A multivalent vaccine and single platform diagnostic for bacterial respiratory diseases of pigs

Lead Research Organisation: Royal Veterinary College
Department Name: Pathology and Pathogen Biology


See main proposal.

Technical Summary

See main proposal.


10 25 50
Description The project has developed new genetic techniques and tools for the manipulation of Mycoplasma hyopneumoniae. Previously, this had not been possible at all but we now have an efficient system to prepare random and site-specific mutants. This has led to the TraDIS analysis of large pools of mutants in vitro and in vivo to determine (1) the genes required for life (essential genome) and (2) those involved in survival and fitness in the body of the animal.

Mutants lacking a key gene required for survival in the body of the pig have been identified from the functional genomic analysis. These have been isolated from our library of mutants and have been examined, using experimental infection in the pig, for safety (ability to cause pneumonia). Selected mutants have then been tested as vaccine candidates in the pig for their ability to cause an immune response to protect against experimental challenge. The most promising of these has now been transmitted to Zoetis for evaluation as a potential commercial vaccine. Other candidates have also been derived and discussions with Pharma for further evaluation and exploitation have been held.

Substantial progress has also been made with Actinobacillus pleuropneumoniae, Haemophilus parasuis and Streptococcus suis, primarily in the laboratories of our collaborators on the grant: Langford, Maskell, Tucker & Wren.
Exploitation Route Live attenuated vaccination of pigs against respiratory disease. Pharmaceutical companies are currently evaluating our results to determine whether they wish to exploit the findings in the development of a new generation of vaccines against pneumonia together with the diagnostic systems developed in the project.
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

URL http://www.rvc.ac.uk/about/our-people/andrew-rycroft
Description The outcomes from this project are in progress. The project has led to new opportunities for vaccination and diagnostic techniques for pig respiratory disease which are being evaluated for development by pharmaceutical companies. Only then will the full impact of these results become manifest. Diagnostic methods are being exploited for the pig industry in Europe by RVC and they are being evaluated as part of a technical package by CEVA. Different novel vaccine opportunities are currently being evaluated by Zoetis. From the RVC, we have offered the option of testing, licencing and registering a live-attenuated enzootic pneumonia vaccine. From other members of the consortium (University of Cambridge) there is the opportunity to exploit results for vaccination against Streptococcus suis meningitis in the pig. Other developments arising from the findings are expected. Intermediate findings in the development of genetic tools for M. hyopneumoniae have allowed functional genomic analysis of the organism. The opportunity to use our data towards a novel vaccine and diagnostic system for M hyo disease are now clear. Research for this is ongoing.
First Year Of Impact 2015
Sector Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

