Mycoplasmas in Swine

Swine can be infected with many different mycoplasmas. Some are important pathogens, causing significant health and welfare issues in pigs and major losses to the swine industry worldwide. Other mycoplasmas are not pathogenic for swine and can be considered commensals. This book provides up-to-date scientific, clinical and practical information of the most important pathogenic mycoplasmas in swine. Most emphasis has been placed on Mycoplasma hyopneumoniae as the most economically important, but other pathogenic species like Mycoplasma hyorhinis, Mycoplasma hyosynoviae and Mycoplasma suis are also discussed.

Written by internationally renowned scientists and clinicians from all over the world, this book draws together in depth knowledge, expertise and experience in swine mycoplasmas to provide an evidence-based, academically rigorous and practical collection. It aims to serve the scientific and veterinary community and the swine industry worldwide.

• Language: English
• Publisher: CAB International
• Release date: Mar 12, 2021
• ISBN: 9781789249965

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Mycoplasmas in swine

Edited by

Dominiek Maes, Marina Sibila & Maria Pieters

Mycoplasmas in swine

© The Authors 2020

All rights reserved. Except as permitted by applicable copyright laws, no part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, duplicating or otherwise, without the prior permission of the copyright owner. Contact ACCO for all permission requests.

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GOLDEN PARTNER

Ceva Santé Animale is a French multinational veterinary pharmaceutical company created in 1999. Ceva specializes in the research, development, production and marketing of pharmaceutical products and vaccines for livestock and companion animals. Ceva is present in 110 countries and employs over 5700 people worldwide.

With 90% of swine farms worldwide affected by respiratory disease and an urgent need to preserve the future of antibiotics by reducing their widespread prophylactic use, the adoption of targeted, preventative health programs has become critical.

Ceva developed a system of recording lung lesions followed by data storage and processing, which is included in Ceva Lung ProgramTM. This way Ceva contributes to continuous improvements in respiratory health of commercial pigs and to the reduction of mass consumption of antimicrobials in swine farms.

Ceva decided to sponsor this book, as supporting the development and dissemination of knowledge about swine health and diseases, including mycoplasmas, is fully in line with the mission of the company.

SILVER PARTNER

BRONZE PARTNERS

Contents

CONTRIBUTORS

Prologue

Abbreviations

CHAPTER 1

Overview of the general characteristics and classification of porcine Mycoplasma species

1.1 Introduction

1.1.1 Characteristics of Mollicutes and mycoplasmas

1.1.2 Phylogenetic relationships of mycoplasmas

1.1.3 Pathogenicity in mycoplasmas

1.2 The mycoplasmas of the pig

1.3 Phylogenetic relationship between porcine mycoplasmas

1.4 Mycoplasma hyopneumoniae

1.4.1 Cultivation of Mycoplasma hyopneumoniae

1.4.2 The discovery of Mycoplasma hyopneumoniae

1.4.3 What is the evidence that Mycoplasma hyopneumoniae is responsible for EP?

1.5 Mycoplasma flocculare

1.6 Mycoplasma hyopharyngis

1.7 Mycoplasma hyosynoviae

1.8 Mycoplasma hyorhinis

1.8.1 What is the evidence that Mycoplasma hyorhinis can also act as the causative agent of EP?

1.9 Mycoplasma suis

CHAPTER 2

Diversity of Mycoplasma hyopneumoniae strains

2.1 Introduction

2.2 Genomic diversity

2.2.1 Mycoplasma hyopneumoniae WGS comparisons

2.2.2 Genetic diversity in Mycoplasma hyopneumoniae field isolates

2.3 Virulence variation

2.4 Antigenic variation

2.4.1 Size variation through VNTRs

2.4.2 Size variation through proteolytic processing

2.5 Diversity in Mycoplasma hyopneumoniae proteomes

2.6 Concluding remarks

CHAPTER 3

Mycoplasma hyopneumoniae pathogenicity: the known and the unknown

3.1 Introduction

3.2 Sequence of pathogenesis

3.3 Adhesion

3.4 Candidate virulence factors

3.5 Immune modulation and Mycoplasma host interaction

3.6 Pathogenicity model

CHAPTER 4

Epidemiology of Mycoplasma hyopneumoniae infections

4.1 Introduction

4.2 Prevalence

4.3 Infection dynamics

4.4 Transmission

4.5 Risk factors for Mycoplasma hyopneumoniae infection

4.6 Molecular epidemiology

CHAPTER 5

Mycoplasma hyopneumoniae clinical signs and gross lung lesions, including monitoring

