Dataset Information

Diagnostic Sequences That Distinguish M. avium Subspecies Strains.

ABSTRACT: Over a decade ago Mycobacterium avium subspecies paratuberculosis (Map) specific genes were initially identified in a whole genome context by comparing draft genome sequences of Map strain K-10 with Mycobacterium avium subspecies hominissuis (Mah) strain 104. This resulted in identification of 32 Map specific genes, not including repetitive elements, based on the two-genome comparison. The goal of this study was to define a more complete catalog of M. avium subspecies-specific genes. This is important for obtaining additional diagnostic targets for Johne's disease detection and for understanding the unique biology, evolution and niche adaptation of these organisms. There are now over 28 complete genome sequences representing three M. avium subspecies, including avium (Maa), Mah, and Map. We have conducted a comprehensive comparison of these genomes using two independent pan genomic comparison tools, PanOCT and Roary. This has led to the identification of more than 250 subspecies defining genes common to both analyses. The majority of these genes are arranged in clusters called genomic islands. We further reduced the number of diagnostic targets by excluding sequences having high BLAST similarity to other mycobacterial species recently added to the National Center for Biotechnology Information database. Genes identified as diagnostic following these bioinformatic approaches were further tested by DNA amplification PCR on an additional 20 M. avium subspecies strains. This combined approach confirmed 86 genes as Map-specific, seven as Maa-specific and three as Mah-specific. A single-tube PCR reaction was conducted as a proof of concept method to quickly distinguish M. avium subspecies strains. With these novel data, researchers can classify isolates in their freezers, quickly characterize clinical samples, and functionally analyze these unique genes.

SUBMITTER: Bannantine JP

PROVIDER: S-EPMC7876471 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Diagnostic Sequences That Distinguish M. avium Subspecies Strains.

Bannantine John P JP Stabel Judith R JR Bayles Darrell O DO Conde Cyril C Biet Franck F

Frontiers in veterinary science 20210128

Over a decade ago Mycobacterium avium subspecies paratuberculosis (Map) specific genes were initially identified in a whole genome context by comparing draft genome sequences of Map strain K-10 with Mycobacterium avium subspecies hominissuis (Mah) strain 104. This resulted in identification of 32 Map specific genes, not including repetitive elements, based on the two-genome comparison. The goal of this study was to define a more complete ca ...[more]

PMID: 33585607

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Project description:BackgroundMycobacterium avium subspecies paratuberculosis (Map) is an infectious enteric pathogen that causes Johne's disease in livestock. Determining genetic diversity is prerequisite to understanding the epidemiology and biology of Map. We performed the first whole genome sequencing (WGS) of 141 global Map isolates that encompass the main molecular strain types currently reported. We investigated the phylogeny of the Map strains, the diversity of the genome and the limitations of commonly used genotyping methods.ResultsSingle nucleotide polymorphism (SNP) and phylogenetic analyses confirmed two major lineages concordant with the former Type S and Type C designations. The Type I and Type III strain groups are subtypes of Type S, and Type B strains are a subtype of Type C and not restricted to Bison species. We found that the genome-wide SNPs detected provided greater resolution between isolates than currently employed genotyping methods. Furthermore, the SNP used for IS1311 typing is not informative, as it is likely to have occurred after Type S and C strains diverged and does not assign all strains to the correct lineage. Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) differentiates Type S from Type C but provides limited resolution between isolates within these lineages and the polymorphisms detected do not necessarily accurately reflect the phylogenetic relationships between strains. WGS of passaged strains and coalescent analysis of the collection revealed a very high level of genetic stability, with the substitution rate estimated to be less than 0.5 SNPs per genome per year.ConclusionsThis study clarifies the phylogenetic relationships between the previously described Map strain groups, and highlights the limitations of current genotyping techniques. Map isolates exhibit restricted genetic diversity and a substitution rate consistent with a monomorphic pathogen. WGS provides the ultimate level of resolution for differentiation between strains. However, WGS alone will not be sufficient for tracing and tracking Map infections, yet importantly it can provide a phylogenetic context for affirming epidemiological connections.