Title Collection and sequencing of 125 strains of Mycoplasma hyopneumoniae. 
Description 125 strains of Mycoplasma hyopneumoniae have been amassed from different geographical locations and from 1969 to 2014. These are not easy to isolate in culture. All strains have been whole genome sequenced. 
Type Of Material Biological samples 
Year Produced 2013 
Provided To Others? Yes  
Impact Understanding the phylogenetic structure and temporal changes in the species Mycoplasma hyopneumoniae. Recognition of unique genes common to the species leading to research and development of diagnostic tools for the recognition of Mycoplasma hyopneumoniae and for the development of improved serological testing of pigs. Antibiotic sensitivity profiling of strains of M hyopneumoniae and publication of this. 
Title Genetic manipulation of Mycoplasma hyopneumoniae 
Description Genetic methods and tools have been developed for the genetic transformation of M. hyopneumoniae. We have used these to demonstrate site-directed mutagenesis of M. hyopneumoniae. Tools have also been developed to derive transposon mutants of M. hyopneumoniae and to use these to design and produce candidate live-attenuated vaccines against enzootic pneumonia in the pig. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact Great interest from the mycoplasma research community. 
Description USDA Ames Iowa 
Organisation U.S. Department of Agriculture USDA
Department National Animal Disease Center
Country United States 
Sector Public 
PI Contribution Contribution at meeting in Ames for design of experiments for pathogenicity testing and vaccination/protection of pigs for Streptocioccus suis and Haemophilus parasuis.
Collaborator Contribution Provision of cesearean-derived, colostrum-deprived piglets for challenge studies with Streptococcus suis. Pathogenicity testing and vaccination studies with S. suis and H. parasuis in piglets.
Impact Submission of grant proposal to USDA (Agriculture and Food Research Initiative, AFRI): Tracy L Nicholson Proposal "US-UK Collaborative: Disease and transmission dynamics of Mycoplasma hyopneumoniae". This was relient entirely upon the technological developments at RVC arising from the BBSRC LoLa grant on pig respiratory vaccination & diagnostics.
Start Year 2012
Description UTS Sydney 
Organisation University of Technology Sydney
Country Australia 
Sector Academic/University 
PI Contribution Hosting of a PhD student in the lab at RVC for 3 months. Transfer of techniques for culture and mutagenesis of Mycoplasma hyopneumoniae. Identification of genes of M. hyopneumoniae as contributing to aspects of pathogenicity and biofilm formation. Transfer of gridded (ordered) library of 5000 transposon mutants (3000 from RVC lab) to UTS Sydney. Contribution, as co-applicant, on a grant proposal to the Australian Research Council 2014 and 2015. Discussions of aberrant (non-conventional) mycoplasmal respiratory disease (fibrinous pleuritis) observed in pigs and cattle.
Collaborator Contribution Seminar by Steve Djordjevic at RVC. Inclusion of ANR and GAM on a poster at 3rd Prato conference on Pathogenicity of Bacteria in Animal Disease (October 2014). Advice on microscopic techniques and stains for Alanah Deeney, PhD student by Ben Raymond. Meetings in Sydney and Melbourne to discuss current understanding of Mycoplasma pathogenicity and future research direction. Discussion of possible visit to UTS by junior staff and students particularly for their microscope expertise and facility.
Impact Poster at 3rd Prato Conference.: Raymond, B., Rohde, M., Padula, M., Maglennon, G.A. Rycroft, A.N. & Djordjevic, S. (2014). Proteins involved in the adherence of Mycoplasma hyopneumoniae to abiotic surfaces and porcine monolayers which may also play a role in biofilm formation. Proceedings of the 2nd Prato Conference on the Pathogenesis of Bacterial Diseases of Animals. Prato, Italy, October 2014. abs #36.
Start Year 2014
Title Methods for genetic manipulation of Mycoplasma hyopneumoniae. 
Description Genetic tools have been developed for the transformation of M. hyopneumoniae and for the mutagenesis of M. hyopneumoniae to derive site-directed mutants of M. hyopneumoniae. Tools have also been developed for efficient transposon mutagenesis of M. hyopneumoniae and to design and produce candidate live-attenuated vaccines. Abstract: A plasmid for transforming Mycoplasma hyopneumoniae comprising an oriC region comprising both AT-rich regions from a predicted oriC region of M. hyopneumoniae 232, or comprising a variant thereof that retains the ability to act as an OriC region in M. hyopneumoniae and has at least 90% sequence identity with a sequence comprising both AT-rich regions from a predicted oriC region of M. hyopneumoniae 232. A M. hyopneumoniae cell transformed with a plasmid of the invention may be useful in a vaccine composition. A nucleic acid construct comprising a nucleic acid sequence encoding a transposase enzyme and a promoter sequence, wherein the promoter sequence is active in M. hyopneumoniae; optionally wherein the promoter sequence comprises a promoter sequence from M. hyopneumoniae, or spiralin gene promoter of Spiroplasma citri or tetM promoter sequence from S. aureus; optionally wherein the promoter sequence from M. hyopneumoniae is a constitutively active promoter sequence, optionally a promoter sequence from the M. hyopneumoniae ldh, P97, secD, Tuf, rpoB, P146, ATP transporter ATP binding protein, Asparagine-tRNA synthetase or Translation elongation factor gene. The transposase enzyme may be a Mariner family transposase, optionally Himar1 transposase or Himar1 C9 mutant transposase. 
IP Reference WO2014174257 
Protection Patent application published
Year Protection Granted 2013
Licensed No
Impact Nil