5.1 Introduction

5.2 Clinical signs

5.2.1 Negative farms (farms with all animals seronegative against Mycoplasma hyopneumoniae)

5.2.2 Epizootically affected farms

5.2.3 Enzootically affected farms

5.3 Gross lung lesions

5.4 Clinical-pathological monitoring

CHAPTER 6

Immune responses against porcine Mycoplasma infections

6.1 Introduction

6.2 Innate immune responses

6.2.1 Overview of innate immune responses

6.2.2 Inflammatory responses after infection of pigs with Mycoplasma hyopneumoniae

6.2.3 Sensing of Mycoplasma by the innate immune system

6.2.4 Contribution of myeloid cells

6.2.5 Complement and other opsonins

6.2.6 Antimicrobial peptides

6.3 Antibody response against Mycoplasma hyopneumoniae

6.3.1 General considerations of antibody responses against Mycoplasma

6.3.2 Kinetics of antibody responses after Mycoplasma hyopneumoniae infection

6.3.3 Role of antibodies in protection against Mycoplasma

6.3.4 Maternally-derived antibodies

6.3.5 Mucosal antibody responses

6.4 T-cell mediated immune responses against Mycoplasma

6.4.1 General considerations of T-cell responses against Mycoplasma

6.4.2 Role of T cells in protective immunity against Mycoplasma infections

6.4.3 Role of different types of T-cell responses

6.5 Conclusions

CHAPTER 7

Interactions of Mycoplasma hyopneumoniae with other pathogens and economic impact

7.1 Introduction

7.2 Impact of Mycoplasma hyopneumoniae interactions with other pathogens on production and economic performance

7.3 Interactions of Mycoplasma hyopneumoniae with bacteria involved in lung diseases

7.3.1 Interaction with Actinobacillus pleuropneumoniae

7.3.2 Interaction with Bordetella bronchiseptica

7.3.3 Interaction with Pasteurella multocida

7.3.4 Interaction with other Mycoplasma species

7.3.5 Interaction with other bacterial species

7.4 Interactions of Mycoplasma hyopneumoniae with viruses involved in lung diseases

7.4.1 Interaction with PRRSV

7.4.2 Interaction with PCV-2

7.4.3 Interaction with swine influenza A viruses

7.4.4 Interaction with other viruses

7.5 Interactions of Mycoplasma hyopneumoniae with parasitic infections and mycotoxins

7.6 Conclusions

CHAPTER 8

Diagnosis of Mycoplasma hyopneumoniae infection and associated diseases

8.1 Introduction

8.2 Clinical-pathological diagnosis

8.2.1 Differential diagnosis

8.3 Detection of the pathogen

8.3.1 Isolation and culturing

8.3.2 Detection and localization of Mycoplasma hyopneumoniae in tissues

8.3.3 Detection of the pathogen by PCR

8.4 Detection of antibodies against Mycoplasma hyopneumoniae infection

8.5 Selecting an adequate sample size

8.6 Conclusions

CHAPTER 9

General control measures against Mycoplasma hyopneumoniae infections

9.1 Introduction

9.2 Production systems

9.2.1 Herd size

9.2.2 Piglet source

9.2.3 Pig flow and batching

9.2.4 Parity one vs. multiparous sows

9.3 Gilt acclimation

9.4 Management

9.4.1 Pre-weaning management

9.4.2 All-in/all-out

9.4.3 Stocking density

9.4.4 Stocking rates

9.4.5 Group size

9.4.6 Other diseases management

9.5 Climate and housing conditions

9.5.1 Seasonality

9.5.2 Thermal sensation (temperature, air speed and humidity)

9.5.3 Air contaminants

9.5.4 Improving air quality

CHAPTER 10

Antimicrobial treatment Mycoplasma hyopneumoniae infections

10.1 Introduction

10.2 Antimicrobial treatments

10.2.1 Antimicrobials

10.2.2 Administration routes of antimicrobials for treatment of respiratory diseases in pigs

10.2.3 Efficacy of several antimicrobials against Mycoplasma hyopneumoniae infections under experimental and field conditions

