Until now, only vaccines consisting of live M. mycoides SC strains have conferred

satisfactory protective immune responses and other vaccines have failed. A vaccine of surface

components that may differ between M. mycoides SC in a host environment and those cultivated in

growth media, such as these putative variable surface proteins, could raise similar responses as a

live vaccine. This hypothesis was investigated in Paper IV.


36

10 Creating a surface protein array assay (Paper II)

10.1 Rationale

The general goal of this study was to expand the cloning efforts and screening for humoral

immune responses in CBPP-affected bovines initiated in Paper I. The primary aim involved an

expansion of the number of recombinant surface proteins and most importantly to develop a

rapid and highly multiplex method for analysis of antibody levels in serum samples from CBPP-

affected bovines against these proteins. The method needed to be verified for protein-specific

characterization of humoral immune responses in individual sera from diseased and healthy

animals. As a second aim, the validation would be expanded to include a comparison of IgG, IgA

and IgM responses in sera from a vaccine study with time series sampling from eight animal

covering pre-vaccination and four months post infection.

10.2 Results

The M. mycoides SC strain PG1 genome (39) was screened using SignalP (211, 212) followed by

TMHMM (213, 214) and BLASTP (222) during the protein selection process. This resulted in a

prediction of 187 genes containing signal peptide sequences that were ranked by ascending

similarity to related mycoplasma species. Transmembrane regions were predicted in 34 of the

corresponding proteins and the number of tryptophan codons per protein (estimating the number

of mutations needed for E. coli expression) ranged from none to 27. Using these data, 64 proteins

were selected based on similarity less than 80% covering 80% of the full-length sequence and

favouring few codons to mutate. Whenever possible, recombinant proteins were designed to

contain the full-length protein to ensure the greatest structural resemblance to the native protein.

Only signal peptides and transmembrane regions that might affect protein expression were

excluded and for the 15 selected proteins that contained transmembrane regions, the largest

extracellular domain was chosen for expression. In the full set of recombinant proteins, the mean

amino acid coverage was 80%. All 64 proteins were successfully cloned and 158 tryptophan

codons (at most 11 per protein) were mutated using an adaptation of the multiple mutation

reaction (223), for higher throughput compared to the commercial kit used in Paper I. Protein

yields from 100 ml culture were 0.2-10.3 mg, which far exceeded the 10 g needed for the selected

CARL HAMSTEN 2009

37

assay format. This set of 64 recombinant surface proteins is a fundamental part of this study and

those presented in Paper III and IV.

Bead-based array systems offer a suitable platform for the desired multiplexed analysis of proteins

using minute serum samples (224) and a system from Luminex Corp. that uses spectrally

distinguishable beads (225) to form an array in suspension was selected for the assay development

this study. It was chosen for its high multiplicability in both samples and analytes as well as the

flexibility to add and remove analytes (recombinant surface proteins). This allowed assay

optimizations in parallel to protein cloning and expression. The bead-based array is analyzed in a

flow cytometer-like instrument and can perform up to 100 simultaneous assays in a single reaction

well. This platform has recently been used to determine binding specificities to antigens produced

in a similar fashion (226) and to profile antibodies in serum towards six antigens of Mycobacterium

tuberculosis (227).

When the assay was optimized for screening of recombinant M. mycoides SC surface proteins, it

enabled separation of CBPP-positive and negative serum samples (Figure 10.1 A) with a window

of detection of median fluorescent intensities from <100 arbitrary units (AU) for the internal

control up to >13,000 AU for the 64 recombinant proteins. The signals were proven to be

protein-specific by competition experiments and signals were also in concordance with Western

blot analysis of selected proteins using two sera (Figure 3.1 B). The serum with the strongest

signals in the bead-based assay generated most bands in blotting as well, but some bands were

missing for both sera in the blotting analysis. This could be due to the different denatured and

non-denatured conditions of the compared assays. Nonetheless, it was proven that the obtained

signals were M. mycoides SC-specific and not due to cross-reactions with the tag, highly protein-

specific and not artefacts of the bead-based assay system.

The high-throughput performance of the assay was evaluated by screening of 51 recombinant

surface proteins (the size of the array at that time) using 242 sera, completed within a week. All

sera could be handled according to a standard procedure and high reproducibility of the serum

specific binding patterns demonstrated the strength of the assay. A cluster analysis of the results

grouped the sera into two major clusters of predominantly CBPP positive and CBPP negative

sera, with uncertain samples evenly distributed among them. A lack of clinical information on the

sample set as well as differences in handling of sera from the sampling event until the present

investigation prevented a full assessment of the assay’s discriminatory power as a diagnostic tool.


38

A true diagnostic evaluation of the array was envisioned to be a reduction of the number of

proteins in the array to select a small set fulfilling a balance between final assay cost, highest CBPP

diagnostic power and least risk of cross-reactions to related diseases. Such work is presented in

Paper III, where nine proteins were identified as candidates for a novel serological diagnostic

method for CBPP and evaluated in a ELISA setup.

Figure 10.1 IgG profiles. Panel A shows IgG binding profiles displayed as bar charts for two CBPP positive sera ( 1MUK15A, 2MUK15A), a CBPP negative serum ( ) and a serum-free control ( ). Each chart shows protein-specific signals to 64 recombinant surface proteins and a control (*; His6ABP, the fusion tag present in all recombinant proteins). Whiskers denote standard deviation in replicate samples and mean intensities for the CBPP positive and negative sera were 1071 and 53 MFI respectively. The binding profiles for the CBPP positive sera were used to select seven recombinant proteins with high and three with low signal intensities for a comparison to Western blot analysis shown in B. Here, binding to selected proteins and the fusion tag as negative control is shown for both CBPP positive sera.

CARL HAMSTEN 2009

39

To fulfil the second aim of the study and to further benchmark the assay, it was used to monitor

IgG, IgA and IgM responses to 64 corresponding native proteins over time in eight cattle from a

previous CBPP vaccine study. Samples were taken during 293 days in groups of animals that were

T1/44 or T1sr vaccinated, intubated with a local M. mycoides SC strain or untreated. All Ig class

responses were monitored successfully and the results were exemplified in graphs showing

antibody titers to all 64 recombinant surface proteins in four animals (Figure 10.2). Here, each line

represented antibody titers to one corresponding native protein during the 293 days. In figure 10.2

A, IgG, IgM and IgA responses were shown for a T1/44 vaccinated animal. The highest antibody

levels were observed for IgG in all animals, while the absolute signal intensities were considerably

lower for IgM and IgA. Figure 10.2 B exemplified monitoring of IgG levels in animals from the

other groups. In general, the intubated animals had the earliest CBPP-specific responses, within

ten days after intubation, while the other groups responded at least a month later. It is also evident

that a more frequent sampling would have been desirable to allow a finer resolution of the

response curves. Recombinant proteins corresponding to MSC_1046, MSC_0209 and MSC_0364

were found to dominant in a rudimentary analysis of protein-specific responses in the different

animals. These results were concordant between the different Ig classes in each animal.

MSC_1046 corresponds to the previously studied immunodominant LppQ (93) and proved

accordance with previous research, MSC_0209 is a lipoprotein and MSC_0364 was among the

proteins studied in Paper I. A deeper analysis was not done, as adequate clinical information

regarding disease outcome of the individual animal was unfortunately not available.


40

Figure 10.2 Time course study. IgG, IgM and IgA responses to the 64 proteins over time were studied using a series of samples collected from eight animals, as exemplified in A for a T1/44 vaccinated animal. Each line represented signals obtained for one protein. All Ig classes showed protein-specific responses both after vaccination and following CBPP exposure. In B, protein-specific IgG responses were further exemplified by data from an intubated bovine, one vaccinated by T1SR and an untreated control animal.

CARL HAMSTEN 2009

41

10.3 Conclusions

This paper presented the cloning and expression of 64 recombinant M. mycoides SC surface

proteins, the development of an assay for high-throughput analysis of CBPP-specific humoral

immune responses against these proteins and a proof-of-concept screening using sera from a

previous CBPP vaccine trial. The assay was proven robust, allowing detection and 20-fold mean

signal separation of CBPP-positive and negative sera. Furthermore, signals were protein-specific

as concluded from inhibition experiments and comparisons to western blot experiments. From

the proof-of-concept study it was concluded that IgG, IgA and IgM responses could be

monitored over time and the identification of immunodominant proteins was possible. All three

Ig classes displayed similar relative antibody titers to the different proteins, implying that IgG

monitoring might be enough for an initial selection of proteins suitable for the development of

novel serological diagnosis. However, the choice of Ig class detection in a final assay might have

an impact on diagnostic sensitivity during CBPP progression in an animal (see chapter 5.3).

Another important conclusion was that the proof-of-concept study was hampered by the need for

better-characterized sera (disease outcome, serological diagnostic results, post-mortem

examinations etc) when using the assay to identify proteins suitable for novel serological diagnosis

(addressed in Paper III) and proteins associated to protective immune responses (addressed in

Paper IV).


42

11 Selection of antigens for a novel serological diagnostic assay

(Paper III)

11.1 Rationale

This study was prompted by the completion of the M. mycoides SC recombinant surface protein

array assay, described in Paper II, and the need for better serological diagnosis of CBPP. In light

of the thorough characterization of LppQ (93, 94) and its use in a diagnostic ELISA (95), there

should be more proteins that elicit humoral immune responses that are useful for diagnostic

applications. Combinations of such antigens could offer higher specificity and sensitivity than

current serological methods (CFT, eELISA, LppQ-ELISA, LATs etc) by adding discriminative

power to the LppQ-ELISA while circumventing cross-reactivity to other mycoplasma species

seen with whole cell based methods. In this study, the array assay was to be used to identify the

most potent diagnostic surface antigens among the 64 recombinant proteins and furthermore

develop an diagnostic ELISA based on these proteins.

11.2 Results

During this investigation, 61 recombinant surface proteins were screened in the bead based assay

presented in Paper II using a total of 115 sera, including bovine sera with well-characterized

CBPP status and 17 hyperimmune sera from calves and rabbits immunized with M. mycoides SC

strain PG1 and closely related mycoplasma species as controls. The results were summarized in a

heat map in combination with a cluster analysis on both serum and protein level (Figure 11.1).

Serum samples separated into five main clusters, denoted I-V. Clusters I, II and V consisted of

mostly CBPP-positive sera (54 out of 61 sera) and contained most of the high signal data points.

All hyperimmune sera, except for the M. mycoides SC PG1 antiserum, clustered together in cluster

III that contained the lowest signals obtained, together with five sera from Swedish bovines

infected with Mycoplasma bovis, one Swedish negative control and one CBPP-positive sample.

Cluster IV consisted of 50 samples whereof 45 were CBPP-negative, four were CBPP-positive

and one was the above mentioned M. mycoides SC PG1 antiserum. When studying the protein

clustering, the cluster consisting nine proteins at the top of the dendrogram appeared to have

diagnostic importance as high antibody titers were detected to these proteins in CBPP-positive

sera.

CARL HAMSTEN 2009

43

Figure 11.1 Cluster analysis of screening data. The heat map displayed data from the bead based screening of 134 samples (115 sera) against 61 recombinant M. mycoides SC surface proteins, where colour intensity denoted signal intensity. Proteins to which high amounts of serum antibodies were detected in CBPP-positive sera, thus suggesting a diagnostic importance, formed a cluster of nine proteins (top vertical dendrogram, the cluster is highlighted in red). Horizontally, sera were clustered into five groups. Cluster I, II and V contained a majority of the CBPP-positive samples (red). Cluster III contained all but one hyperimmune sera (yellow) which displayed signals of low intensities, while cluster IV contained mostly CBPP-negative bovine sera (blue) as well as the positive control antiserum to M. mycoides SC PG1.


44

The Welch two sample t-test was used to select the most immunogenic antigens in CBPP-affected

cattle among the 61 recombinant surface proteins and was performed on a reduced data set

containing 76 sera excluding blanks, replicates, hyperimmune sera and Swedish control sera. The

Swedish control samples were excluded as the statistical analyses showed significant differences

between the Swedish negative controls and the African negative controls. The Swedish sera

appeared to have a lower general antibody titer compared to the African field sera, probably due

to differences in exposure to infectious agents and other environmental factors. However, the

same top candidate diagnostic proteins were identified regardless if the Swedish samples were

included or not.

The results, displayed as a volcano plot in Figure 11.2, revealed eight highly significant proteins (p-

values ranging from 10-6 to 10-18) as the theoretically most potent diagnostic antigens. These were

R1046, R0136, R0653, R0431, R0816, R0813, R0240 as well as R0397 and represent all but one of

the proteins within the important cluster shown in figure 11.1. R0079 was cloned at a later stage

and displayed diagnostic potential in Paper II; hence it was included in the following evaluation

and ELISA development. Three of the final nine selected proteins had been previously studied;

R1046 corresponding to LppQ (93), R0816 corresponding to a putative variable surface protein

(Paper I and II) and R0397 corresponding to a putative lipoprotein investigated in a study aiming

for a DNA vaccine against CBPP (209). Also, the gene MSC_0136 corresponding to R0136 had

previously been used in a phylogenetic study of the Mycoplasma mycoides cluster (228).

Cross-reactivity to closely related mycoplasma species was evaluated with hyperimmune sera that

revealed signal patterns that were different compared to the African bovine sera. Signals were

obtained to few recombinant proteins only; those were also among the most immunopotent

surface proteins identified in the analysis of CBPP-positive sera. It might therefore be reasonable

to believe that the high signal intensity values obtained were representative for immune responses

in a case of the corresponding infection, but the “artificial” immunization gave a more selective

response. As judged from these results, field samples from natural outbreaks of other

mycoplasmoses would offer a preferable study cohort. Nonetheless, R1046 cross-reacted to

antisera for the same species as had been shown to cross-react with its corresponding native

protein LppQ (Mycoplasma mycoides subsp. capri, Mycoplasma capricolum subsp. capripneumoniae, and

Mycoplasma leachii (93)).