Project description:Mycobacterium avium comprises four subspecies that contain both human and veterinary pathogens. At the inception of this study, twenty-eight M. avium genomes had been annotated as RefSeq genomes, facilitating direct comparisons. These genomes represent strains from around the world and provided a unique opportunity to examine genome dynamics in this species. Each genome was confirmed to be classified correctly based on SNP genotyping, nucleotide identity and presence/absence of repetitive elements or other typing methods. The Mycobacterium avium subspecies paratuberculosis (Map) genome size and organization was remarkably consistent, averaging 4.8 Mb with a variance of only 29.6 kb among the 13 strains. Comparing recombination events along with the larger genome size and variance observed among Mycobacterium avium subspecies avium (Maa) and Mycobacterium avium subspecies hominissuis (Mah) strains (collectively termed non-Map) suggests horizontal gene transfer occurs in non-Map, but not in Map strains. Overall, M. avium subspecies could be divided into two major sub-divisions, with the Map type II (bovine strains) clustering tightly on one end of a phylogenetic spectrum and Mah strains clustering more loosely together on the other end. The most evolutionarily distinct Map strain was an ovine strain, designated Telford, which had >1,000 SNPs and showed large rearrangements compared to the bovine type II strains. The Telford strain clustered with Maa strains as an intermediate between Map type II and Mah. SNP analysis and genome organization analyses repeatedly demonstrated the conserved nature of Map versus the mosaic nature of non-Map M. avium strains. Finally, core and pangenomes were developed for Map and non-Map strains. A total of 80% Map genes belonged to the Map core genome, while only 40% of non-Map genes belonged to the non-Map core genome. These genomes provide a more complete and detailed comparison of these subspecies strains as well as a blueprint for how genetic diversity originated.

Project description:BACKGROUND: Mycobacterium avium subspecies paratuberculosis (Map) is the aetiological agent of Johne's disease or paratuberculosis and is included within the Mycobacterium avium complex (MAC). Map strains are of two major types often referred to as 'Sheep' or 'S-type' and 'Cattle' or 'C-type'. With the advent of more discriminatory typing techniques it has been possible to further classify the S-type strains into two groups referred to as Type I and Type III. This study was undertaken to genotype a large panel of S-type small ruminant isolates from different hosts and geographical origins and to compare them with a large panel of well documented C-type isolates to assess the genetic diversity of these strain types. Methods used included Mycobacterial Interspersed Repetitive Units - Variable-Number Tandem Repeat analysis (MIRU-VNTR), analysis of Large Sequence Polymorphisms by PCR (LSP analysis), Single Nucleotide Polymorphism (SNP) analysis of gyr genes, Pulsed-Field Gel Electrophoresis (PFGE) and Restriction Fragment Length Polymorphism analysis coupled with hybridization to IS900 (IS900-RFLP) analysis. RESULTS: The presence of LSP(A)4 and absence of LSP(A)20 was confirmed in all 24 Map S-type strains analysed. SNPs within the gyr genes divided the S-type strains into types I and III. Twenty four PFGE multiplex profiles and eleven different IS900-RFLP profiles were identified among the S-type isolates, some of them not previously published. Both PFGE and IS900-RFLP segregated the S-type strains into types I and III and the results concurred with those of the gyr SNP analysis. Nine MIRU-VNTR genotypes were identified in these isolates. MIRU-VNTR analysis differentiated Map strains from other members of Mycobacterium avium Complex, and Map S-type from C-type but not type I from III. Pigmented Map isolates were found of type I or III. CONCLUSION: This is the largest panel of S-type strains investigated to date. The S-type strains could be further divided into two subtypes, I and III by some of the typing techniques (IS900-RFLP, PFGE and SNP analysis of the gyr genes). MIRU-VNTR did not divide the strains into the subtypes I and III but did detect genetic differences between isolates within each of the subtypes. Pigmentation is not exclusively associated with type I strains.