10.3 In vitro determination of antimicrobial activity against Mycoplasma hyopneumoniae

10.3.1 Minimal inhibitory concentration (MIC) determination

10.3.2 Minimal bactericidal concentration (MBC) determination

10.4 In vitro activities of antibiotics against Mycoplasma hyopneumoniae

10.5 Mycoplasma hyopneumoniae resistance to antimicrobials

10.5.1 Resistance to macrolides

10.5.2 Resistance to fluoroquinolones

10.5.3 Resistance to other antimicrobials

10.6 Conclusions

CHAPTER 11

Vaccines and vaccination against Mycoplasma hyopneumoniae

11.1 Introduction

11.2 Commercial vaccines against Mycoplasma hyopneumoniae

11.3 Mechanisms of protection

11.4 Effects of vaccination

11.5 Vaccination strategies

11.5.1 Piglet vaccination

11.5.2 Breeding gilt vaccination

11.5.3 Sow vaccination

11.5.4 Administration routes

11.6 Factors influencing efficacy of vaccination

11.6.1 Stress factors

11.6.2 Infections with other pathogens at the moment of Mycoplasma hyopneumoniae vaccination

11.6.3 Co-infections with other pathogens involved in PRDC

11.6.4 Diversity of Mycoplasma hyopneumoniae strains

11.6.5 Maternally derived immunity

11.7 Experimental vaccines

CHAPTER 12

Eradication of Mycoplasma hyopneumoniae IURP SLJ KHUGV

12.1 Introduction

12.2 Mycoplasma hyopneumoniae eradication protocols

12.2.1 Depopulation/Repopulation

12.2.2 Swiss method

12.2.3 Herd closure and whole herd medication

12.2.4 Whole herd medication without closure

12.2.5 Other protocols

12.3 The value of Mycoplasma hyopneumoniae eradication

12.4 Eradication economics

12.5 Mycoplasma hyopneumoniae eradication trends

CHAPTER 13

Mycoplasma hyorhinis and Mycoplasma hyosynoviae in pig herds

13.1 Mycoplasma hyorhinis

13.1.1 Etiology

13.1.2 Epidemiology

13.1.3 Pathogenesis

13.1.4 Clinical-pathological presentation

13.1.5 Diagnosis

13.1.6 Therapy

13.1.7 Control and prevention

13.2 Mycoplasma hyosynoviae

13.2.1 Etiology

13.2.2 Epidemiology

13.2.3 Pathogenesis

13.2.4 Clinical signs

13.2.5 Diagnosis

13.2.6 Therapy and control

CHAPTER 14

Mycoplasma suis infections in pigs

14.1 Pathogen history

14.2 Pathogen characteristics

14.3 Epidemiology: prevalence and transmission

14.4 Pathogenesis

14.4.1 Adhesion and invasion

14.4.2 Nutrient scavenging

14.4.3 Eryptosis

14.4.4 Immunopathology

14.4.5 Endothelial targeting

14.5 Incubation period and clinical signs

14.6 Socio-economic impact

14.7 Diagnostics

14.8 Treatment, general control measures and vaccination

References

CONTRIBUTORS

Editors

Dominiek Maes

Faculty of Veterinary Medicine

Ghent University

Ghent, Belgium

[e-mail]

Marina Sibila

Centre de Recerca en Sanitat Animal (CReSA)

Institut de Recerca i Tecnologia Agroalimentàries (IRTA)

Campus de la Universitat Autònoma de Barcelona

Bellaterra, Spain [e-mail]

Maria Pieters

College of Veterinary Medicine

University of Minnesota

St. Paul, USA

[e-mail]

Authors

Alyssa Betlach

College of Veterinary Medicine

University of Minnesota

St. Paul, USA

Swine Vet Center, P.A.

St. Peter, USA

Anne Gautier-Bouchardon

Ploufragan-Plouzané-Niort Laboratory

French Agency for Food, Environmental and Occupational Health and Safety (Anses)

Ploufragan, France

[e-mail]

Filip Boyen

Faculty of Veterinary Medicine

Ghent University

Ghent, Belgium

[e-mail]

John Carr

College of Public Health, Medical and Veterinary Sciences

James Cook University

Queensland, Australia

[e-mail]

Chanhee Chae

College of Veterinary Medicine

Seoul National University

Seoul, Republic of Korea [e-mail]

Céline Deblanc

Swine Virology and Immunology Unit

French Agency for Food, Environmental and Occupational Health and Safety (Anses)

Ploufragan, France

[e-mail]