CARL HAMSTEN 2009

45

Figure 11.2 Statistical selection of immunogenic antigens. The significance test of the log2-transformed screening data is visualized here, where eight proteins (black) showed signal fold changes between the CBPP-positive and the CBPP-negative populations that were greater than three and significance p-values ranging from 10-6 to 10-18. Four additional proteins (grey) displayed significance exceeding the p-value limit of 0.01, but were deemed with less discriminative importance than the top eight. Based on this test, the top eight proteins were selected as candidates for an antigen cocktail ELISA.

To avoid having to monitor potential cross reactions to the His6ABP-tag present in each

recombinant surface protein, the selected proteins were re-cloned with a His6-tag only in

preparation for ELISA development. This was successful for eight proteins that were rerun with

and without tag on the suspension bead array assay to confirm that separation between CBPP

positive and CBPP negative sera was unaffected by the presence or absence of the ABP part. An

ELISA was then developed and optimized using a cocktail of equal amounts of the eight proteins

as antigen. In the final set-up, results were analyzed in comparison to a reference serum and

signals reported as percent positivity. Reproducibility was determined to an average intra assay

coefficient of variation (CV) of 3% and average inter assay CV of 8%. A proof-of-concept study

including 47 sera revealed a five-fold signal ratio separation between CBPP positive and CBPP

negative sera (105 and 22 % positivity respectively, see Figure 11.3). Receiver operator


46

characteristics of the assay showed a high diagnostic capability since the area under the curve was

calculated to 0.991. An initial cut-off of 50% positivity resulted in no false positives and two false

negatives with about 30% positivity. However, these two sera were supplied with inconsistent

results from previous serological tests. Two negative sera had a positivity of 48% and 49%

respectively which is close to the cut-off and would prompt an additional analysis in a diagnostic

situation. Nonetheless, 96% of the samples were diagnosed correctly proving this ELISA setup

promising and suitable for further evaluation.

11.3 Conclusions

This paper presented the selection and evaluation of recombinant surface proteins useful for

CBPP diagnosis. The suspension bead array assay presented in Paper II was used to select eight

proteins by statistic analysis of a screening using better-characterized CBPP positive and negative

sera. Using hyperimmune sera to evaluate potential cross-reactivity with related mycoplasma

species resulted in distinctly different binding patterns from the surface protein array assay. It was

concluded that the hyperimmunizations raised a more “narrow” response as antibody titers were

detected for the most immunodominant proteins only. Although these results are useful and

reflect potential cross-reactions, sera from natural infections are preferable. The eight selected

proteins were supplemented with a ninth not available for the screening and eight of these were

successfully cloned without the ABP fusion partner. These were then combined in an antigen

cocktail used to build an ELISA for the diagnosis of M. mycoides SC infection. The final ELISA

supplied a discriminative power that enabled a 96% correct classification of sera from CBPP-

affected and CBPP-free bovines. Thus a promising ELISA setup emerged from the screening

assay. However, to become a solid diagnostic ELISA, future evaluation on much larger sets of

sera from herds of various geographical origins are needed to optimize positivity thresholds and

to determine the true sensitivity and specificity. It is preferred to conduct future studies in

facilities performing routine CBPP diagnosis to allow a direct comparison between the cocktail

ELISA and other available tests. The set of proteins and technologies presented in this paper offer

a powerful combination to drive and further improve serological assays towards reliable, simple

and cost-effective diagnostics of CBPP.

CARL HAMSTEN 2009

47

Figure 11.3 Proof of concept antigen cocktail ELISA. A) The antigen cocktail ELISA was evaluated using twenty-four CBPP-positive (grey) and 23 CBPP-negative (white) bovine sera analyzed twice. The optic density values were transformed to percent positivity of a reference sample and presented as an average of the two experiments. A suggested cut-off value of 50% resulted in two false negatives and no false positives, thus assigning 96% of the samples correctly. The average percent positivity of the CBPP-positive sera was 105% compared to 22% for CBPP-negative sera, which translated to a positive to negative signal ratio of 4.8. B) Receiver operator characteristics of the ELISA results with an area under the curve (AUC) of 0.991 indicate a high diagnostic capability.


48

12 Analysis of a CBPP vaccine trial (Paper IV)

12.1 Rationale

This paper reports in-depth analysis of a CBPP vaccine trial held in collaboration with the

Veterinary Laboratories Agency in the UK and the Central Veterinary Laboratory in Namibia. The

participation in the trial was prompted by the possibility to test a recombinant protein vaccine in

five animals, in parallel to other novel vaccines. At the time of the vaccine trial, the six

recombinant variable surface proteins reported in Paper I were cloned and expressed in E. coli

unlike the remaining 59 recombinant proteins used in Papers II, III and the current Paper. Even

so, five of these variable surface proteins were selected as vaccine components first and foremost

on the basis to make a vaccine of surface components that may differ between M. mycoides SC in a

host environment and those cultivated in growth media, since so far only vaccines of live M.

mycoides SC strains have conferred satisfactory protective immune responses. Variable surface

proteins are generally believed to enhance colonization of the host tissue and help evade host

immune responses by antigenic variation, as detailed in chapter 3.3. It was believed that a subunit

vaccine with these components could trigger immune responses that inhibit or prevent the above-

mentioned mechanisms. However, the clinical results of the vaccine trial clearly showed that the

recombinant vaccine did not confer protection against CBPP.

In light of these results, the aims of this study were to further evaluate the candidate recombinant

vaccine and more importantly to find novel protein targets to include in a second-generation

recombinant vaccine. This was to be accomplished by protein-specific analysis of humoral

immune responses in animals of the different vaccine groups using the surface protein array assay

presented in Paper II.

CARL HAMSTEN 2009

49

12.2 Results

The clinical outcome of the vaccine trial has been reported1, but results for the cattle included in

this study were restated to aid in interpreting the measured protein-specific humoral responses.

Responses in three groups of five cattle that were either vaccinated with the currently approved

T1/44 vaccine, vaccinated with the recombinant protein vaccine (denoted Pur. Protein) or

untreated in contact infection controls were compared. The clinical results for those groups were

summarized in Table 12.1. The T1/44 vaccine conferred some protection, although one animal

revealed a sequestrum at post-mortem examination. The recombinant vaccine and control (In

contact) groups were indistinguishable, manifesting acute and/or subacute CBPP lesions at post-

mortem examinations.

Animals that had received the recombinant vaccine had no persisting antibody titers to the five

vaccine proteins at day 0, showing that vaccination administered in this way did not raise a

persisting humoral immune response. The groups of animals vaccinated with the recombinant

vaccine candidate (Figure 12.1 B) and untreated controls (Figure 12.1 C) showed similar

responses, characterized by a drastic increase in antibody titers to many M. mycoides SC

recombinant surface proteins in the latter part of the trial. As all these animals had CBPP lesions

including sequestra at post mortem examination (Table 12.1), this elevated M. mycoides SC-specific

response was interpreted as the onset of clinical CBPP.

The results were also visualized in a heat map where proteins and sera were hierarchically

clustered based on Euclidian distances of log2-transformed signals (see Paper IV). In brief, the

proteins formed two major clusters. The first contained presumably immunodominant surface

proteins that had evoked strong humoral immune responses shown by overall medium to high

signals. The two proteins MSC_1046 (LppQ) and MSC_0816 displayed high antibody titers in

most samples from the diseased as well as the T1/44 vaccinated cattle and these proteins had also

been identified as immunodominant in several other screening analyses of CBPP diseased

bovines, including (93) and Papers I-III. In the second major protein cluster, proteins that may be

1 G. Tjipura-Zaire, L. McAuliffe, C. Churchward, R.A.J. Nicholas, M.D. Fielder, O.J.B. Hübschle¸M. Scacchia, R. Gonçalves, A. Persson, C. Hamsten and R. D. Ayling (2009). Comparison of T1/44 Vaccine and Two Novel Vaccines to Protect Against an Experimental Infection of Contagious Bovine Pleuropneumonia and Comparative Pathogenicity of Planktonic and Biofilm Grown Mycoplasma mycoides subsp. mycoides Small Colony Cells in Cattle. Manuscript


50

poor immunogens or at least have triggered low titers of IgG were found. Judging from this

analysis, the CBPP diseased bovines had evoked strong humoral immune responses to about half

(36 of 65) of the native counterparts to the proteins included in the array.

Animal Group Temp.a/cough CFT at withdrawalb Pathology

146 T1/44 0/Yes 0 PF, PCF

178 T1/44 0/No 0 PF

179 T1/44 0/No 0 PF, HPZ/M-DL

181 T1/44 0/Yes 0 PF, PCF

192 T1/44 1/No 160 PF, SS-CL, AD

150 Pur. Protein 1/No 2560 PF, SS-DLx2, HLN, AD

155 Pur. Protein 12/Yes 1280 PF, LS-CL, AD, PCF

161 Pur. Protein 13/Yes 2560 PF, LS-AL/CL/DL, AD

162 Pur. Protein 9/No 320 HLN, LS-DL, AD

189 Pur. Protein 10/No 320 PF, SS-AL, AD

145 In contact 2/No 640 PF, HPZ/M-AL/DL/CL, LS-DL, HLN, AD

165 In contact 1/Yes 320 PF, SS-DL

175 In contact 1/Yes 160 PF, SS-AL, SS-CL, AD

177 In contact 8/Yes 5120 PF, LS-AL/CL, AD, PCF

188 In contact 11/No 1280 LS-DL, HLN, AD

Table 12.1 A clinical summary of the vaccine trial. Disease records obtained during CBPP vaccine trial or at post-mortem exam with CFT expressed as reciprocal of serum dilution. Key: aDays above 39 oC .bOne week prior to withdrawal for in contact group ,PF - pleural fluid, LS - large sequester, SS - small sequester, AL - apical lobe, DL - diaphragmatic lobe, HLN - hyperplastic lymph nodes, HPZ/M - hepatization/marbling, AD - pleural adhesion, CL - cardiac lobe, PCF – pericardic fluid.

CARL HAMSTEN 2009

51

Figure 12.1 Time line analysis. Line charts displaying protein-specific IgG titers to 65 recombinant surface proteins monitored during 125 days from when vaccinated cattle were put in contact with CBPP carriers. Panel A displays five animals vaccinated with the T1/44 vaccine, panel B five animals vaccinated with a recombinant protein candidate vaccine and panel C five untreated, in contact control animals. Dashed lines mark titers to R1046, corresponding to LppQ (MSC_1046), in light gray and R816 (MSC_0816) in dark grey. These proteins are highly immunogenic and initial and persistent IgG titers to LppQ are induced by T1/44 vaccination. The raised titers to several proteins seen during the later stages of the trial in B) and C) coincide with CFT titers of 1/320 or more, indicating onset of clinical CBPP.


52

As a way to identify novel vaccine components and expand the knowledge of immunity to CBPP,

humoral responses specific for the protected animals in the T1/44 group were identified by

comparing protein-specific differences in the IgG levels. The dataset was reduced for this analysis

by removing time points corresponding to clinical CBPP in the purified protein vaccine group,

controls and T1/44 vaccinated animal 192 (individual time points for each animal). The Welch

two sample t-test was used to identify significant mean signal differences for each protein and the

results were visualized in a Volcano plot (Figure 12.2). The analysis identified four or five proteins

depending on the cutoff for signal differences between the groups. A minimum of three-fold

higher signals in T1/44 vaccinated animals was imposed in recognition of the small sample set of

five animals per group and to isolate proteins with largely increased IgG titers. The identified

proteins were designed on the following native counterparts, in descending order: MSC_1046,

MSC_0271, MSC_0136, MSC_0079 and MSC_0431.

Figure 12.2 Significant differences in responses of T1/44 vaccinated animals compared to other groups. The Welch two sample t-test was used to determine significant differences in protein-specific mean IgG titers and the results were displayed as a volcano plot seen here. Dashed lines form two upper-right sections of proteins with p-values less than 0.05 or 0.01 and over three-fold differences in antibody titer of T1/44 vaccinated animals. Four proteins meet these criteria, one with a p-value less than 0.01. These are marked in grey; 1 – R1046, 2 – R271, 3 – R0136 and 4 – R0079. A fifth protein is significant (p<0.05) but barely misses the signal fold criteria (5 – R0431). The recombinant fusion partner of all proteins, His6ABP, marked in solid black.

CARL HAMSTEN 2009

53

Of all proteins included in the assay, MSC_1046 (LppQ) seemed to be most important in the

T1/44-induced humoral response. It had the highest significance (p<0.01), the largest signal

differences (an average of 5800 AU to 400 AU) and as seen in Figure 12.1 A, antibody titers to

MSC_1046 were high throughout the trial with the exception of animal 192, as discussed earlier.

In this animal and other animals (Figure 12.1 B and C), high antibody titers to R1046 concurred

with the onset of clinical CBPP. Hence, an initial strong antibody response to LppQ seemed to be

an indicator in creating a protective immune respons. However, earlier studies have questioned

the usefulness of LppQ in vaccines (Chapter 7.1, (96, 97)). It is important to remember that the

presented results were protein-specific systemic IgG responses associated to T1/44 induced

immunity. Whether these humoral immune responses are protective is yet to be determined. Since

cell-mediated immunity also appears to be important, further studies are needed (see chapter 6.1).