Project description:Mycobacterium avium subspecies paratuberculosis (MAP) is the aetiological agent of Johne's disease (JD), a chronic enteritis that causes major losses to the global livestock industry. Further, it has been associated with human Crohn's disease. Several strains of MAP have been identified, the two major groups being sheep strain MAP, which includes the Type I and Type III sub-lineages, and the cattle strain or Type II MAP lineage, of which bison strains are a sub-grouping. Major genotypic, phenotypic and pathogenic variations have been identified in prior comparisons, but the research has predominately focused on cattle strains of MAP. In countries where the sheep industries are more prevalent, however, such as Australia and New Zealand, ovine JD is a substantial burden. An information gap exists regarding the genomic differences between sheep strain sub-lineages and the relevance of Type I and Type III MAP in terms of epidemiology and/or pathogenicity. We therefore investigated sheep MAP isolates from Australia and New Zealand using whole genome sequencing. For additional context, sheep MAP genome datasets were downloaded from the Sequence Read Archive and GenBank. The final dataset contained 18 Type III and 16 Type I isolates and the K10 cattle strain MAP reference genome. Using a pan-genome approach, an updated global phylogeny for sheep MAP from de novo assemblies was produced. When rooted with the K10 cattle reference strain, two distinct clades representing the lineages were apparent. The Australian and New Zealand isolates formed a distinct sub-clade within the type I lineage, while the European type I isolates formed another less closely related group. Within the type III lineage, isolates appeared more genetically diverse and were from a greater number of continents. Querying of the pan-genome and verification using BLAST analysis revealed lineage-specific variations (n = 13) including genes responsible for metabolism and stress responses. The genetic differences identified may represent important epidemiological and virulence traits specific to sheep MAP. This knowledge will potentially contribute to improved vaccine development and control measures for these strains.

Project description:The genetic similarity between Mycobacterium avium subsp. paratuberculosis and other mycobacterial species has confounded the development of M. avium subsp. paratuberculosis-specific diagnostic reagents. Random shotgun sequencing of the M. avium subsp. paratuberculosis genome in our laboratories has shown >98% sequence identity with Mycobacterium avium subsp. avium in some regions. However, an in silico comparison of the largest annotated M. avium subsp. paratuberculosis contigs, totaling 2,658,271 bp, with the unfinished M. avium subsp. avium genome has revealed 27 predicted M. avium subsp. paratuberculosis coding sequences that do not align with M. avium subsp. avium sequences. BLASTP analysis of the 27 predicted coding sequences (genes) shows that 24 do not match sequences in public sequence databases, such as GenBank. These novel sequences were examined by PCR amplification with genomic DNA from eight mycobacterial species and ten independent isolates of M. avium subsp. paratuberculosis. From these analyses, 21 genes were found to be present in all M. avium subsp. paratuberculosis isolates and absent from all other mycobacterial species tested. One region of the M. avium subsp. paratuberculosis genome contains a cluster of eight genes, arranged in tandem, that is absent in other mycobacterial species. This region spans 4.4 kb and is separated from other predicted coding regions by 1,408 bp upstream and 1,092 bp downstream. The gene upstream of this eight-gene cluster has strong similarity to mycobacteriophage integrase sequences. The GC content of this 4.4-kb region is 66%, which is similar to the rest of the genome, indicating that this region was not horizontally acquired recently. Southern hybridization analysis confirmed that this gene cluster is present only in M. avium subsp. paratuberculosis. Collectively, these studies suggest that a genomics approach will help in identifying novel M. avium subsp. paratuberculosis genes as candidate diagnostic sequences.

Dataset Information

Diagnostic Sequences That Distinguish M. avium Subspecies Strains.

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Diagnostic Sequences That Distinguish <i>M. avium</i> Subspecies Strains.

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