Odir Antonio Dellagostin

Unit of Biotechnology

Federal University of Pelotas

Pelotas, Brazil

[e-mail]

Steven Djordjevic

The ithree institute

University of Technology Sydney

Sydney, Australia

[e-mail]

Christelle Fablet

Ploufragan-Plouzané-Niort Laboratory

French Agency for Food, Environmental and Occupational Health and Safety (Anses)

Ploufragan, France

[e-mail]

João Carlos Gomes Neto

Nebraska Innovation Campus

University of Nebraska-Lincoln

Lincoln, USA

[e-mail]

Freddy Haesebrouck

Faculty of Veterinary Medicine

Ghent University

Ghent, Belgium

[e-mail]

Katharina Hoelzle

Department Behavioral physiology of livestock

University of Hohenheim

Stuttgart, Germany

[e-mail]

Ludwig Hoelzle

Department Livestock infectiology and environmental hygiene

University of Hohenheim

Stuttgart, Germany

[e-mail]

Sam Holst

Swine Vet Center, P.A.

St. Peter, USA

[e-mail]

Derald Holtkamp

College of Veterinary Medicine

Iowa State University

Ames, USA

[e-mail]

Veronica Jarocki

The ithree institute

University of Technology Sydney

Sydney, Australia

[e-mail]

Jörg Jores

Vetsuisse Faculty

University of Bern

Bern, Switzerland

[e-mail]

Peter Kuhnert

Vetsuisse Faculty

University of Bern

Bern, Switzerland [e-mail]

Enrique Marco

Marco VetGrup SLP

Barcelona, Spain

[e-mail]

Corinne Marois

Ploufragan-Plouzané-Niort Laboratory

French Agency for Food, Environmental and Occupational Health & Safety (Anses)

Ploufragan, France

[e-mail]

Heiko Nathues

Vetsuisse Faculty

University of Bern

Bern, Switzerland

[e-mail]

Tanja Opriessnig

College of Veterinary Medicine

Iowa State University, USA

The Roslin Institute and The Royal (Dick) School of Veterinary Studies

University of Edinburgh, UK

[e-mail]

Andreas Palzer

Veterinary Pig Practice Scheidegg

Scheidegg, Germany

[e-mail]

Mathias Ritzmann

Faculty of Veterinary Medicine

Ludwig-Maximilians-Universität München

München, Germany

[e-mail]

Andrew Rycroft

Department of Pathobiology & Population Sciences

Royal Veterinary College

London, UK

[e-mail]

Joaquim Segalés

Facultat de Veterinària (Universitat Autònoma de Barcelona)

Centre de Recerca en Sanitat Animal (CReSA)-Institut de Recerca i

Tecnologia Agroalimentàries (IRTA)

Campus de la Universitat Autonoma de Barcelona

Bellaterra, Spain

[e-mail]

Guoqing Shao

Institute of veterinary science

Jiangsu Academy of Agriculture Sciences

Nanjing, Jiangsu, China

[e-mail]

Joachim Spergser

University of Veterinary Medicine Vienna

Vienna, Austria

[e-mail]

Institute of Virology and Immunology

Faculty of Veterinary Medicine

University of Bern

Bern, Switzerland

[e-mail]

Paul Yeske

Swine Vet Center, P.A.

St. Peter, USA

[e-mail]

Reviewers

Rachel Derscheid

Veterinary Diagnostic Laboratory

Iowa State University

Ames, USA

[e-mail]

Mathias Devreese

Faculty of Veterinary Medicine

Ghent University

Ghent, Belgium

[e-mail]

Bert Devriendt

Faculty of Veterinary Medicine

Ghent University

Ghent, Belgium

[e-mail]

Jeroen Dewulf

Faculty of Veterinary Medicine

Ghent University

Ghent, Belgium

[e-mail]

Marcelo Gottschalk

Faculty of Veterinary Medicine

University of Montreal

Québec, Canada

[e-mail]

Roberto Maurício Carvalho Guedes

Veterinary School

Universidade Federal de Minas Gerais

Belo Horizonte, Brazil

[e-mail]

Luís Guilherme de Oliveira

School of Agricultural and Veterinarian Sciences

São Paulo State University (Unesp)

Jaboticabal, Brazil

[e-mail]

Isabel Hennig-Pauka

Field Station for Epidemiology

University of Veterinary Medicine Hannover

Bakum, Germany

[e-mail]