The recombinant proteins produced for the current assay could of course also be used for

analyzing protein-specific cellular responses. Each protein would then be separately incubated

with viable lymphocytes from cattle (CBPP-infected, vaccinated etc) and responses measured

using lymphocyte proliferation/stimulation assays. The four other identified proteins were not as

evident as LppQ in the time line graphs (Figure 12.1) since their relative antibody titers were lower

(an average of <1700 AU) in the T1/44 vaccinated group. None of these proteins had been

extensively studied before, MSC_0079 is a putative phosphonate ABC transporter and MSC_0136

is a putative protein which corresponding gene has been used in a phylogenetic study of the

Mycoplasma mycoides cluster (229). The remaining two are lipoproteins (MSC_0431 and MSC_0271).

Three of these proteins (except MSC_0271) and MSC_1046 were suggested as parts of the novel

CBPP vaccine presented in Paper III.

To investigate whether the failure of the recombinant protein vaccine was due to poor vaccination

or non-protective immune responses, antibody titers to the subcomponents of the vaccine were

examined. MSC_1033 had uniformly low titers throughout the vaccine trial (signals of ±1000 AU)

and was excluded from the analysis. Results were summarized as box plots showing results from

all time points grouped as before and during CBPP infection (Figure 12.3). For one protein,

MSC_0847, significantly (p<0.05) higher mean IgG titers were observed in the recombinant

vaccine group compared to the controls before onset of CBPP. During the manifested disease,

mean titers to MSC_0117 were significantly (p<0.05) higher in the purified protein vaccine group

and MSC_0364 had seemingly higher mean titers. Judging from these results, the vaccination

induced a stronger immune response to three of the five proteins but it was far from the high IgG

titer to MSC_1046 seen throughout the study induced by T1/44 vaccination (Figure 12.1 A).


54

Disregarding whether this response could confer immunity or not, a successful vaccination should

elicit a stronger response upon contact with the pathogen. In this sense, the vaccine concentration

needs to be titrated in order to generate stronger immune responses that might be protective. The

1 mg/ml, single dose vaccine protocol used for both novel vaccines in this trial was based on

results from an previous M. bovis vaccine (230) and a implication that a TH2 immune response

might be protective (173), however it clearly needs revision. Booster vaccinations or other

adjuvants should be considered in this optimization process, which probably is necessary

regardless of the recombinant protein components in the final vaccine.

12.3 Conclusions

This study added to the knowledge on protective immune responses to CBPP. The identification

of several proteins (LppQ, MSC_0271, MSC_0136, MSC_0079 and MSC_0431) that were

associated with immunity for CBPP was a significant step towards a second-generation

recombinant protein vaccine for CBPP. However, further evaluation of these candidates is needed

and a suggested next step is to evaluate cellular immune responses to these proteins. A careful

selection of adjuvant and administration protocol is also needed to stimulate strong and protective

immune responses, which was evident in the analysis of the recombinant protein vaccine tested in

the trial. Significant differences were seen for one protein prior to the onset of the disease and for

one protein during clinical CBPP, implying that the recombinant vaccine triggered a weak immune

response. However, it is still unclear whether the recombinant protein vaccine failed due to its

protein components or an insufficient vaccine response under the present adjuvant and

administration conditions.

CARL HAMSTEN 2009

55

Figure 12.3 Responses to subcomponents of the recombinant protein vaccine. Box plots displaying the diverse results of four vaccine components in the three animal groups before (b) and during (d) CBPP infection. Only two proteins had significantly (p<0.05) different titers between the purified protein vaccine group and the two other groups, as measured by average responses and marked by *. The bold line represents the median signal, the boxes comprise 50% of the dataset and the whiskers extend to the furthest data point within 1.5 times of the box length. Circles mark outliers. †Only one animal in the T1/44 group developed CBPP.


56

13 Concluding remarks and future perspectives

The work presented in this thesis is centred on recombinantly expressed surface proteins of M.

mycoides SC, analysis of humoral immune responses to the native counterparts of these proteins in

cattle infected by M. mycoides SC and the use of these proteins in the development of novel

diagnostic methods and vaccines for the prevention of CBPP.

In Paper I, five putative variable surface proteins were expressed and analyzed by blotting

methods using sera from CBPP-affected cattle and polyclonal antibodies were raised to each

protein. Sera form the CBPP-affected animals contained antibodies to these proteins or their

homologues indicating that they were expressed in vivo during M. mycoides SC infection and for

MSC_0364, variable expression in vitro was shown. With these results in mind and the notion that

a vaccine containing the variable surface protein repertoire should inhibit the colonization

enhancing and host immune response evading effects of antigenic variation, the five novel

putative variable surface proteins were tested as a CBPP vaccine. As reported in Paper IV, the

vaccine conferred no protection to CBPP and the protein-specific analysis revealed that the

vaccine only triggered weak immune responses during the trial. This implies two major challenges

for future work on a recombinant protein vaccine for CBPP. First of all, the selection of adjuvant

and/or administration protocol needs revision. A TH2-stimulating adjuvant was used in this study

based on previous work and implications that a TH2 response might be protective (231), but as

detailed in chapter 6.1, TH1 responses have since been suggested as more important (98, 207). Of

course, this has a huge impact on adjuvant selection. A better understanding of the pathogenesis

of CBPP and the protein-specific protective immune responses, as exemplified by Paper IV and

(98), will benefit the selection of a better adjuvant. Nonetheless, further animal trials are needed

for the development of a vaccine and since unfortunately no good small animal model is available

for CBPP (98), these studies will have to rely on induced mycoplasmaemia in mice or cattle

experiments. In the development of a second-generation recombinant protein vaccine, it would be

suitable with an initial trial in mice to evaluate the adjuvant and get indications of protection

manifested as reduced mycoplasmaemia, ahead of a trial in cattle.

The second challenge for future work on a recombinant vaccine is the selection of protein

components. This process is greatly benefited by the work presented in Paper II where the

recombinant expression of 64 surface proteins of M. mycoides SC in E. coli and an assay for

CARL HAMSTEN 2009

57

screening of specific humoral immune responses against their native counterparts with 20 fold

signal separation between CBPP-affected animals and healthy controls was presented. The assay

allows monitoring of protein-specific IgG, IgA and IgM titers in individual animals over time, as

demonstrated in Paper II. This can greatly enhance the analysis of a CBPP vaccine trial, which was

done in Paper IV. Here it was also used to suggest proteins for use in novel vaccines, as humoral

immune responses to proteins MSC_1046 (LppQ), MSC_0271, MSC_0136, MSC_0079 and

MSC_0431 were found to be associated to protection induced by the T1/44 vaccine. Whether

immune responses to these proteins alone are protective against CBPP, part of a more complex

immune response or even just an artefact of T1/44 immunization remains to be determined. The

last question should be resolved by analyzing sera from a larger set of T1/44-vaccinated cattle,

including individuals where the vaccination failed, to discriminate responses related to the

immunization per se from protective immune responses. Aside from this type of experiments,

gaining additional knowledge from earlier studies, the selected proteins need to be evaluated in

new cattle experiments as a next step in determining if immune responses to these proteins confer

protection against CBPP. Nonetheless, the association of LppQ to T1/44-induced protection

somewhat contradicts the failure of a previous LppQ-based vaccine (96). This failure has on the

other hand been suggested to be due to a lack of major T-cell epitopes in LppQ (98). Once again,

until the nature of a protective immune response to CBPP is known, one should assume that both

humoral and cellular immune responses are important. The strength of the assay presented in

Paper II is the streamlined analysis of hundreds of sera and currently 65 recombinant surface

proteins in parallel, and to my knowledge no assay comparable in easy of use and throughput is

currently available for measuring cellular immune responses. Even though measuring of protein-

specific cellular immune responses using all 65 recombinant proteins would be preferable to

expand the results in Paper IV and suggest further candidates for a vaccine, at least the four other

proteins identified in this paper should be analyzed similarly to LppQ (98).

A complementary means of finding candidates for a recombinant protein vaccine is to rationally

select essential surface proteins from M. mycoides SC, such as membrane transport proteins, with

the aim to block their function with humoral responses. Such proteins are not expected to be

immunodominant and might not be identified using methods described in Papers II and IV.

These proteins could be targeted by preferably cloning and fusing all surface exposed loops of

these transmembrane proteins and using the “string of loops” in the vaccine. Raising polyclonal

antibodies against these recombinant proteins could be used for initial verification of terminal or

growth inhibiting effect on M. mycoides SC in pure culture. This line of investigation is currently


58

being pursued in collaboration with the Clinical Immunology and Allergy Unit at the Karoliska

Institute.

The use of the recombinant surface proteins in the development of novel diagnostics for CBPP

was explored in Paper III. Here, a selection was made among 61 proteins using well-characterized

sera from CBPP-affected and healthy cattle to find antigens suitable for use in serological

diagnosis of CBPP. Eight proteins showing strong responses in CBPP-affected sera and minimal

reactivity in negative control sera were identified using statistical analysis. A total of nine proteins

were further evaluated using the suspension bead array assay as well as in a cocktail ELISA built

on these antigens. In the end, a proof-of-concept ELISA using equal amounts of eight

recombinant proteins was presented. In an initial evaluation using 47 sera, 96% of the test samples

were assigned correctly. This prompts further evaluation of the true specificity and sensitivity

using many more samples and direct comparisons to other serological diagnosis methods. There is

also room for improvement in the design of the recombinant proteins. These could for example

be shortened from full-length to remove regions with epitopes causing serological cross-reactions,

such as the C-terminal domain of LppQ (93). The proteins could also be chained together by

flexible linkers in order to for a large recombinant protein as a means to simplify production and

reduce final assay cost.

There are also advantages to be found in switching diagnostic assay platform. A path towards an

inexpensive pen side test would be to evaluate the proteins in a latex agglutination test assay. Such

tests have already been proven useful in diagnosis of M. mycoides SC (178, 179) and Mycoplasma

capricolum subsp. capripneumoniae (232). These tests are based on capsular polysaccharides or

antibodies and there is a need for improved sensitivity and specificity (see chapter 5.4), which

could be addressed by using the recombinant surface proteins. Even more advantageous would be

to use an assay that allows retaining specific detection of the individual protein components, in a

microarray-like format. This is of course offered by the suspension bead array assay (Paper II, IV),

but at a higher cost and in a laboratory environment. A less expensive alternative would be to

develop a lateral flow microarray assay.

CARL HAMSTEN 2009

59

ACKNOWLEDGEMENTS

Nu när den här resan börjar nå sitt slut är det dags att tacka alla som gjort de senaste åren så

roliga, utmanande, spännande och ganska krävande. Förutom att ha lärt känna massa nya vänner

och kollegor har jag lärt mig lite nya saker på vägen!

Jag vill givetvis börja med att tacka Anja som introducerade mig till den spännande mykoplasma-

världen och höll mig på kurs genom åren. Våra trevliga (och långa) diskussioner om aktuella

projekt, forskningsvärlden och livet därutanför har varit den stora inspirationen och kraftkällan i

doktorerandet!

Dessutom har jag haft privilegiet att ha ytterligare två eminenta handledare till hjälp under åren:

Mathias, tack för inspirerande och målinriktade möten och diskussioner. Din entusiasm, din

förmåga att fokusera på det viktigaste och din enorma energi är en tillgång för oss alla på plan 3!

Per-Åke, vårt affibodyprojekt nådde aldrig mål men lurpassar i frysen för senare tillfälle. Tack för

alla tillfällen då mina ”enkla och snabba” frågor resulterat i långa diskussioner med helt nya

infallsvinklar!

Tack alla övriga Professorer och PIs för att ni med kunskap och engagemang leder arbetet på

avdelningen och skapar en trevlig atmosfär att jobba i. Givetvis har alla på plan 3 och i HPR

också en stor del i detta, tack för stämningen där alla hjälps åt (för det mesta), där inga frågor går

utan svar och där den sociala biten utanför jobbet inte glöms bort!

Vissa av er har jag haft förmånen att jobba närmare med i olika projekt:

Maja, den dagen du började blev mykoplasmagänget färggladare och mer entusiastiskt, det har

hållit i sig sen dess! Tack för all hjälp med luminexandet, all peppning och det goda kaffet! Jesper,

tack för ditt kämpande med arrayerna och diagnostiken! Rätt som det var hade du gjort massa

försök en sen kväll eller helg! Jochen, vår guide i luminex-världen! Utan dig hade Maja och jag

inte hunnit lika långt så snabbt! Tack även till Tommy, min första exjobbare som fick ”lära” mig

bli handledare.

Tack till alla samarbetspartners utanför KTH: Göran, tack för att vi fick komma till SVA och köra

otaliga koloniblottar! Att vi dessutom fått ta del av din serumbank har varit ovärdeligt! Tack även


60

till Hans, Kurt, Erik och Mamma på KI. Per-Åke föreslog att vi skulle samarbeta och även om

projektet inte är klart än har det varit kul och lärorikt för oss alla!

I would also like to thank all international collaborators and friends. Roger, thanks for all support

and interesting collaborations! I hope we continue to meet in places like Great Britain, Sweden,

Namibia, China and the Canary Islands! I thank You, Laura, Robin, Massimo, Georgina and

Rosario for letting us participate in your trial and letting me join you in Namibia! I also thank

John for letting us come to Scotland and get aliquots from your serum bank.

Tack till alla rumskamrater genom åren: Åsa, Cristina, Lisa, Johan R, Pawel, Mats, Lotta,

Afshin, Emelie, Torun och nuvarande gänget Sara, Patrik, Per-Åke, John och Jojje. Tack vare

er har dagarna fyllts av roliga diskussioner, mest viktiga saker men ibland även arbetsrelaterade.

