Final Report Summary - MAPSEQ (High-throughput sequencing to analyse genome variation and evolution in Map strains.)
Project context
Mycobacterium avium subspecies paratuberculosis (Map) causes infectious chronic enteritis of principally ruminants (Johnes disease or paratuberculosis) and is responsible for significant global economic losses to the livestock and associated industries. Controversially, Map has been associated with Crohns disease in humans. Since the organism is found in milk, blood and muscle of infected animals it can enter the food chain and has been detected in retail milk and milk products, which if ingested could potentially lead to infection in humans. Paratuberculosis is therefore an animal health and welfare problem and potential zoonosis and as such precautionary steps need to be taken to eliminate Map from the food chain. However, control of the disease is problematic and there is a lack of understanding of the epidemiology of paratuberculosis, virulence of strain types, interspecies transmission and the role of wildlife reservoirs. This information is paramount for designing and implementing efficient control programmes.
Project objectives
This project analysed genome sequences from 125 Map isolates to study genetic diversification and evolution. An understanding of the evolutionary dynamics of Map populations has important implications for the control of paratuberculosis, and genome information can provide insights into virulence, pathogenicity and host preference.
Project work
A panel of Map strains was carefully selected from the unique, international collection maintained at the Moredun Research Institute (MRI) in Scotland. The panel comprised isolates selected from different geographic regions and from different hosts, where possible. The majority of isolates were from European countries but a few from other continents were included to give a global dimension to the analyses. Where possible, isolates with known typing profiles were selected. There were representative isolates from recognised strain groups (Type I or S Type; Type II or C Type; Type III and Bison-type), isolates from the ATCC isolated from human Crohn's patients, pigmented isolates and Map strains used in the former vaccine preparations. The isolates were grown in Middlebrook based media with the appropriate supplements and DNA extracted at MRI. The DNA samples were sequenced at the Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK using the Illumina/Solexa Genome Analyzer.
The first objective was to assemble and annotate the genome sequence of a Type I pigmented Map strain. This was necessary to provide a scaffold for the assembly of the Solexa reads from the Type I strains and subsequent SNP analysis. Due to the relatively short read lengths and presence of repeat sequences and insertion sequences in the genome, it was not possible to close the genome sequence within the time constraint of the project. However, although a complete and fully annotated Type I sequence was not obtained, the assembled sequence data proved extremely valuable for follow-on studies with Type I strains.
The second objective was to analyse the genome sequence data for single nucleotide polymorphisms (SNPs) that could differentiate between the different Map strain types and between isolates from Crohns patients and livestock. The work is ongoing but at the end of the project period a significant number of SNPs (~3276) were identified that could differentiate between Types I/III and Type II. Additional SNPs could differentiate Type I strains (~1193) and Type III strains (~868). No SNPs were found that could differentiate the human Map isolates from animal isolates.
The final objective was to perform phylogenetic analysis to provide insights into the evolution and diversification of Map strains. Analyses confirmed that there are two major groups of Map strains: Type I/III corresponding to the previously described 'S' or 'sheep' type and Type II synonymous with the 'C' or 'cattle' type. The Type I and III strains were more closely related to each other than to the Type II strains. The human isolates clustered with the Type II strains and in general human and animal isolates from the same region clustered together. There were no SNPs associated with host species, including bison. Bison-type strains included isolates from cow, goat, moufflon and buffalo. No SNPs were identified associated with a particular country.
Project outcomes
Few whole genome intraspecies comparisons of pathogenic bacteria have been done on this scale and this is the first of such studies in mycobacteria. The project has generated important data. The results have clarified the relationships between the different Map strain types. As there does not appear to be any SNPs to differentiate Crohns isolates from livestock isolates, this suggests that the strains found in humans are the same as those found in livestock, which has important implications for public health. SNPs can be used to distinguish between isolates and will lead to improved reliable epidemiological tracing, monitoring and surveillance, which will assist in the control of paratuberculosis. The project is ongoing and the genome data is being mined for information relating to virulence, pathogenicity, host preference and other phenotypic characteristics, which will facilitate the design of vaccines and novel diagnostic markers and identify drug targets for treatment and control of paratuberculosis.
Socio-economic impact
The ultimate socio-economic impact of this project will be improved health and welfare of livestock and the quality and safety of dairy and meat produce. This will lead to greater efficiency and cost savings which will improve the competitiveness of the European livestock and dairy industries in internal and global markets and, in turn, lead to increased employment opportunities and wealth creation. The production of healthier, safer and higher-quality dairy and meat produce will enhance consumer satisfaction and EU citizens' quality of life. The improvement of agricultural systems will help support sustainable development policies and the quality of the environment. Epidemiological monitoring, surveillance and control of paratuberculosis will lead to improvements in the natural environment by limiting spread of the disease to wildlife.