Paolo Martelli

Department of Veterinary Science

University of Parma

Parma, Italy [e-mail]

Guy-Pierre Martineau

National Veterinary School of Toulouse

Toulouse, France

[e-mail]

Chris Minion

Veterinary Medicine

Iowa State University

Ames, USA

[e-mail]

Jens Peter Nielsen

Det Sundhedsvidenskabelige Fakultet

Københavns Universitet

Copenhagen, Denmark

[e-mail]

Katharina Stärk

Royal Veterinary College

London, UK

[e-mail]

Karine Ludwig Takeuti

Federal University of Rio Grande do Sul, Brazil

Porto Alegre, Brazil [e-mail]

Pablo Tamiozzo

Facultad de Agronomía y Veterinaria

Universidad Nacional de Río Cuarto

Río Cuarto, Argentina

[e-mail]

Tijs Tobias

Faculty of Veterinary Medicine

Utrecht University

Utrecht, The Netherlands

[e-mail]

Per Wallgren

Dept of Animal Health and Antimicrobial Strategies

National Veterinary Institute

Uppsala, Sweden

[e-mail]

PROLOGUE

Glenn F. Browning

Mycoplasmas are among the most important bacterial pathogens of pigs. They establish chronic infections that are difficult to eliminate, from the pig and the farm. They result in significant economic loss, predispose pigs to disease caused by more acute pathogens, and have an impact on the welfare of the pigs.

Control of mycoplasmosis in the intensive chicken industry has reached a point where antimicrobial therapy for mycoplasmas is rarely required in many countries. This has been achieved by establishing mycoplasma-free breeding stock, by development of accurate diagnostic tests, implementation of regular testing, development of vaccines that limit infection, eliminate its effects and reduce transmission, and by implementing rigorous biosecurity protocols. Control of mycoplasmoses is an important goal for the global pig industry, and recent eradication efforts attest to the level of determination in some countries to achieve this goal. However, the limitations we still face in effectively controlling porcine mycoplasmoses is one of the leading reasons for using antimicrobials in pig farms. Total disease control should be achievable with sufficient ongoing research and development to enhance our tools for control and their application in the field.

Better management of the mycoplasmoses in pigs requires a comprehensive approach. We need better understanding of many fundamental aspects of the biology of these pathogens. While there are several pathogenic mycoplasmas of pigs, Mycoplasma hyopneumoniae, is undoubtedly the most important. M. hyopneumoniae was first identified in the 1960s (Goodwin et al., 1967; Goodwin and Whittlestone, 1963; 1966; Mare and Switzer, 1966), although the disease it causes, enzootic pneumonia, was recognized as a distinct entity in the early 1950s (and named viral pneumonia of pigs at the time) (Gulrajani and Beveridge, 1951).

However, our understanding of it has tended to lag behind that which we have of many other important pathogenic mycoplasmas, in part because it has more fastidious growth requirements than many of the mycoplasmas. Recent work exploring the mechanisms used by M. hyopneumoniae to generate surface diversity has demonstrated how complex this bacterium is, in spite of its apparent genomic simplicity. Improvements in tools for control of this pathogen will require a fuller understanding of how this organism interacts with its host, including the interplay between its adhesins and their receptors on the ciliary mucosa. In most mycoplasmoses, the major cause of damage in infected tissues is immunopathology, so a full appreciation of the interactions with the immune system is critical to understanding the diseases we wish to prevent.

While a better understanding of the organism and its interactions with its host will assist in the development of better tools for control, the optimal application of current and future tools depends on our knowledge of the clinical picture in affected pigs and, crucially, of the epidemiology of the pathogen and the disease it causes. A major contributor to the impact of M. hyopneumoniae is its interactions with other bacterial and viral pathogens, as co-infections with respiratory pathogens are the norm in large populations of animals, and frequently lead to much more severe outcomes than single infections with these pathogens.

Finally, while we are waiting for novel tools for improving our control of M. hyopneumoniae, it is essential that we understand how best to use those diagnostic tests, vaccines and antimicrobial drugs we currently have available. This will reduce production losses, minimize less effective use of antimicrobial drugs, and reduce the selection for resistance in M. hyopneumoniae itself, as well as in other pathogens in pigs.