Jag vill även tacka gänget som gjort labkursen i molekylär bioteknik så avslappnad, ibland stressig

med alltid lyckad i slutändan! Under ledning av Per-Åke eller Patrik har vi varit ett stort gäng

genom åren (ingen nämnd, ingen glömd…)

HPR molbio, ni har alltid varit min andra ”familj” och tagit hand om mig när jag var ensam i

mykoplasma-gruppen, Tack! HPR Profab, ni har hjälpt mig massor genom åren och det har alltid

funnits möjlighet att låna er utrustning och expertis, tack! HPR Immunotech, jag fick äran att

jobba med er ett tag och har alltid fått låna bra-att-ha-saker av er, tack!

Jag vill även tacka Erik P, Mattias och Christian för trevliga luncher och annat umgänge genom

åren.

Innan jag kommer till de viktiga människorna i mitt liv utanför KTH finns det några i gråzonen

där emellan: Jojje, tack för alla skratt och upptåg de senaste åren. Jag hade inte trivts lika bra utan

dig i rummet och labkursen hade kraschat för flera år sen! Kom ihåg, snart är det fredag!! Erik M,

vi har hängt i nio år på KTH men faktiskt lämnat KTH-livet utanför den mesta av tiden, tack! Jag

vet inte var jag ska börja eller sluta, vi har gjort så mycket och träffats nästan varje dag. Tack för

alla skratt, långa sessioner med en padda i handen och allmänna konstigheter. Kattis, tack för alla

roliga stunder och för att du låter mig ”låna” Erik så ofta!

CARL HAMSTEN 2009

61

Tack till alla vänner utanför jobbet som har klargjort vad som är viktigast i livet: Tack Johan W

för lång vänskap, Mac-diskussioner, effektivitetstips och allt mellan himmel och jord! Anders,

tack för vänskap, Guitar Hero-mangel och annat kul! Jakob, Tack för alla ”temahelger” i

Hässleholm! Peter, tack för London- och Skåne-perspektivet på livet!

Tack Mamma och Pappa för allt stöd och vetenskapliga diskussioner men framförallt tack för att

ni alltid finns där för mig! Tack till min kära syster Tessan som alltid ställer upp när tiden inte

räcker till!

Tack Annika, Carl-Henrik, Anna, Gustav och Sofia för alla trevliga stunder de senaste åren!

Tack även till min åländska familj Cai-Eric, Sussi, Eva och Jonny som alltid ser till att jag

glömmer bort allt som har med doktorerande att göra för en helg eller längre!

Mest av allt vill jag tacka Marica! Utan din hjälp och stöd hade jag aldrig fått ihop mina projekt

och än mindre avhandlingen! Du betyder allt för mig! Tillsammans med dig har även det bästa i

mitt liv skett: Linnea, tack för att du står ut med pappas försök att avsluta avhandlingen under

föräldraledigheten!

Dessutom vill jag tacka Leia, Mimmi och alla åländska fyrbeningar som dagligen får mig att

släppa jobb och allt annat för lite välbehövlig motion!

Du som läser detta och känner dig bortglömd, sorry! Det här är inte på något sett en komplett

lista över alla som på något sätt hjälpt och stöttat mig under dessa år… Tack för allt!


62

LIST OF ABBREVIATIONS

AU Arbitrary units

CBPP Contagious bovine pleuropneumonia

CFT Complement fixation test

ConA Concanavalin A

CPS Capsular polysaccharide

CsA Cyclosporine A

CV Coefficient of variation

ECaNEp Embryonic calf nasal epithelial

ELISA Enzyme-linked immunosorbent assay

FAO Food and Agriculture Organization of the United Nations

IBT Immunoblotting test

IFN- Interferon gamma

Ig Immunoglobulin

IL Interleukin

ISCOM Immunostimulating complex

LAT Latex agglutination test

Lpp Lipoprotein

mAb Monoclonal antibody

M. mycoides SC Mycoplasma mycoides subsp. mycoides small colony type

NGU Nongonococcal urethritis

OIE Office International des Epizooties /World organization for Animal Health

pAb Polyclonal antibody

PBMC Peripheral blood mononuclear cell

PHA Phytohemagglutinin

SNP Single nucleotide polymorphism

SRBC Sheep red blood cell

Subsp. Subspecies

CARL HAMSTEN 2009

63

REFERENCES

1. OIE (2009) Handistatus II, Multiannual animal disease status. OIE, Paris. 2. Scudamore, J. M. (1995) Contagious bovine pleuropneumonia. State Veterinary Journal 5, 13-16. 3. Brandão, E. (1995) Isolation and identification of Mycoplasma mycoides subspecies mycoides SC

strains in sheep and goats. The Veterinary record 136, 98-99. 4. Masiga, W. N., Domenech, J., and Windsor, R. S. (1996) Manifestation and epidemiology of

contagious bovine pleuropneumonia in Africa. Revue scientifique et technique (International Office of Epizootics) 15, 1283-1308.

5. Kusiluka, L. J., and Sudi, F. F. (2003) Review of successes and failures of contagious bovine pleuropneumonia control strategies in Tanzania. Preventive veterinary medicine 59, 113-123.

6. EMPRES (2002) Recognizing Contagious bovine pleuropneumonia : FAO Animal Health Manual, FAO, Rome.

7. Egwu, G. O., Nicholas, R. A. J., Ameh, J. A., and Bashiruddin, J. B. (1996) Contagious Bovine Pleuroneumonia : An Update. Veterinary Bulletin 66, 875-888.

8. Nicholas, R., Bashiruddin, J., Ayling, R., and Miles, R. (2000) Contagious bovine pleuropneumonia: a review of recent developments. Veterinary Bulletin 70, 827-838.

9. Nicholas, R., and Palmer, N. (1994) Contagious bovine pleuropneumonia in Europe. State Veterinary Journal 4, 14-16.

10. Nicholas, R. A., Santini, F. G., Clark, K. M., Palmer, N. M., De Santis, P., and Bashiruddin, J. B. (1996) A comparison of serological tests and gross lung pathology for detecting contagious bovine pleuropneumonia in two groups of Italian cattle. The Veterinary record 139, 89-93.

11. Regalla, J., Caporale, V., Giovannini, A., Santini, F., Martel, J. L., and Goncalves, A. P. (1996) Manifestation and epidemiology of contagious bovine pleuropneumonia in Europe. Revue scientifique et technique (International Office of Epizootics) 15, 1309-1329.

12. ter Laak, E. A. (1992) Contagious bovine pleuropneumonia. A review. The Veterinary quarterly 14, 104-110.

13. Food and Agriculture Organization of the United Nations (2003) Contagious bovine pleuropneumonia. EMPRESS Transboundary Anim. Dis. Bull. 24, 2-7.

14. Windsor, R. S. (2000) The eradication of contagious bovine pleuropneumonia from south western Africa. A plan for action. Ann N Y Acad Sci 916, 326-332.

15. OIE (2009) World Animal Health Information Database (WAHID). OIE, Paris. 16. Blancou, J. (1996) Early methods of surveillance and control for contagious bovine

pleuropneumonia. Revue scientifique et technique (International Office of Epizootics) 15, 1241-1282. 17. Provost, A., Perreau, P., Beard, A., Le Goff, C., Martel, J. L., and Cottew, G. S. (1987)

Contagious Bovine Pleuropneumonia. Rev. Sci. Tech. Off. Int. Epiz. 6, 625-679. 18. Nocard, E., and Roux, E. (1898) Le microbe de la péripneumonie. Annas de l'institute Pasteur 12,

240-262. 19. Edward, D. G., and Freundt, E. A. (1956) The classification and nomenclature of organisms

of the pleuropneumonia group. Journal of general microbiology 14, 197-207. 20. Cottew, G. S., and Yeats, F. R. (1978) Subdivision of Mycoplasma mycoides subsp. mycoides from

cattle and goats into two types. Australian veterinary journal 54, 293-296. 21. Edward, D. G., Freundt, E. A., Chanock, R. M., Fabricant, J., Hayflick, L., Lemcke, R. M.,

Rezin, S., Somerson, N. L., and Wittler, R. G. (1967) Recommendations on nomenclature of the order Mycoplasmatales. Science (New York, N.Y 155, 1694-1696.

22. Razin, S., Yogev, D., and Naot, Y. (1998) Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev 62, 1094-1156.


64

23. Murray, P. R., Rosenthal, K. S., and Pfaller, M. A. (2009) Medical microbiology, 6th ed., Mosby Elsevier, Philadelphia.

24. Waites, K. B., Katz, B., and Schelonka, R. L. (2005) Mycoplasmas and ureaplasmas as neonatal pathogens. Clinical microbiology reviews 18, 757-789.

25. Christensen, N. M., Axelsen, K. B., Nicolaisen, M., and Schulz, A. (2005) Phytoplasmas and their interactions with hosts. Trends in plant science 10, 526-535.

26. Nicholas, R. A., and Ayling, R. D. (2003) Mycoplasma bovis: disease, diagnosis, and control. Res Vet Sci 74, 105-112.

27. Johansson, K.-E., and Pettersson, B. (2002) Taxonomy of Mollicutes. In: Razin, S., and Herrmann, R., eds. Molecular Biology and Pathogenicity of Mycoplasmas, pp. 1-29, Kluwer, London.

28. Weisburg, W. G., Tully, J. G., Rose, D. L., Petzel, J. P., Oyaizu, H., Yang, D., Mandelco, L., Sechrest, J., Lawrence, T. G., Van Etten, J., and et al. (1989) A phylogenetic analysis of the mycoplasmas: basis for their classification. Journal of bacteriology 171, 6455-6467.

29. Heldtander, M., Pettersson, B., Tully, J. G., and Johansson, K. E. (1998) Sequences of the 16S rRNA genes and phylogeny of the goat mycoplasmas Mycoplasma adleri, Mycoplasma auris, Mycoplasma cottewii and Mycoplasma yeatsii. International journal of systematic bacteriology 48 Pt 1, 263-268.

30. Pettersson, B., Leitner, T., Ronaghi, M., Bölske, G., Uhlén, M., and Johansson, K. E. (1996) Phylogeny of the Mycoplasma mycoides cluster as determined by sequence analysis of the 16S rRNA genes from the two rRNA operons. Journal of bacteriology 178, 4131-4142.

31. Cottew, G. S., Breard, A., DaMassa, A. J., Erno, H., Leach, R. H., Lefevre, P. C., Rodwell, A. W., and Smith, G. R. (1987) Taxonomy of the Mycoplasma mycoides cluster. Israel journal of medical sciences 23, 632-635.

32. Manso-Silvan, L., Vilei, E. M., Sachse, K., Djordjevic, S. P., Thiaucourt, F., and Frey, J. (2009) Mycoplasma leachii sp. nov. as a new species designation for Mycoplasma sp. bovine group 7 of Leach, and reclassification of Mycoplasma mycoides subsp. mycoides LC as a serovar of Mycoplasma mycoides subsp. capri. International journal of systematic and evolutionary microbiology 59, 1353-1358.

33. Rurangirwa, F. R., Shompole, P. S., Wambugu, A. N., and McGuire, T. C. (2000) Monoclonal antibody differentiation of Mycoplasma mycoides subsp. mycoides small-colony strains causing contagious bovine pleuropneumonia from less important large-colony strains. Clinical and diagnostic laboratory immunology 7, 519-521.

34. Nicholas, R. A., and Bashiruddin, J. B. (1995) Mycoplasma mycoides subspecies mycoides (small colony variant): the agent of contagious bovine pleuropneumonia and member of the "Mycoplasma mycoides cluster". Journal of comparative pathology 113, 1-27.

35. Pilo, P., Frey, J., and Vilei, E. M. (2007) Molecular mechanisms of pathogenicity of Mycoplasma mycoides subsp. mycoides SC. Vet J 174, 513-521.

36. Woese, C. R., Maniloff, J., and Zablen, L. B. (1980) Phylogenetic analysis of the mycoplasmas. Proceedings of the National Academy of Sciences of the United States of America 77, 494-498.

37. Fraser, C. M., Gocayne, J. D., White, O., Adams, M. D., Clayton, R. A., Fleischmann, R. D., Bult, C. J., Kerlavage, A. R., Sutton, G., Kelley, J. M., Fritchman, R. D., Weidman, J. F., Small, K. V., Sandusky, M., Fuhrmann, J., Nguyen, D., Utterback, T. R., Saudek, D. M., Phillips, C. A., Merrick, J. M., Tomb, J. F., Dougherty, B. A., Bott, K. F., Hu, P. C., Lucier, T. S., Peterson, S. N., Smith, H. O., Hutchison, C. A., 3rd, and Venter, J. C. (1995) The minimal gene complement of Mycoplasma genitalium. Science (New York, N.Y 270, 397-403.

38. Sasaki, Y., Ishikawa, J., Yamashita, A., Oshima, K., Kenri, T., Furuya, K., Yoshino, C., Horino, A., Shiba, T., Sasaki, T., and Hattori, M. (2002) The complete genomic sequence of Mycoplasma penetrans, an intracellular bacterial pathogen in humans. Nucleic acids research 30, 5293-5300.

CARL HAMSTEN 2009

65

39. Westberg, J., Persson, A., Holmberg, A., Goesmann, A., Lundeberg, J., Johansson, K. E., Pettersson, B., and Uhlén, M. (2004) The genome sequence of Mycoplasma mycoides subsp. mycoides SC type strain PG1T, the causative agent of contagious bovine pleuropneumonia (CBPP). Genome Res 14, 221-227.