Mycobacterium avium subspecies paratuberculosis (Map) causes infectious chronic enteritis of principally ruminants (Johnes disease or paratuberculosis) and is responsible for significant global economic losses to the livestock and associated industries. Controversially, Map has been associated with Crohns disease in humans. Since the organism is found in milk, blood and muscle of infected animals it can enter the food chain and has been detected in retail milk and milk products, which if ingested could potentially lead to infection in humans. Paratuberculosis is therefore an animal health and welfare problem and potential zoonosis and as such precautionary steps need to be taken to eliminate Map from the food chain. However, control of the disease is problematic and there is a lack of understanding of the epidemiology of paratuberculosis, virulence of strain types, interspecies transmission and the role of wildlife reservoirs. This information is paramount for designing and implementing efficient control programmes.
Project objectives
This project analysed genome sequences from 125 Map isolates to study genetic diversification and evolution. An understanding of the evolutionary dynamics of Map populations has important implications for the control of paratuberculosis, and genome information can provide insights into virulence, pathogenicity and host preference.
Project work
A panel of Map strains was carefully selected from the unique, international collection maintained at the Moredun Research Institute (MRI) in Scotland. The panel comprised isolates selected from different geographic regions and from different hosts, where possible. The majority of isolates were from European countries but a few from other continents were included to give a global dimension to the analyses. Where possible, isolates with known typing profiles were selected. There were representative isolates from recognised strain groups (Type I or S Type; Type II or C Type; Type III and Bison-type), isolates from the ATCC isolated from human Crohn's patients, pigmented isolates and Map strains used in the former vaccine preparations. The isolates were grown in Middlebrook based media with the appropriate supplements and DNA extracted at MRI. The DNA samples were sequenced at the Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK using the Illumina/Solexa Genome Analyzer.
The first objective was to assemble and annotate the genome sequence of a Type I pigmented Map strain. This was necessary to provide a scaffold for the assembly of the Solexa reads from the Type I strains and subsequent SNP analysis. Due to the relatively short read lengths and presence of repeat sequences and insertion sequences in the genome, it was not possible to close the genome sequence within the time constraint of the project. However, although a complete and fully annotated Type I sequence was not obtained, the assembled sequence data proved extremely valuable for follow-on studies with Type I strains.
The second objective was to analyse the genome sequence data for single nucleotide polymorphisms (SNPs) that could differentiate between the different Map strain types and between isolates from Crohns patients and livestock. The work is ongoing but at the end of the project period a significant number of SNPs (~3276) were identified that could differentiate between Types I/III and Type II. Additional SNPs could differentiate Type I strains (~1193) and Type III strains (~868). No SNPs were found that could differentiate the human Map isolates from animal isolates.
The final objective was to perform phylogenetic analysis to provide insights into the evolution and diversification of Map strains. Analyses confirmed that there are two major groups of Map strains: Type I/III corresponding to the previously described 'S' or 'sheep' type and Type II synonymous with the 'C' or 'cattle' type. The Type I and III strains were more closely related to each other than to the Type II strains. The human isolates clustered with the Type II strains and in general human and animal isolates from the same region clustered together. There were no SNPs associated with host species, including bison. Bison-type strains included isolates from cow, goat, moufflon and buffalo. No SNPs were identified associated with a particular country.
Project outcomes
Few whole genome intraspecies comparisons of pathogenic bacteria have been done on this scale and this is the first of such studies in mycobacteria. The project has generated important data. The results have clarified the relationships between the different Map strain types. As there does not appear to be any SNPs to differentiate Crohns isolates from livestock isolates, this suggests that the strains found in humans are the same as those found in livestock, which has important implications for public health. SNPs can be used to distinguish between isolates and will lead to improved reliable epidemiological tracing, monitoring and surveillance, which will assist in the control of paratuberculosis. The project is ongoing and the genome data is being mined for information relating to virulence, pathogenicity, host preference and other phenotypic characteristics, which will facilitate the design of vaccines and novel diagnostic markers and identify drug targets for treatment and control of paratuberculosis.
Socio-economic impact
The ultimate socio-economic impact of this project will be improved health and welfare of livestock and the quality and safety of dairy and meat produce. This will lead to greater efficiency and cost savings which will improve the competitiveness of the European livestock and dairy industries in internal and global markets and, in turn, lead to increased employment opportunities and wealth creation. The production of healthier, safer and higher-quality dairy and meat produce will enhance consumer satisfaction and EU citizens' quality of life. The improvement of agricultural systems will help support sustainable development policies and the quality of the environment. Epidemiological monitoring, surveillance and control of paratuberculosis will lead to improvements in the natural environment by limiting spread of the disease to wildlife.