While much attention has rightly been focussed on M. hyopneumoniae, it is important to recognize that there are other significant mycoplasmal pathogens of pigs. Mycoplasma hyorhinis and Mycoplasma hyosynoviae are important causes of infectious arthritis in pigs, a disease problem that also results in considerable economic loss and that adversely affects the welfare of growing pigs, while Mycoplasma suis, a parasite of erythrocytes, can also cause economic losses in growing pigs.

The following chapters of this book address all these issues, and many others. The authors are international experts and offer a global perspective on this important group of pathogens. They have succinctly summarized our current knowledge and highlighted the gaps we need to fill to further improve the welfare and productivity of the animals under our care.

GLENN F. BROWNING

Asia-Pacific Centre for Animal Health, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria 3010, Australia.

ABBREVIATIONS

CHAPTER 1

OVERVIEW OF THE GENERAL CHARACTERISTICS AND CLASSIFICATION OF PORCINE MYCOPLASMA SPECIES

Andrew Rycroft¹

1.1 Introduction

1.1.1 Characteristics of Mollicutes and mycoplasmas

The bacteria which permanently lack cell wall peptidoglycan are placed in the class Mollicutes (Latin for soft skin). This is a distinct class of prokaryotes which includes genera such as Mycoplasma and Ureaplasma. They are free-living prokaryotes with small cells (0.2 – 0.4 μm) and are pleomorphic because they have no shape-defining cell wall. They also tend to produce very small colonies in culture. These bacteria carry a small genome (usually between 700 and 1000 kbp). This presumably confers only a relatively limited anabolic and catabolic ability compared to many bacteria. This may reflect the failure to acquire or evolve more functions or, alternatively, the loss of genetic capacity as the organism has degenerated to rely on its close association with the host.

The first mollicute to be successfully cultured was Mycoplasma mycoides, the organism we now recognize as the causative agent of bovine pleuropneumonia. Later isolations of mycoplasmas or other Mollicutes resembling M. mycoides were referred to as pleuropneumonia-like organisms or PPLO. This name was widely used but was gradually replaced by the term Mycoplasma for all bacteria of this type causing disease in humans and animals. The term Mollicutes was agreed for the entire class of organisms in 1967 but the word "Mycoplasma" that in the correct sense only refers to the genus, still persists among biologists as the general or trivial term for these wall-less bacteria in many situations today.

1.1.2 Phylogenetic relationships of mycoplasmas

Until the advent of molecular (DNA-based) investigations, the classification of Mollicutes was reliant on culture-based characteristics and serology. Often growth requirements in culture were difficult to determine because successful culture usually requires animal serum. Since serum contains a wide variety of undefined chemicals and nutrients, it was usually not possible to recognize which components were essential requirements and which were not. The growth-inhibition test became widely established as the method of choice for recognition and classification of Mollicutes. This used antibody in hyperimmune serum, usually applied to an absorbent paper disc, to inhibit the growth of a mollicute on a culture plate. This activity was specific and only caused the inhibitory effect on strains of the same species or those very closely related to the strain used as antigen to raise the hyperimmune serum. Unfortunately, the method depended on the sharing of sera and was not always reproducible between laboratories. It was also dependent upon the key antigen or antigens for inhibition being present on all members of the species but not on any other species. This worked well within the boundaries of a laboratory dealing with a relatively restricted range of organisms and with known anti-sera. But as the number of possibilities increased, the number of sera needed also increased, as did the possibility of growth-restricting antigens being shared with unrelated organisms.

With the availability of DNA sequence analysis, particularly that encoding ribosomal RNA, estimates could be made of evolutionary distance between species. A number of attempts to do this have been made, perhaps with increasing accuracy, and these have greatly helped our understanding of the relationships between Mollicutes (Toth et al., 1994).

Mollicutes are sometimes described in the literature as Gram-negative, but the reason for this is unclear, and it is mistaken, since they possess neither outer membrane nor lipopolysaccharide, the key elements of a Gram-negative cell envelope. Almost certainly, Mollicutes are distantly related to the Gram-positive bacteria (Firmicutes, Latin: firm skin). Evidence for this was first published by Woese et al. (1980) using 16S rRNA sequence analysis. They concluded that the Mollicutes (Mycoplasma, Spiroplasma and Acholeplasma) arose by degenerative or regressive evolution. This apparently happened in a branch of Prokaryotes related to ancestors of Clostridium that led to the genera Bacillus and Lactobacillus. There remained considerable uncertainty from this study and the authors concluded that the Mollicutes were not a phylogenetically coherent group but they were all related to the family of Gram-positive bacteria, the Bacillaceae.