40. Sirand-Pugnet, P., Lartigue, C., Marenda, M., Jacob, D., Barre, A., Barbe, V., Schenowitz, C., Mangenot, S., Couloux, A., Segurens, B., de Daruvar, A., Blanchard, A., and Citti, C. (2007) Being pathogenic, plastic, and sexual while living with a nearly minimal bacterial genome. PLoS genetics 3, e75.

41. Rosengarten, R., Citti, C., Glew, M., Lischewski, A., Droesse, M., Much, P., Winner, F., Brank, M., and Spergser, J. (2000) Host-pathogen interactions in mycoplasma pathogenesis: virulence and survival strategies of minimalist prokaryotes. Int J Med Microbiol 290, 15-25.

42. Wise, K. S., Foecking, M. F., Röske, K., Lee, Y. J., Lee, Y. M., Madan, A., and Calcutt, M. J. (2006) Distinctive repertoire of contingency genes conferring mutation- based phase variation and combinatorial expression of surface lipoproteins in Mycoplasma capricolum subsp. capricolum of the Mycoplasma mycoides phylogenetic cluster. Journal of bacteriology 188, 4926-4941.

43. Rottem, S. (2003) Interaction of mycoplasmas with host cells. Physiological reviews 83, 417-432. 44. Pollack, J. D., Williams, M. V., and McElhaney, R. N. (1997) The comparative metabolism of

the mollicutes (Mycoplasmas): the utility for taxonomic classification and the relationship of putative gene annotation and phylogeny to enzymatic function in the smallest free-living cells. Critical reviews in microbiology 23, 269-354.

45. Glass, J. I., Assad-Garcia, N., Alperovich, N., Yooseph, S., Lewis, M. R., Maruf, M., Hutchison, C. A., 3rd, Smith, H. O., and Venter, J. C. (2006) Essential genes of a minimal bacterium. Proceedings of the National Academy of Sciences of the United States of America 103, 425-430.

46. Hutchison, C. A., Peterson, S. N., Gill, S. R., Cline, R. T., White, O., Fraser, C. M., Smith, H. O., and Venter, J. C. (1999) Global transposon mutagenesis and a minimal Mycoplasma genome. Science (New York, N.Y 286, 2165-2169.

47. Mushegian, A. R., and Koonin, E. V. (1996) A minimal gene set for cellular life derived by comparison of complete bacterial genomes. Proceedings of the National Academy of Sciences of the United States of America 93, 10268-10273.

48. Suthers, P. F., Dasika, M. S., Kumar, V. S., Denisov, G., Glass, J. I., and Maranas, C. D. (2009) A genome-scale metabolic reconstruction of Mycoplasma genitalium, iPS189. PLoS computational biology 5, e1000285.

49. Gibson, D. G., Benders, G. A., Andrews-Pfannkoch, C., Denisova, E. A., Baden-Tillson, H., Zaveri, J., Stockwell, T. B., Brownley, A., Thomas, D. W., Algire, M. A., Merryman, C., Young, L., Noskov, V. N., Glass, J. I., Venter, J. C., Hutchison, C. A., 3rd, and Smith, H. O. (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science (New York, N.Y 319, 1215-1220.

50. Lartigue, C., Glass, J. I., Alperovich, N., Pieper, R., Parmar, P. P., Hutchison, C. A., 3rd, Smith, H. O., and Venter, J. C. (2007) Genome transplantation in bacteria: changing one species to another. Science (New York, N.Y 317, 632-638.

51. Himmelreich, R., Hilbert, H., Plagens, H., Pirkl, E., Li, B. C., and Herrmann, R. (1996) Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae. Nucleic acids research 24, 4420-4449.

52. Chambaud, I., Heilig, R., Ferris, S., Barbe, V., Samson, D., Galisson, F., Moszer, I., Dybvig, K., Wróblewski, H., Viari, A., Rocha, E. P., and Blanchard, A. (2001) The complete genome sequence of the murine respiratory pathogen Mycoplasma pulmonis. Nucleic acids research 29, 2145-2153.

53. Papazisi, L., Gorton, T. S., Kutish, G., Markham, P. F., Browning, G. F., Nguyen, D. K., Swartzell, S., Madan, A., Mahairas, G., and Geary, S. J. (2003) The complete genome


66

sequence of the avian pathogen Mycoplasma gallisepticum strain R(low). Microbiology (Reading, England) 149, 2307-2316.

54. Jaffe, J. D., Stange-Thomann, N., Smith, C., DeCaprio, D., Fisher, S., Butler, J., Calvo, S., Elkins, T., FitzGerald, M. G., Hafez, N., Kodira, C. D., Major, J., Wang, S., Wilkinson, J., Nicol, R., Nusbaum, C., Birren, B., Berg, H. C., and Church, G. M. (2004) The complete genome and proteome of Mycoplasma mobile. Genome Res 14, 1447-1461.

55. Minion, F. C., Lefkowitz, E. J., Madsen, M. L., Cleary, B. J., Swartzell, S. M., and Mahairas, G. G. (2004) The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis. Journal of bacteriology 186, 7123-7133.

56. Vasconcelos, A. T., Ferreira, H. B., Bizarro, C. V., Bonatto, S. L., Carvalho, M. O., Pinto, P. M., Almeida, D. F., Almeida, L. G., Almeida, R., Alves-Filho, L., Assuncao, E. N., Azevedo, V. A., Bogo, M. R., Brigido, M. M., Brocchi, M., Burity, H. A., Camargo, A. A., Camargo, S. S., Carepo, M. S., Carraro, D. M., de Mattos Cascardo, J. C., Castro, L. A., Cavalcanti, G., Chemale, G., Collevatti, R. G., Cunha, C. W., Dallagiovanna, B., Dambros, B. P., Dellagostin, O. A., Falcao, C., Fantinatti-Garboggini, F., Felipe, M. S., Fiorentin, L., Franco, G. R., Freitas, N. S., Frias, D., Grangeiro, T. B., Grisard, E. C., Guimaraes, C. T., Hungria, M., Jardim, S. N., Krieger, M. A., Laurino, J. P., Lima, L. F., Lopes, M. I., Loreto, E. L., Madeira, H. M., Manfio, G. P., Maranhao, A. Q., Martinkovics, C. T., Medeiros, S. R., Moreira, M. A., Neiva, M., Ramalho-Neto, C. E., Nicolas, M. F., Oliveira, S. C., Paixao, R. F., Pedrosa, F. O., Pena, S. D., Pereira, M., Pereira-Ferrari, L., Piffer, I., Pinto, L. S., Potrich, D. P., Salim, A. C., Santos, F. R., Schmitt, R., Schneider, M. P., Schrank, A., Schrank, I. S., Schuck, A. F., Seuanez, H. N., Silva, D. W., Silva, R., Silva, S. C., Soares, C. M., Souza, K. R., Souza, R. C., Staats, C. C., Steffens, M. B., Teixeira, S. M., Urmenyi, T. P., Vainstein, M. H., Zuccherato, L. W., Simpson, A. J., and Zaha, A. (2005) Swine and poultry pathogens: the complete genome sequences of two strains of Mycoplasma hyopneumoniae and a strain of Mycoplasma synoviae. Journal of bacteriology 187, 5568-5577.

57. Dybvig, K., Zuhua, C., Lao, P., Jordan, D. S., French, C. T., Tu, A. H., and Loraine, A. E. (2008) Genome of Mycoplasma arthritidis. Infection and immunity 76, 4000-4008.

58. Glass, J. I., Lefkowitz, E. J., Glass, J. S., Heiner, C. R., Chen, E. Y., and Cassell, G. H. (2000) The complete sequence of the mucosal pathogen Ureaplasma urealyticum. Nature 407, 757-762.

59. Oshima, K., Kakizawa, S., Nishigawa, H., Jung, H. Y., Wei, W., Suzuki, S., Arashida, R., Nakata, D., Miyata, S., Ugaki, M., and Namba, S. (2004) Reductive evolution suggested from the complete genome sequence of a plant-pathogenic phytoplasma. Nature genetics 36, 27-29.

60. Bai, X., Zhang, J., Ewing, A., Miller, S. A., Jancso Radek, A., Shevchenko, D. V., Tsukerman, K., Walunas, T., Lapidus, A., Campbell, J. W., and Hogenhout, S. A. (2006) Living with genome instability: the adaptation of phytoplasmas to diverse environments of their insect and plant hosts. Journal of bacteriology 188, 3682-3696.

61. Tran-Nguyen, L. T., Kube, M., Schneider, B., Reinhardt, R., and Gibb, K. S. (2008) Comparative genome analysis of "Candidatus Phytoplasma australiense" (subgroup tuf-Australia I; rp-A) and "Ca. Phytoplasma asteris" Strains OY-M and AY-WB. Journal of bacteriology 190, 3979-3991.

62. Kube, M., Schneider, B., Kuhl, H., Dandekar, T., Heitmann, K., Migdoll, A. M., Reinhardt, R., and Seemuller, E. (2008) The linear chromosome of the plant-pathogenic mycoplasma 'Candidatus Phytoplasma mali'. BMC genomics 9, 306.

63. Dallo, S. F., Lazzell, A. L., Chavoya, A., Reddy, S. P., and Baseman, J. B. (1996) Biofunctional domains of the Mycoplasma pneumoniae P30 adhesin. Infection and immunity 64, 2595-2601.

64. Su, C. J., Tryon, V. V., and Baseman, J. B. (1987) Cloning and sequence analysis of cytadhesin P1 gene from Mycoplasma pneumoniae. Infection and immunity 55, 3023-3029.

CARL HAMSTEN 2009

67

65. Tryon, V. V., and Baseman, J. B. (1992) Pathogenic determinants and mechanisms. In: Maniloff, J., McElhaney, R. N., Finch, L. R., and Baseman, J. B., eds. Mycoplasmas: molecular biology and pathogenesis, pp. 457-472, American society for microbiology, Washington D. C.

66. Miles, R. J., Taylor, R. R., and Varsani, H. (1991) Oxygen uptake and H2O2 production by fermentative Mycoplasma spp. Journal of medical microbiology 34, 219-223.

67. Abdo el, M., Nicolet, J., Miserez, R., Goncalves, R., Regalla, J., Griot, C., Bensaide, A., Krampe, M., and Frey, J. (1998) Humoral and bronchial immune responses in cattle experimentally infected with Mycoplasma mycoides subsp. mycoides small colony type. Vet Microbiol 59, 109-122.

68. Rice, P., Houshaymi, B. M., Abu-Groun, E. A., Nicholas, R. A., and Miles, R. J. (2001) Rapid screening of H(2)O(2) production by Mycoplasma mycoides and differentiation of European subsp. mycoides SC (small colony) isolates. Vet Microbiol 78, 343-351.

69. Houshaymi, B. M., Miles, R. J., and Nicholas, R. A. (1997) Oxidation of glycerol differentiates African from European isolates of Mycoplasma mycoides subspecies mycoides SC (small colony). The Veterinary record 140, 182-183.

70. Pilo, P., Vilei, E. M., Peterhans, E., Bonvin-Klotz, L., Stoffel, M. H., Dobbelaere, D., and Frey, J. (2005) A metabolic enzyme as a primary virulence factor of Mycoplasma mycoides subsp. mycoides small colony. Journal of bacteriology 187, 6824-6831.

71. Vilei, E. M., and Frey, J. (2001) Genetic and biochemical characterization of glycerol uptake in Mycoplasma mycoides subsp. mycoides SC: its impact on H(2)O(2) production and virulence. Clinical and diagnostic laboratory immunology 8, 85-92.

72. Vilei, E. M., Abdo, E. M., Nicolet, J., Botelho, A., Goncalves, R., and Frey, J. (2000) Genomic and antigenic differences between the European and African/Australian clusters of Mycoplasma mycoides subsp. mycoides SC. Microbiology (Reading, England) 146 ( Pt 2), 477-486.

73. Bischof, D. F., Janis, C., Vilei, E. M., Bertoni, G., and Frey, J. (2008) Cytotoxicity of Mycoplasma mycoides subsp. mycoides small colony type to bovine epithelial cells. Infection and immunity 76, 263-269.

74. Bischof, D. F., Vilei, E. M., and Frey, J. (2009) Functional and antigenic properties of GlpO from Mycoplasma mycoides subsp. mycoides SC: characterization of a flavin adenine dinucleotide-binding site deletion mutant. Veterinary research 40, 35.

75. Vilei, E. M., and Frey, J. (2004) Differential clustering of Mycoplasma mycoides subsp. mycoides SC strains by PCR-REA of the bgl locus. Vet Microbiol 100, 283-288.

76. Vilei, E. M., Correia, I., Ferronha, M. H., Bischof, D. F., and Frey, J. (2007) Beta-D-glucoside utilization by Mycoplasma mycoides subsp. mycoides SC: possible involvement in the control of cytotoxicity towards bovine lung cells. BMC microbiology 7, 31.

77. Hudson, J. R., Buttery, S., and Cottew, G. S. (1967) Investigations into the influence of the galactan of Mycoplasma mycoides on experimental infection with that organism. The Journal of pathology and bacteriology 94, 257-273.

78. Buttery, S. H., Lloyd, L. C., and Titchen, D. A. (1976) Acute respiratory, circulatory and pathological changes in the calf after intravenous injections of the galactan from Mycoplasma mycoides subsp. mycoides. Journal of medical microbiology 9, 379-391.

79. March, J. B., and Brodlie, M. (2000) Comparison of the virulence of European and African isolates of Mycoplasma mycoides subspecies mycoides small colony type. The Veterinary record 147, 20-21.

