3 research outputs found
Actinobacillus pleuropneumoniae serovar 8 predominates in England and Wales
This work was supported by a Longer and Larger (LoLa) grant from the Biotechnology and Biological Sciences Research Council (BBSRC grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1 and BB/G018553/1) and Zoetis (formerly Pfizer Animal Health) awarded to the Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) Consortium
Study of papillomavirus latent infection in an animal model
Papillomaviruses cause a wide spectrum of benign and neoplastic diseases
in humans and animals. They may also cause infections that are
characterised by the absence of clinical signs of disease and some of these
may represent viral latency. Many viruses have a latent stage of their life
cycle and papillomaviruses appear to be no different. For example, some
low-risk human papillomavirus types such as HPV-11 and HPV-6 can cause
infections that resemble latency. It has recently been shown that rabbit oral
papillomavirus (ROPV) is an appropriate model of these virus types.
Infection of the tongue mucosa with ROPV leads to the formation of benign
papillomas that form and regress within a matter of weeks.
In this thesis, we show that ROPV is a suitable model system for the study of
latent papillomavirus infections. The regression of ROPV papillomas is
followed by the persistence of viral DNA and RNA in the absence of clinical
signs of disease. Persistence of ROPV DNA is generally restricted to basal
epithelial cells at sites of previous infection. Low copy numbers of viral DNA
in the basal layer are compatible with infection remaining in only a subset of
these cells (possibly epithelial stem cells). Typically there is no amplification
of viral DNA in the upper layers of the epithelium. ROPV proteins are
undetectable during latency suggesting that the productive stages of the life
cycle are not completed. Low levels of ROPV early transcripts are
detectable and it is possible that early proteins are necessary to allow stable
maintenance of viral episomes in basal epithelial cells.
We attempt to demonstrate the ability of latent ROPV infection to reactivate
to form clinical disease. Evidence of spontaneous reactivation was seen on
one occasion, but efforts to initiate reactivation by immunosuppressing
rabbits were hampered by the toxicity of the drugs used. However, our
preliminary data suggest that immunosuppression of rabbits can cause
reactivation of latent ROPV
Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS).
Comprehensive identification of conditionally essential genes requires efficient tools for generating high-density transposon libraries that, ideally, can be analysed using next-generation sequencing methods such as Transposon Directed Insertion-site Sequencing (TraDIS). The Himar1 (mariner) transposon is ideal for generating near-saturating mutant libraries, especially in AT-rich chromosomes, as the requirement for integration is a TA dinucleotide, and this transposon has been used for mutagenesis of a wide variety of bacteria. However, plasmids for mariner delivery do not necessarily work well in all bacteria. In particular, there are limited tools for functional genomic analysis of Pasteurellaceae species of major veterinary importance, such as swine and cattle pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, respectively. Here, we developed plasmids, pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the transposase gene), that allow delivery of mariner into both these pathogens, but which should also be applicable to a wider range of bacteria. Using the pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant libraries in both A. pleuropneumoniae and P. multocida that showed a near random distribution of insertions around the respective chromosomes as detected by TraDIS. A preliminary screen of 5000 mutants each identified 8 and 14 genes, respectively, that are required for growth under anaerobic conditions. Future high-throughput screening of the generated libraries will facilitate identification of mutants required for growth under different conditions, including in vivo, highlighting key virulence factors and pathways that can be exploited for development of novel therapeutics and vaccines