Revision and refinement of this analysis of 16S rRNA sequence by Weisburg et al. (1989) and Manilov (1992) from 48 different species of Mollicutes led to the recognition of 5 distinct phylogenetic groups or clades. These were the Spiroplasma, Hominis and Pneumoniae groupings together with the Anaeroplasma group (which included Acholeplasma) and a separate group containing the single species Asteroleplasma anaeroboium.

Phylogenetic relatedness based on the analysis of 16S rRNA is not reflected in the characteristics most easily seen by a bacteriologist. Thus, the site of disease, the ease and speed of growth in culture, nutritional requirements and the colonial appearance have all been thought of as meaningful in showing the similarity or differences between species of Mollicutes. Nevertheless, the DNA evidence must carry considerable weight and the superficial phenotypic features that were previously considered important in classifying these organisms are now seen to be of little significance in the phylogenetic relationships of these organisms (Figure 1.1).

Figure 1.1. Simplified groupings of porcine Mycoplasma species based on the 16S rRNA derived groups and clusters of Weisburg et al. (1989) but modified to include results of the hemotropic mycoplasmas from Peters et al. (2008) and Siqueira et al. (2013)

The Spiroplasma group consists of four clusters, one of which is the agent of contagious bovine pleuropneumonia (CBPP) M. mycoides, and another is M. capricolum. The majority of members known to be in this group are the helical spiroplasmas: usually found on plants, insects and arachnids.

The Pneumoniae group includes the human pathogen M. pneumoniae and the avian respiratory pathogen M. gallisepticum among others.

The Hominis group includes many of the mollicute animal pathogens including the porcine mycoplasmas.

It has been suggested that the evolutionary distance from Gram-positive bacteria must be very great. One characteristic of mycoplasmas that speaks volumes is the differential use of the TGA codon. Bacteria of almost all types use TGA (UGA) as a stop codon while all mycoplasmas (excluding acholeplasmas and phytoplasmas) use this to code for the amino acid tryptophan. Such a fundamental difference that is seen so widely among the Mollicutes indicates a long isolation of these organisms from other bacteria. However, Weisburg et al. (1989) proposed that from the molecular evidence of DNA sequence, Mollicutes appear to be normal bacteria and they were described as having an unspectactular phylogenetic position. Despite the very different phenotypic appearance arising from the lack of peptidoglycan, the evidence suggests that this is not because Mollicutes are phylogenetically distant from other eubacteria.

Another characteristic of Mollicutes that has come to light with genome sequencing and annotation, is the apparent lack of pseudogenes or intermittent junk DNA. The presence of rusting hulks of working genes, as Steve Jones refers to them (Jones, 1993), might be expected if a proportion of the genetic makeup of Gram-positive bacteria was no longer required and had become redundant in Mollicutes. Perhaps, and we can only speculate, genome sized reductions have allowed removal of unnecessary, non-functional DNA more efficiently than appears to happen in eukaryotes particularly.

Yet another characteristic of Mollicutes is their inclusion of cholesterol in their cytoplasmic membrane. This is thought to give some stability to the membrane. Most Mollicutes acquire the cholesterol from an exogenous source. Acholeplasmas (which also lack peptidoglycan) do not require exogenous cholesterol because they have the capacity to synthesize it and incorporate this in their membrane for stability (Khan et al., 1981).

Finally, a characteristic of mycoplasmas is their very close association, in many cases, with other organisms (Razin et al., 1998). Their niche is to have an intimate contact with the mucosal surfaces of animals and plants. It is this very close association with host cells, of which we understand relatively little, that is the key to understanding mycoplasmas. These organisms have evolved alongside animals and plants to be quiet intruders: living, like many commensal organisms, without prompting a response from the host nor causing it perceptible damage. Some, of course, do prompt an inflammatory response and these we see as the pathogens. The reason for those mycoplasmas causing a response may be to assist in their spread to other animals or it may be accidental as environments change or the genetics and physiology of the host alters in some way. It is the subtleties of this close association between host and Mycoplasma, and perturbation of the balance in that close relationship, that will provide the solution to understanding mycoplasmas and Mycoplasma pathogenicity in the future.