80. March, J. B., Clark, J., and Brodlie, M. (2000) Characterization of strains of Mycoplasma mycoides subsp. mycoides small colony type isolated from recent outbreaks of contagious bovine pleuropneumonia in Botswana and Tanzania: evidence for a new biotype. Journal of clinical microbiology 38, 1419-1425.

81. Marshall, A. J., Miles, R. J., and Richards, L. (1995) The phagocytosis of mycoplasmas. Journal of medical microbiology 43, 239-250.


68

82. Calcutt, M. J., Kim, M. F., Karpas, A. B., Mühlradt, P. F., and Wise, K. S. (1999) Differential posttranslational processing confers intraspecies variation of a major surface lipoprotein and a macrophage-activating lipopeptide of Mycoplasma fermentans. Infection and immunity 67, 760-771.

83. Belloy, L., Vilei, E. M., Giacometti, M., and Frey, J. (2003) Characterization of LppS, an adhesin of Mycoplasma conjunctivae. Microbiology (Reading, England) 149, 185-193.

84. Marie, C., Roman-Roman, S., and Rawadi, G. (1999) Involvement of mitogen-activated protein kinase pathways in interleukin-8 production by human monocytes and polymorphonuclear cells stimulated with lipopolysaccharide or Mycoplasma fermentans membrane lipoproteins. Infection and immunity 67, 688-693.

85. Brenner, C., Wróblewski, H., Le Henaff, M., Montagnier, L., and Blanchard, A. (1997) Spiralin, a mycoplasmal membrane lipoprotein, induces T-cell-independent B-cell blastogenesis and secretion of proinflammatory cytokines. Infection and immunity 65, 4322-4329.

86. Herbelin, A., Ruuth, E., Delorme, D., Michel-Herbelin, C., and Praz, F. (1994) Mycoplasma arginini TUH-14 membrane lipoproteins induce production of interleukin-1, interleukin-6, and tumor necrosis factor alpha by human monocytes. Infection and immunity 62, 4690-4694.

87. Mühlradt, P. F., and Frisch, M. (1994) Purification and partial biochemical characterization of a Mycoplasma fermentans-derived substance that activates macrophages to release nitric oxide, tumor necrosis factor, and interleukin-6. Infection and immunity 62, 3801-3807.

88. Chambaud, I., Wróblewski, H., and Blanchard, A. (1999) Interactions between mycoplasma lipoproteins and the host immune system. Trends in microbiology 7, 493-499.

89. Cheng, X., Nicolet, J., Miserez, R., Kuhnert, P., Krampe, M., Pilloud, T., Abdo, E. M., Griot, C., and Frey, J. (1996) Characterization of the gene for an immunodominant 72 kDa lipoprotein of Mycoplasma mycoides subsp. mycoides small colony type. Microbiology (Reading, England) 142 ( Pt 12), 3515-3524.

90. Monnerat, M. P., Thiaucourt, F., Nicolet, J., and Frey, J. (1999) Comparative analysis of the lppA locus in Mycoplasma capricolum subsp. capricolum and Mycoplasma capricolum subsp. capripneumoniae. Vet Microbiol 69, 157-172.

91. Monnerat, M. P., Thiaucourt, F., Poveda, J. B., Nicolet, J., and Frey, J. (1999) Genetic and serological analysis of lipoprotein LppA in Mycoplasma mycoides subsp. mycoides LC and Mycoplasma mycoides subsp. capri. Clinical and diagnostic laboratory immunology 6, 224-230.

92. Pilo, P., Martig, S., Frey, J., and Vilei, E. M. (2003) Antigenic and genetic characterisation of lipoprotein LppC from Mycoplasma mycoides subsp. mycoides SC. Veterinary research 34, 761-775.

93. Abdo, E. M., Nicolet, J., and Frey, J. (2000) Antigenic and genetic characterization of lipoprotein LppQ from Mycoplasma mycoides subsp. mycoides SC. Clinical and diagnostic laboratory immunology 7, 588-595.

94. Bonvin-Klotz, L., Vilei, E. M., Kühni-Boghenbor, K., Kapp, N., Frey, J., and Stoffel, M. H. (2008) Domain analysis of lipoprotein LppQ in Mycoplasma mycoides subsp. mycoides SC. Antonie van Leeuwenhoek 93, 175-183.

95. Bruderer, U., Regalla, J., Abdo el, M., Huebschle, O. J., and Frey, J. (2002) Serodiagnosis and monitoring of contagious bovine pleuropneumonia (CBPP) with an indirect ELISA based on the specific lipoprotein LppQ of Mycoplasma mycoides subsp. mycoides SC. Vet Microbiol 84, 195-205.

96. Nicholas, R. A. J., Tjipura-Zaire, G., Scacchia, M., Frey, J., and Hübschle, O. J. B. (2004) An inactivated whole cell vaccine and LppQ subunit vaccine appear to exacerbate the effects of CBPP in adult cattle. Towards Sustainable CBPP Control Programmes for Africa, pp. 91-97, FAO, Rome.

97. Janis, C., Bischof, D., Gourgues, G., Frey, J., Blanchard, A., and Sirand-Pugnet, P. (2008) Unmarked insertional mutagenesis in the bovine pathogen Mycoplasma mycoides subsp.

CARL HAMSTEN 2009

69

mycoides SC: characterization of a lppQ mutant. Microbiology (Reading, England) 154, 2427-2436.

98. Dedieu, L., Totte, P., Rodrigues, V., Vilei, E. M., and Frey, J. (2009) Comparative analysis of four lipoproteins from Mycoplasma mycoides subsp. mycoides Small Colony identifies LppA as a major T-cell antigen. Comparative immunology, microbiology and infectious diseases

99. Persson, A., Jacobsson, K., Frykberg, L., Johansson, K. E., and Poumarat, F. (2002) Variable surface protein Vmm of Mycoplasma mycoides subsp. mycoides small colony type. Journal of bacteriology 184, 3712-3722.

100. Rosengarten, R., and Wise, K. S. (1990) Phenotypic switching in mycoplasmas: phase variation of diverse surface lipoproteins. Science (New York, N.Y 247, 315-318.

101. Citti, C., and Rosengarten, R. (1997) Mycoplasma genetic variation and its implication for pathogenesis. Wiener klinische Wochenschrift 109, 562-568.

102. Sachse, K., Helbig, J. H., Lysnyansky, I., Grajetzki, C., Muller, W., Jacobs, E., and Yogev, D. (2000) Epitope mapping of immunogenic and adhesive structures in repetitive domains of Mycoplasma bovis variable surface lipoproteins. Infection and immunity 68, 680-687.

103. Citti, C., Kim, M. F., and Wise, K. S. (1997) Elongated versions of Vlp surface lipoproteins protect Mycoplasma hyorhinis escape variants from growth-inhibiting host antibodies. Infection and immunity 65, 1773-1785.

104. Denison, A. M., Clapper, B., and Dybvig, K. (2005) Avoidance of the host immune system through phase variation in Mycoplasma pulmonis. Infection and immunity 73, 2033-2039.

105. Theiss, P., and Wise, K. S. (1997) Localized frameshift mutation generates selective, high-frequency phase variation of a surface lipoprotein encoded by a mycoplasma ABC transporter operon. Journal of bacteriology 179, 4013-4022.

106. Gaurivaud, P., Persson, A., Grand, D. L., Westberg, J., Solsona, M., Johansson, K. E., and Poumarat, F. (2004) Variability of a glucose phosphotransferase system permease in Mycoplasma mycoides subsp. mycoides Small Colony. Microbiology (Reading, England) 150, 4009-4022.

107. Markham, P. F., Glew, M. D., Whithear, K. G., and Walker, I. D. (1993) Molecular cloning of a member of the gene family that encodes pMGA, a hemagglutinin of Mycoplasma gallisepticum. Infection and immunity 61, 903-909.

108. Noormohammadi, A. H., Markham, P. F., Whithear, K. G., Walker, I. D., Gurevich, V. A., Ley, D. H., and Browning, G. F. (1997) Mycoplasma synoviae has two distinct phase-variable major membrane antigens, one of which is a putative hemagglutinin. Infection and immunity 65, 2542-2547.

109. Yogev, D., Rosengarten, R., Watson-McKown, R., and Wise, K. S. (1991) Molecular basis of Mycoplasma surface antigenic variation: a novel set of divergent genes undergo spontaneous mutation of periodic coding regions and 5' regulatory sequences. The EMBO journal 10, 4069-4079.

110. Glew, M. D., Baseggio, N., Markham, P. F., Browning, G. F., and Walker, I. D. (1998) Expression of the pMGA genes of Mycoplasma gallisepticum is controlled by variation in the GAA trinucleotide repeat lengths within the 5' noncoding regions. Infection and immunity 66, 5833-5841.

111. Washburn, L. R., Weaver, K. E., Weaver, E. J., Donelan, W., and Al-Sheboul, S. (1998) Molecular characterization of Mycoplasma arthritidis variable surface protein MAA2. Infection and immunity 66, 2576-2586.

112. Winner, F., Markova, I., Much, P., Lugmair, A., Siebert-Gulle, K., Vogl, G., Rosengarten, R., and Citti, C. (2003) Phenotypic switching in Mycoplasma gallisepticum hemadsorption is governed by a high-frequency, reversible point mutation. Infection and immunity 71, 1265-1273.


70

113. Zhang, Q., and Wise, K. S. (1997) Localized reversible frameshift mutation in an adhesin gene confers a phase-variable adherence phenotype in mycoplasma. Molecular microbiology 25, 859-869.

114. Boguslavsky, S., Menaker, D., Lysnyansky, I., Liu, T., Levisohn, S., Rosengarten, R., Garcia, M., and Yogev, D. (2000) Molecular characterization of the Mycoplasma gallisepticum pvpA gene which encodes a putative variable cytadhesin protein. Infection and immunity 68, 3956-3964.

115. Flitman-Tene, R., Mudahi-Orenstein, S., Levisohn, S., and Yogev, D. (2003) Variable lipoprotein genes of Mycoplasma agalactiae are activated in vivo by promoter addition via site-specific DNA inversions. Infection and immunity 71, 3821-3830.

116. Bhugra, B., Voelker, L. L., Zou, N., Yu, H., and Dybvig, K. (1995) Mechanism of antigenic variation in Mycoplasma pulmonis: interwoven, site-specific DNA inversions. Molecular microbiology 18, 703-714.

117. Glew, M. D., Marenda, M., Rosengarten, R., and Citti, C. (2002) Surface diversity in Mycoplasma agalactiae is driven by site-specific DNA inversions within the vpma multigene locus. Journal of bacteriology 184, 5987-5998.

118. Noormohammadi, A. H., Markham, P. F., Kanci, A., Whithear, K. G., and Browning, G. F. (2000) A novel mechanism for control of antigenic variation in the haemagglutinin gene family of Mycoplasma synoviae. Molecular microbiology 35, 911-923.

119. Lysnyansky, I., Ron, Y., and Yogev, D. (2001) Juxtaposition of an active promoter to vsp genes via site-specific DNA inversions generates antigenic variation in Mycoplasma bovis. Journal of bacteriology 183, 5698-5708.

120. Chopra-Dewasthaly, R., Citti, C., Glew, M. D., Zimmermann, M., Rosengarten, R., and Jechlinger, W. (2008) Phase-locked mutants of Mycoplasma agalactiae: defining the molecular switch of high-frequency Vpma antigenic variation. Molecular microbiology 67, 1196-1210.

121. Lysnyansky, I., Rosengarten, R., and Yogev, D. (1996) Phenotypic switching of variable surface lipoproteins in Mycoplasma bovis involves high-frequency chromosomal rearrangements. Journal of bacteriology 178, 5395-5401.

122. Lysnyansky, I., Sachse, K., Rosenbusch, R., Levisohn, S., and Yogev, D. (1999) The vsp locus of Mycoplasma bovis: gene organization and structural features. Journal of bacteriology 181, 5734-5741.

123. Citti, C., Watson-McKown, R., Droesse, M., and Wise, K. S. (2000) Gene families encoding phase- and size-variable surface lipoproteins of Mycoplasma hyorhinis. Journal of bacteriology 182, 1356-1363.

124. Boesen, T., Emmersen, J., Jensen, L. T., Ladefoged, S. A., Thorsen, P., Birkelund, S., and Christiansen, G. (1998) The Mycoplasma hominis vaa gene displays a mosaic gene structure. Molecular microbiology 29, 97-110.

125. Lysnyansky, I., Ron, Y., Sachse, K., and Yogev, D. (2001) Intrachromosomal recombination within the vsp locus of Mycoplasma bovis generates a chimeric variable surface lipoprotein antigen. Infection and immunity 69, 3703-3712.

126. Zhang, Q., and Wise, K. S. (1996) Molecular basis of size and antigenic variation of a Mycoplasma hominis adhesin encoded by divergent vaa genes. Infection and immunity 64, 2737-2744.

127. Zheng, X., Teng, L. J., Watson, H. L., Glass, J. I., Blanchard, A., and Cassell, G. H. (1995) Small repeating units within the Ureaplasma urealyticum MB antigen gene encode serovar specificity and are associated with antigen size variation. Infection and immunity 63, 891-898.

128. Zhang, Q., and Wise, K. S. (2001) Coupled phase-variable expression and epitope masking of selective surface lipoproteins increase surface phenotypic diversity in Mycoplasma hominis. Infection and immunity 69, 5177-5181.

CARL HAMSTEN 2009

71

129. Röske, K., Blanchard, A., Chambaud, I., Citti, C., Helbig, J. H., Prevost, M. C., Rosengarten, R., and Jacobs, E. (2001) Phase variation among major surface antigens of Mycoplasma penetrans. Infection and immunity 69, 7642-7651.

130. Horino, A., Sasaki, Y., Sasaki, T., and Kenri, T. (2003) Multiple promoter inversions generate surface antigenic variation in Mycoplasma penetrans. Journal of bacteriology 185, 231-242.

131. Glew, M. D., Papazisi, L., Poumarat, F., Bergonier, D., Rosengarten, R., and Citti, C. (2000) Characterization of a multigene family undergoing high-frequency DNA rearrangements and coding for abundant variable surface proteins in Mycoplasma agalactiae. Infection and immunity 68, 4539-4548.

132. Shen, X., Gumulak, J., Yu, H., French, C. T., Zou, N., and Dybvig, K. (2000) Gene rearrangements in the vsa locus of Mycoplasma pulmonis. Journal of bacteriology 182, 2900-2908.

133. Baseggio, N., Glew, M. D., Markham, P. F., Whithear, K. G., and Browning, G. F. (1996) Size and genomic location of the pMGA multigene family of Mycoplasma gallisepticum. Microbiology (Reading, England) 142 ( Pt 6), 1429-1435.

134. Brocchi, E., Gamba, D., Poumarat, F., Martel, J. L., and De Simone, F. (1993) Improvements in the diagnosis of contagious bovine pleuropneumonia through the use of monoclonal antibodies. Revue scientifique et technique (International Office of Epizootics) 12, 559-570.

135. Windsor, R. S., and Wood, A. (1998) Contagious bovine pleuropneumonia. The costs of control in Central/southern Africa. Ann N Y Acad Sci 849, 299-306.

136. Jores, J., Nkando, I., Sterner-Kock, A., Haider, W., Poole, J., Unger, H., Muriuki, C., Wesonga, H., and Taracha, E. L. (2008) Assessment of in vitro interferon-gamma responses from peripheral blood mononuclear cells of cattle infected with Mycoplasma mycoides ssp. mycoides small colony type. Veterinary immunology and immunopathology 124, 192-197.

137. Boonstra, E., Lindbaek, M., Fidzani, B., and Bruusgaard, D. (2001) Cattle eradication and malnutrition in under five's: a natural experiment in Botswana. Public Health Nutr 4, 877-882.

138. March, J. B. (2004) Improved formulations for existing CBPP vaccines--recommendations for change. Vaccine 22, 4358-4364.

139. OIE (2008) OIE Terrestrial Animal Health Code 2008 ed., OIE, Paris. 140. Nicholas, R. A., Ayling, R. D., and McAuliffe, L. (2009) Vaccines for Mycoplasma diseases in

animals and man. Journal of comparative pathology 140, 85-96. 141. Mbulu, R. S., Tjipura-Zaire, G., Lelli, R., Frey, J., Pilo, P., Vilei, E. M., Mettler, F., Nicholas,

R. A., and Huebschle, O. J. (2004) Contagious bovine pleuropneumonia (CBPP) caused by vaccine strain T1/44 of Mycoplasma mycoides subsp. mycoides SC. Vet Microbiol 98, 229-234.

142. Thiaucourt, F., Lorenzon, S., David, A., Tulasne, J. J., and Domenech, J. (1998) Vaccination against contagious bovine pleuropneumonia and the use of molecular tools in epidemiology. Ann N Y Acad Sci 849, 146-151.

143. Thiaucourt, F., Yaya, A., Wesonga, H., Hübschle, O. J., Tulasne, J. J., and Provost, A. (2000) Contagious bovine pleuropneumonia. A reassessment of the efficacy of vaccines used in Africa. Ann N Y Acad Sci 916, 71-80.

144. Tulasne, J. J., Litamoi, J. K., Morein, B., Dedieu, L., Palya, V. J., Yami, M., Abusugra, I., Sylla, D., and Bensaid, A. (1996) Contagious bovine pleuropneumonia vaccines: the current situation and the need for improvement. Revue scientifique et technique (International Office of Epizootics) 15, 1373-1396.

145. Provost, A. (1996) [Strategies for prevention and eradication of contagious bovine pleuropneumonia with or without vaccination]. Revue scientifique et technique (International Office of Epizootics) 15, 1355-1371.

146. Amanfu, W. (2006) The use of antibiotics for CBPP control: the challenges. CBPP control: Antibiotics to the resuce? FAO-OIE-AU/IBAR-IAEA Consultative Group Meeting on CBPP in Africa, pp. 7-11, FAO, Rome.


72

147. Hübschle, O. J., Ayling, R. D., Godinho, K., Lukhele, O., Tjipura-Zaire, G., Rowan, T. G., and Nicholas, R. A. (2006) Danofloxacin (Advocin) reduces the spread of contagious bovine pleuropneumonia to healthy in-contact cattle. Res Vet Sci 81, 304-309.

148. Anonymous (2006) Summary of recommendations. CBPP control: Antibiotics to the resuce? FAO-OIE-AU/IBAR-IAEA Consultative Group Meeting on CBPP in Africa, pp. 13-15, FAO, Rome.

149. Benkarine, A. (1998) Role of chemotherapy in CBPP. Meeting of the FAO/OIE/OAU-IBAR Consultative Group on CBPP, pp. 97-101, FAO, Rome.

150. Anonymous (2003) Recommendations. Third meeting of the FAO-OIE-AU/IBAR-IAEA Consultative Group on Contagious Bovine Pleuropneumonia, pp. 24-29, FAO, Rome.

151. Ayling, R. D., Baker, S. E., Nicholas, R. A., Peek, M. L., and Simon, A. J. (2000) Comparison of in vitro activity of danofloxacin, florfenicol, oxytetracycline, spectinomycin and tilmicosin against Mycoplasma mycoides subspecies mycoides small colony type. The Veterinary record 146, 243-246.

152. Ayling, R. D., Bisgaard-Frantzen, S., March, J. B., Godinho, K., and Nicholas, R. A. (2005) Assessing the in vitro effectiveness of antimicrobials against Mycoplasma mycoides subsp. mycoides small-colony type to reduce contagious bovine pleuropneumonia infection. Antimicrobial agents and chemotherapy 49, 5162-5165.

153. Niang, M., Sery, A., Cissé, O., Diallo, M., Doucouré, M., Koné, M., Simbé, C. F., Amanfu, W., and Thiaucourt, F. (2006) Effect of antibiotic therapy on the pathogenesis of CBPP. CBPP control: Antibiotics to the rescue? FAO-OIE-AU/IBAR-IAEA Consultative Group Meeting on CBPP in Africa, pp. 25-32, FAO.

154. OIE (2008) Manual of diagnostic test and vaccines for terrestial animals (Terrestial manual) OIE, Paris.

155. Rice, P., Houshaymi, B. M., Nicholas, R. A., and Miles, R. J. (2000) A rapid biochemical test to aid identification of Mycoplasma mycoides subsp. mycoides small colony (SC) strains. Letters in applied microbiology 30, 70-74.

156. Brooks, C., Finlay, D., Blackburn, P., and Ball, H. J. (2009) Detection of Mycoplasma mycoides sub-species mycoides small colony by a specific capture/enrichment monoclonal antibody-based sandwich ELISA. Res Vet Sci

157. Rodriguez, F., Ball, H. J., Finlay, D., Campbell, D., and Mackie, D. P. (1996) Detection of Mycoplasma mycoides subspecies mycoides by monoclonal antibody-based sandwich ELISA. Vet Microbiol 51, 69-76.

158. Bashiruddin, J. B., Taylor, T. K., and Gould, A. R. (1994) A PCR-based test for the specific identification of Mycoplasma mycoides subspecies mycoides SC. J Vet Diagn Invest 6, 428-434.

159. Bashiruddin, J. B., De Santis, P., Vacciana, A., and Santini, F. G. (1999) Detection of Mycoplasma mycoides subspecies mycoides SC in clinical material by a rapid colorimetric PCR. Molecular and cellular probes 13, 23-28.

160. Dedieu, L., Mady, V., and Lefevre, P. C. (1994) Development of a selective polymerase chain reaction assay for the detection of Mycoplasma mycoides subsp. Mycoides S.C. (contagious bovine pleuropneumonia agent). Vet Microbiol 42, 327-339.

161. Hotzel, H., Sachse, K., and Pfutzner, H. (1996) A PCR scheme for differentiation of organisms belonging to the Mycoplasma mycoides cluster. Vet Microbiol 49, 31-43.

162. Miserez, R., Pilloud, T., Cheng, X., Nicolet, J., Griot, C., and Frey, J. (1997) Development of a sensitive nested PCR method for the specific detection of Mycoplasma mycoides subsp. mycoides SC. Molecular and cellular probes 11, 103-111.

163. Persson, A., Pettersson, B., Bölske, G., and Johansson, K. E. (1999) Diagnosis of contagious bovine pleuropneumonia by PCR-laser- induced fluorescence and PCR-restriction endonuclease analysis based on the 16S rRNA genes of Mycoplasma mycoides subsp. mycoides SC. Journal of clinical microbiology 37, 3815-3821.

CARL HAMSTEN 2009

73

164. Lorenzon, S., David, A., Nadew, M., Wesonga, H., and Thiaucourt, F. (2000) Specific PCR identification of the T1 vaccine strains for contagious bovine pleuropneumonia. Molecular and cellular probes 14, 205-210.

165. Lorenzon, S., Manso-Silván, L., and Thiaucourt, F. (2008) Specific real-time PCR assays for the detection and quantification of Mycoplasma mycoides subsp. mycoides SC and Mycoplasma capricolum subsp. capripneumoniae. Molecular and cellular probes 22, 324-328.

166. Bashiruddin, J. B., de Santis, P., Persson, A., Ball, H., and Regalla, J. (2005) Detection of Mycoplasma mycoides subspecies mycoides SC in bovine lung and lymph node tissues by culture, sandwich ELISA and polymerase chain reaction systems. Res Vet Sci 78, 199-205.

167. Le Grand, D., Saras, E., Blond, D., Solsona, M., and Poumarat, F. (2004) Assessment of PCR for routine identification of species of the Mycoplasma mycoides cluster in ruminants. Veterinary research 35, 635-649.

168. Fitzmaurice, J., Sewell, M., Manso-Silvan, L., Thiaucourt, F., McDonald, W. L., and O'Keefe, J. S. (2008) Real-time polymerase chain reaction assays for the detection of members of the Mycoplasma mycoides cluster. New Zealand veterinary journal 56, 40-47.

169. Gorton, T. S., Barnett, M. M., Gull, T., French, R. A., Lu, Z., Kutish, G. F., Adams, L. G., and Geary, S. J. (2005) Development of real-time diagnostic assays specific for Mycoplasma mycoides subspecies mycoides Small Colony. Vet Microbiol 111, 51-58.

170. Bellini, S., Giovannini, A., di Francesco, C., Tittarelli, M., and Caporale, V. (1998) Sensitivity and specificity of serological and bacteriological tests for contagious bovine pleuropneumonia. Revue scientifique et technique (International Office of Epizootics) 17, 654-659.

171. Stärk, K. D., Vicari, A., Kihm, U., and Nicolet, J. (1995) Surveillance of contagious bovine pleuropneumonia in Switzerland. Revue scientifique et technique (International Office of Epizootics) 14, 621-629.

172. Campbell, A. D., and Turner, A. W. (1953) Studies on contagious bovine pleuropneumonia: IV. An improved complement-fixation test. Australian veterinary journal 154-163.

173. Anonymous (2001) Diagnostic tests for contagious bovine pleuropneumonia. Report of the Scientific Committee on Animal Health and Animal Welfare. Commission of the European Community.

174. Le Goff, C., and Thiaucourt, F. (1998) A competitive ELISA for the specific diagnosis of contagious bovine pleuropneumonia (CBPP). Vet Microbiol 60, 179-191.

175. Amanfu, W., Sediadie, S., Masupu, K. V., Raborokgwe, M. V., Benkirane, A., Geiger, R., and Thiaucourt, F. (2000) Comparison between c-ELISA and CFT in detecting antibodies to Mycoplasma mycoides mycoides biotype SC in cattle affected by CBPP in Botswana. Ann N Y Acad Sci 916, 364-369.

176. Goldsby, R. A., Kindt, T. J., Osbourne, B. A., and Kuby, J. (2003) Immunology, 5th ed., WH Freeman, New York.

177. Niang, M., Diallo, M., Cisse, O., Kone, M., Doucoure, M., Roth, J. A., Balcer-Rodrigues, V., and Dedieu, L. (2006) Pulmonary and serum antibody responses elicited in zebu cattle experimentally infected with Mycoplasma mycoides subsp. mycoides SC by contact exposure. Veterinary research 37, 733-744.

178. Ayling, R. D., Regalla, J., and Nicholas, R. A. J. (1999) A field test for detecting antibodies to Mycoplasma mycoides subsp. mycoides small colony type using the latex slide agglutination test. In: Stipkovits, L., Rosengarten, R., and Frey, J., eds. Mycoplasmas of ruminants: pathogenicity, diagostics, epidemology and molecular genetics Vol III, pp. 155-158, Office for official publications of the European Communities, Luxembourg.

179. March, J. B., Kerr, K., and Lema, B. (2003) Rapid detection of contagious bovine pleuropneumonia by a Mycoplasma mycoides subsp. mycoides SC capsular polysaccharide-specific antigen detection latex agglutination test. Clinical and diagnostic laboratory immunology 10, 233-240.


74

180. Goncalves, R., Regalla, J., Nicolet, J., Frey, J., Nicholas, R., Bashiruddin, J., de Santis, P., and Goncalves, A. P. (1998) Antigen heterogeneity among Mycoplasma mycoides subsp. mycoides SC isolates: discrimination of major surface proteins. Vet Microbiol 63, 13-28.