1.1.3 Pathogenicity in mycoplasmas

Specific aspects of pathogenicity of porcine mycoplasmas will be dealt with in later chapters. In general, however, the mechanisms by which mycoplasmas cause disease often remain either obscure or speculative. Because mycoplasmas are so different from many other pathogens studied there is limited opportunity to recognize homologues of genes known in other pathogens. It is not surprising that biosynthetic genes for amino acids and growth factors are missing; but global regulatory genes such as the sigma factor rpoS, transcriptional repressor crp and the leucine-responsive regulatory protein lrp, are also absent (Salyers and Whitt, 2002). It is therefore likely that mycoplasmas have independently developed their own regulatory systems. Similarly, they could have as yet unrecognized functions acting to engage with host cells, enable their survival in vivo and promote transmission between hosts.

It appears clear that adhesion to mucosal surfaces is an important aspect of colonization of host tissues. Mycoplasmas are extracellular surface parasites although some have been reported to get inside host cells (Yavlovich et al., 2004; McGowin et al., 2009; Burki et al., 2015). Because mycoplasmas lack the peptidoglycan of a cell wall or the lipopolysaccharide of the outer membrane, they present a very different exposed surface to that of most bacteria. The antigenic composition and topography of the single plasma membrane must be critical in the avoidance of recognition and evasion of damage from the innate immune response of the host. Some important progress has been made in understanding the role of this membrane surface (as will be detailed in later sections) but investigations of the host-pathogen relationship of mycoplasmas and their animal hosts is difficult and much remains to be understood at the cellular and molecular level.

Early suggestions of one or more toxins produced by M. hyopneumoniae (Debey and Ross, 1994) have not yet been corroborated. It was suggested that cytopathological effects observed from the human pathogen M. pneumoniae, including loss of cilia, was likely to be related to elaboration of a toxic substance or enzyme by Gabridge et al. (1974). However, the existence of any mycoplasma-derived protein exotoxins is not yet established. In contrast, the involvement of small molecules such as H2O2 in cellular damage has gained some support, at least in specific (bovine) mycoplasmas but also in porcine mycoplasmas (Galvao Ferrarini et al., 2018). In contrast, mutants of M. gallisepticum unable to produce H2O2 from glycerol were consistently virulent in the respiratory tracts of experimental chickens implying that H2O2 is simply not required for this organism to cause disease, at least in some circumstances (Szczepanek et al., 2014). The role of membrane phospholipases, perhaps acting on the host cell membrane, is still unclear (Shibata et al., 1995; Rottem and Naot, 1998).

What is clear is that mycoplasmas are stealthy. They are able to persist in the body of the host, either on the mucosal surface or even, it appears, in major organs, without alerting the innate immune system to their presence. Or perhaps the immune system is alerted, but then suppressed or silenced. This is seen in the poor immunological responses to infection and in the relatively limited inflammatory response. Related to this is the antigenic variation displayed by many Mycoplasma organisms such as M. hyorhinis (Wise et al., 1992). The importance of altering surface antigens over time and the role it might play in evasion of the host immune defenses remains to be seen.

For many years it had been recognized that mycoplasmas bind immunoglobulin onto their surface. This was considered likely to be a passive means of coating the bacteria with self antigen and thereby evading recognition by the innate immune system. More recently, a two-protein immunoglobulin (Ig) binding system was described by Arfi et al. (2016). This was originally found in the ruminant mycoplasma M. mycoides subsp. capri. One protein, the mycoplasma immunoglobulin binding protein (MIB), captures antibody molecules with high affinity while the second, a serine protease known as mycoplasma Ig protease (MIP), cleaves the heavy chain of the Ig molecule. Analysis of genomes suggests that this system is widespread among the Mycoplasma pathogens and may contribute to avoidance of recognition and consequent immune evasion by mycoplasmas invading the body.

One important aspect of Mycoplasma pathogenicity that has become apparent is the lack of ability of phagocytic cells (neutrophils and macrophages) to eliminate these pathogens in the body. The reason for this is not yet known but mycoplasmas appear to be hiding effectively from the cells that are intended to destroy them. In some cases there is a clear cytokine response to Mycoplasma infection (Rottem and Noat, 1998). Lymphocytes are activated but sometimes in an inappropriate manner such that B cells are non-specifically stimulated and generate ineffective antibodies. Perhaps this is a deliberate attempt to subvert the immune response as can be seen in other bacterial pathogens. In Staphylococcus aureus and Streptococcus equi superantigens such as TSST-1 are produced. These directly activate T cells without the requirement for an antigen to be presented in the context of Class II