181. Regalla, J., Goncalves, R., Riberio, R., Duarte, L., and Goncalves, A. P. (1996) Use of immunoblotting test for carrier state definition in contagious bovine pleuropneumonia. In: Frey, J., and Sarris, K., eds. Mycoplasmas of Ruminants: pathogenicity, diagnostics, epidemiology and molecular genetics, pp. 72-74, European Commisson, Luxembourg.

182. Regalla, J., Goncalves, R., Niza Riberio, J., Duarte, L., Nicholas, R., Bashiruddin, J., De Santis, P., Garrido Abellan, F., and Goncalves, A. P. (1999) Development of immunoblotting as a diagnostic tool for contagious bovine pleuropneumonia. International Symposium COST Action 826: Mycoplasmas of Ruminants: pathogenicity, diagostics, epidemiology and molecular genetics. Joint Workshops of E.U. Projects, Toulouse, France.

183. Roberts, D. H., Windsor, R. S., Masiga, W. N., and Kariavu, C. G. (1973) Cell-mediated immune response in cattle to Mycoplasma mycoides var. mycoides. Infection and immunity 8, 349-354.

184. Dedieu, L., Balcer-Rodrigues, V., Yaya, A., Hamadou, B., Cisse, O., Diallo, M., and Niang, M. (2005) Gamma interferon-producing CD4 T-cells correlate with resistance to Mycoplasma mycoides subsp. mycoides S.C. infection in cattle. Veterinary immunology and immunopathology 107, 217-233.

185. Sasaki, Y., Blanchard, A., Watson, H. L., Garcia, S., Dulioust, A., Montagnier, L., and Gougeon, M. L. (1995) In vitro influence of Mycoplasma penetrans on activation of peripheral T lymphocytes from healthy donors or human immunodeficiency virus-infected individuals. Infection and immunity 63, 4277-4283.

186. Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin, M. A., and Coffman, R. L. (1986) Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 136, 2348-2357.

187. Brown, W. C., Rice-Ficht, A. C., and Estes, D. M. (1998) Bovine type 1 and type 2 responses. Veterinary immunology and immunopathology 63, 45-55.

188. Estes, D. M., and Brown, W. C. (2002) Type 1 and type 2 responses in regulation of Ig isotype expression in cattle. Veterinary immunology and immunopathology 90, 1-10.

189. Mena, A., Ioannou, X. P., Van Kessel, A., Van Drunen Little-Van Den Hurk, S., Popowych, Y., Babiuk, L. A., and Godson, D. L. (2002) Th1/Th2 biasing effects of vaccination in cattle as determined by real-time PCR. Journal of immunological methods 263, 11-21.

190. Dedieu, L., Balcer-Rodrigues, V., Cisse, O., Diallo, M., and Niang, M. (2006) Characterisation of the lymph node immune response following Mycoplasma mycoides subsp. Mycoides SC infection in cattle. Veterinary research 37, 579-591.

191. Totté, P., Rodrigues, V., Yaya, A., Hamadou, B., Cisse, O., Diallo, M., Niang, M., Thiaucourt, F., and Dedieu, L. (2008) Analysis of cellular responses to Mycoplasma mycoides subsp. mycoides small colony biotype associated with control of contagious bovine pleuropneumonia. Veterinary research 39, 8.

192. Hübschle, O. J., Tjipura-Zaire, G., Abusugra, I., di Francesca, G., Mettler, F., Pini, A., and Morein, B. (2003) Experimental field trial with an immunostimulating complex (ISCOM) vaccine against contagious bovine pleuropneumonia. Journal of veterinary medicine 50, 298-303.

193. Scacchia, M., Sacchini, F., Filipponi, G., Luciani, M., Lelli, R., Tjipura-Zaire, G., Di Provvido, A., Shiningwane, A., Ndiipanda, F., Pini, A., Caporale, V., and Hübschle, O. J. (2007) Clinical, humoral and IFNgamma responses of cattle to infection with Mycoplasma mycoides var. mycoides small colony and attempts to condition the pathogenesis of the infection. The Onderstepoort journal of veterinary research 74, 251-263.

CARL HAMSTEN 2009

75

194. Naessens, J., Scheerlinck, J. P., De Buysscher, E. V., Kennedy, D., and Sileghem, M. (1998) Effective in vivo depletion of T cell subpopulations and loss of memory cells in cattle using mouse monoclonal antibodies. Veterinary immunology and immunopathology 64, 219-234.

195. Dedieu, L., Chapey, E., and Balcer-Rodrigues, V. (2005) Mycoplasma mycoides ssp. mycoides biotype small colony-secreted components induce apoptotic cell death in bovine leucocytes. Scandinavian journal of immunology 62, 528-538.

196. Dedieu, L., and Balcer-Rodrigues, V. (2006) Viable Mycoplasma mycoides ssp. mycoides small colony-mediated depression of the bovine cell responsiveness to the mitogen concanavalin A. Scandinavian journal of immunology 64, 376-381.

197. Janeway, C. A., Travers, P., Walport, M., and Capra, J. D. (1999) Immunobiology, The immune system in health and disease. 4th Ed., Current Biology Publications, London.

198. Thomas, C. B., Mettler, J., Sharp, P., Jensen-Kostenbader, J., and Schultz, R. D. (1990) Mycoplasma bovis suppression of bovine lymphocyte response to phytohemagglutinin. Veterinary immunology and immunopathology 26, 143-155.

199. Vanden Bush, T. J., and Rosenbusch, R. F. (2004) Characterization of a lympho-inhibitory peptide produced by Mycoplasma bovis. Biochemical and biophysical research communications 315, 336-341.

200. Romero-Rojas, A., Reyes-Esparza, J., Estrada-Parra, S., and Hadden, J. W. (2001) Immunomodulatory properties of Mycoplasma pulmonis. III. Lymphocyte stimulation and cytokine production by Mycoplasma pulmonis products. International immunopharmacology 1, 1699-1707.

201. Teh, H. S., Ho, M., and Williams, L. D. (1988) Suppression of cytotoxic responses by a supernatant factor derived from Mycoplasma hyorhinis-infected mammalian cell lines. Infection and immunity 56, 197-203.

202. Tsunekawa, H., Takagi, E., Kishimoto, H., and Shimokata, K. (1987) Depressed cellular immunity in Mycoplasma pneumoniae pneumonia. European journal of respiratory diseases 70, 293-299.

203. Muneta, Y., Minagawa, Y., Shimoji, Y., Ogawa, Y., Hikono, H., and Mori, Y. (2008) Immune response of gnotobiotic piglets against Mycoplasma hyopneumoniae. The Journal of veterinary medical science / the Japanese Society of Veterinary Science 70, 1065-1070.

204. Shibata, K., and Watanabe, T. (1997) Mycoplasma fermentans enhances concanavalin A-induced apoptosis of mouse splenic T cells. FEMS immunology and medical microbiology 17, 103-109.

205. Abusugra, I., Wolf, G., Bölske, G., Thiaucourt, F., and Morein, B. (1997) ISCOM vaccine against contagious bovine pleuropneumonia (CBPP). 1. Biochemical and immunological characterization. Veterinary immunology and immunopathology 59, 31-48.

206. Gerdts, V., Mutwiri, G. K., Tikoo, S. K., and Babiuk, L. A. (2006) Mucosal delivery of vaccines in domestic animals. Veterinary research 37, 487-510.

207. Dedieu-Engelmann, L. (2008) Contagious bovine pleuropneumonia: A rationale for the development of a mucosal sub-unit vaccine. Comparative immunology, microbiology and infectious diseases 31, 227-238.

208. Waite, E. R., and March, J. B. (2002) Capsular polysaccharide conjugate vaccines against contagious bovine pleuropneumonia: Immune responses and protection in mice. Journal of comparative pathology 126, 171-182.

209. March, J. B., Jepson, C. D., Clark, J. R., Totsika, M., and Calcutt, M. J. (2006) Phage library screening for the rapid identification and in vivo testing of candidate genes for a DNA vaccine against Mycoplasma mycoides subsp. mycoides small colony biotype. Infection and immunity 74, 167-174.

210. Jepson, C. D., and March, J. B. (2004) Bacteriophage lambda is a highly stable DNA vaccine delivery vehicle. Vaccine 22, 2413-2419.


76

211. Nielsen, H., Engelbrecht, J., Brunak, S., and von Heijne, G. (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10, 1-6.

212. Nielsen, H., and Krogh, A. (1998) Prediction of signal peptides and signal anchors by a hidden Markov model. Proc Int Conf Intell Syst Mol Biol 6, 122-130.

213. Krogh, A., Larsson, B., von Heijne, G., and Sonnhammer, E. L. (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305, 567-580.

214. Sonnhammer, E. L., von Heijne, G., and Krogh, A. (1998) A hidden Markov model for predicting transmembrane helices in protein sequences. Proc Int Conf Intell Syst Mol Biol 6, 175-182.

215. Juncker, A. S., Willenbrock, H., Von Heijne, G., Brunak, S., Nielsen, H., and Krogh, A. (2003) Prediction of lipoprotein signal peptides in Gram-negative bacteria. Protein Sci 12, 1652-1662.

216. Steen, J., Uhlén, M., Hober, S., and Ottosson, J. (2006) High-throughput protein purification using an automated set-up for high-yield affinity chromatography. Protein Expr Purif 46, 173-178.

217. Nygren, P. Å., Eliasson, M., Abrahmsen, L., Uhlén, M., and Palmcrantz, E. (1988) Analysis and use of the serum albumin binding domains of streptococcal protein G. J Mol Recognit 1, 69-74.

218. Libon, C., Corvaia, N., Haeuw, J. F., Nguyen, T. N., Ståhl, S., Bonnefoy, J. Y., and Andreoni, C. (1999) The serum albumin-binding region of streptococcal protein G (BB) potentiates the immunogenicity of the G130-230 RSV-A protein. Vaccine 17, 406-414.

219. Sjölander, A., Nygren, P. Å., Ståhl, S., Berzins, K., Uhlén, M., Perlmann, P., and Andersson, R. (1997) The serum albumin-binding region of streptococcal protein G: a bacterial fusion partner with carrier-related properties. Journal of immunological methods 201, 115-123.

220. Bölske, G., Msami, H. M., Gunnarsson, A., Kapaga, A. M., and Loomu, P. M. (1995) Contagious bovine pleuropneumonia in northern Tanzania, culture confirmation and serological studies. Trop Anim Health Prod 27, 193-201.

221. Nilsson, P., Paavilainen, L., Larsson, K., Ödling, J., Sundberg, M., Andersson, A. C., Kampf, C., Persson, A., Al-Khalili Szigyarto, C., Ottosson, J., Björling, E., Hober, S., Wernerus, H., Wester, K., Pontén, F., and Uhlén, M. (2005) Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling. Proteomics 5, 4327-4337.

222. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) Basic local alignment search tool. J Mol Biol 215, 403-410.

223. Hames, C., Halbedel, S., Schilling, O., and Stülke, J. (2005) Multiple-mutation reaction: a method for simultaneous introduction of multiple mutations into the glpK gene of Mycoplasma pneumoniae. Applied and environmental microbiology 71, 4097-4100.

224. Templin, M. F., Stoll, D., Schwenk, J. M., Potz, O., Kramer, S., and Joos, T. O. (2003) Protein microarrays: promising tools for proteomic research. Proteomics 3, 2155-2166.

225. Fulton, R. J., McDade, R. L., Smith, P. L., Kienker, L. J., and Kettman, J. R., Jr. (1997) Advanced multiplexed analysis with the FlowMetrix system. Clinical chemistry 43, 1749-1756.

226. Schwenk, J. M., Lindberg, J., Sundberg, M., Uhlén, M., and Nilsson, P. (2007) Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics. Mol Cell Proteomics 6, 125-132.

227. Khan, I. H., Ravindran, R., Yee, J., Ziman, M., Lewinsohn, D. M., Gennaro, M. L., Flynn, J. L., Goulding, C. W., DeRiemer, K., Lerche, N. W., and Luciw, P. A. (2008) Profiling antibodies to Mycobacterium tuberculosis by multiplex microbead suspension arrays for serodiagnosis of tuberculosis. Clin Vaccine Immunol 15, 433-438.

CARL HAMSTEN 2009

77

228. Poumarat, F., and Solsona, M. (1995) Molecular epidemiology of Mycoplasma mycoides subsp. mycoides biotype Small Colony, the agent of contagious bovine pleuropneumonia. Vet Microbiol 47, 305-315.

229. Thiaucourt, F., Lorenzon, S., David, A., and Breard, A. (2000) Phylogeny of the Mycoplasma mycoides cluster as shown by sequencing of a putative membrane protein gene. Vet Microbiol 72, 251-268.

230. Nicholas, R. A., Ayling, R. D., and Stipkovits, L. P. (2002) An experimental vaccine for calf pneumonia caused by Mycoplasma bovis: clinical, cultural, serological and pathological findings. Vaccine 20, 3569-3575.

231. Martel, J. L., Nicholas, R., Noordhuizen, J., Pini, A., and Regalla, J. (2001) Diagnostic tests for contagious bovine pleuropneumonia. Report of the Scientific Committee on Animal Health and Animal Welfare. Commission of the European Community.

232. March, J. B., Gammack, C., and Nicholas, R. (2000) Rapid detection of contagious caprine pleuropneumonia using a Mycoplasma capricolum subsp. capripneumoniae capsular polysaccharide-specific antigen detection latex agglutination test. Journal of clinical microbiology 38, 4152-4159.

protein based approaches to understand and prevent contagious …235819/fulltext02.pdf · 2009. 9....

Documents