Search

ASMOHEJP 2020 POSTER PRESENTATION PRAGUE (CZECH REPUBLIC) &nbsp

ASMOHEJP2022 poster &nbsp

International audience; Bovine Tuberculosis (bTB) is a zoonotic disease due to Mycobacterium bovis (M. bovis). France has a bTB-free status, but the disease has not been eradicated yet and a worryingly steady increase of bTB outbreaks has been observed in some regions (1). This could be explained by the detection of bTB in wildlife that spills it back to livestock in the same territories. The transmission link within these multi-host systems remains difficult to establish given that they share the same M. bovis genotypes (2). Obtaining new reference genomes for each of these genotypes could improve the knowledge of clonal groups and refine molecular field epidemiology studies based on whole genome sequencing.

International audience; Mycobacterium avium subsp. paratuberculosis is the etiological agent of Johne's disease in ruminants. Here, we report the annotated draft genome sequences of 142 M. avium subsp. paratuberculosis strains that were isolated from dairy cattle in France between 2014 and 2018. The genomes of these strains were sequenced using Illumina technology.

International audience; Introduction: Mycobacterium avium subsp. paratuberculosis (Map) is the etiological agent of paratuberculosis or Johne’s disease that causes chronic intestinal inflammation in ruminants. The IS900 insertion sequence, specific for Map, hasbeen used widely as an epidemiological marker and target for qPCR diagnosis. Recently, thanks to third generation sequencing technologies, the complete genomes of strains belonging to the major phylogenetic lineages of Maphave been made available. This has allowed us to investigate the distribution of IS900 in this slow growing bacterium. The objective of this study is to characterize the distribution of the IS900 element and how it affects genomicevolution and gene function of Map. A secondary goal is to develop automated in silico restriction fragment length polymorphism (RFLP) analysis using IS900.Materials and methods: Complete genomes from the major phylogenetic lineages known as C-type and S-type (including subtypes I and III), were chosen to represent the genetic diversity of Map. Computer analysis included software located IS900 using BLAST and determined fragments from complete genome (FASTA) developed with Biopython. Digital representation was provided using matplotlib. Profile comparisons were carried out using Bionumerics software. Upstream and downstream genomic regions flanking each IS900 copy were extracted and used to identify orthologous insertion site across genomes.Results, discussion and conclusion: The program developed in this study allowed automated location of IS900 sequences to identify their positions and their number. Between 16 to 22 copies of the IS900 sequence were found in the genomes studied. Nine IS900 insertion site locations were conserved across all genomes studied while smaller subsets were unique to a particular lineage. An in silico RFLP analysis was developed to obtain the exact size of the DNA fragments carrying a copy of the IS900. IS900 RFLP profiles were compared by digital visualization of the restriction fragments separated. This study provided a program making it possible to automate IS900 distribution analysis in Map genomes to enrich our knowledge on the dynamics of distribution of this IS for epidemiological purposes, for understanding the evolution within the Map species and studying the biological implication of the presence of IS900.

International audience; Introduction: Paratuberculosis is a chronic infection of the intestine, mainly the ileum, caused by Mycobacterium avium subsp. paratuberculosis (Map) in cattle and other ruminants. This enzootic disease is present worldwide and has a strong impact on the dairy cattle industry. For this species, the typing tools do not make it possible to investigate the genetic diversity of the strains. These limitations can be overcome by the application of Whole Genome Sequencing (WGS), particularly for clonal populations such as Map. WGS analyses can provide comprehensive genetic information, including information on genome evolution, discrimination of closely related strains and virulence determinants.The purpose of the present study was to undertake a whole-genome analysis of Map strains to identify accurate phylogenetic relationships between isolates and establish correlations between genomic traits and epidemiologicaldata within a population of well documented-strains.Materials and methods: A set of 200 animal field strains were selected based on a previous large-scale longitudinal study representative of the French Map population circulating in the West of France. Strains were isolated from bovine of breed Prim’Holstein or Normande naturally infected by Map. For each strain isolated, all information about the animal is available including: breed, herd prevalence, locations, serological status and excretion level. The genomic sequences of Map strains were obtained by Illumina MiSeq sequencing. Genomic sequences were assessed for potential contamination using Kraken2. Cleaned reads were aligned to the reference genome K-10 and SNP were called using FreeBayes. IQ-Tree was used to infer a SNP-based phylogeny.Results, discussion and conclusion: This study provided 200 new genomes of French strains isolates from naturally infected animals. Pangenome analysis of this panel confirmed the degree of Map clonality. SNP analyzes provided accurate phylogeny able to distinguish each strain divided into 4 clusters independently of the breed of the cattle. Interestingly, the phylogeny clearly distinct the two major MLVA profiles. A phylogeny was inferred with French Map isolates and with other Map isolates found across the world. Relations between genetic traits and epidemiological data will be investigated to better understand the transmission dynamics of the disease

International audience; Mycobacterium avium subsp. paratuberculosis ( Map ) is the etiological agent of Johne’s disease in ruminants. The IS 900 insertion sequence (IS) has been used widely as an epidemiological marker and target for PCR diagnosis. Updated DNA sequencing technologies have led to a rapid increase in available Map genomes, which makes it possible to analyze the distribution of IS 900 in this slow-growing bacterium. The objective of this study is to characterize the distribution of the IS 900 element and how it affects genomic evolution and gene function of Map . A secondary goal is to develop automated in silico restriction fragment length polymorphism (RFLP) analysis using IS 900 . Complete genomes from the major phylogenetic lineages known as C-type and S-type (including subtypes I and III), were chosen to represent the genetic diversity of Map . IS 900 elements were located in these genomes using BLAST software and the relevant fragments extracted. An in silico RFLP analysis using the Bst EII restriction site was performed to obtain exact sizes of the DNA fragments carrying a copy of IS 900 and the resulting RFLP profiles were analyzed and compared by digital visualization of the separated restriction fragments. The program developed for this study allowed automated localization of IS 900 sequences to identify their position within each genome along with the exact number of copies per genome. The number of IS 900 copies ranged from 16 in the C-type isolate to 22 in the S-type subtype I isolate. A loci-by-loci sequence alignment of all IS 900 copies within the three genomes revealed new sequence polymorphisms that define three sequevars distinguishing the subtypes. Nine IS 900 insertion site locations were conserved across all genomes studied while smaller subsets were unique to a particular lineage. Preferential insertion motif sequences were identified for IS 900 along with genes bordering all IS 900 insertions. Rarely did IS 900 insert within coding sequences as only three genes were disrupted in this way. This study makes it possible to automate IS 900 distribution in Map genomes to enrich knowledge on the distribution dynamics of this IS for epidemiological purposes, for understanding Map evolution and for studying the biological implications of IS 900 insertions.

National audience; Introduction and objectives: Mycobacterium avium subsp. paratuberculosis (Map) is the etiological agent of paratuberculosis or Johne’s disease that causes chronic intestinal inflammation in ruminants. The IS900 insertion sequence, specific for Map, has been used widely as an epidemiological marker and target for qPCR diagnosis. Thanks to the development of sequencing technologies, the number of available Map genomes is increasing rapidly, which makes it possible to analyze the distribution of IS900 in this very slow-growing bacterium. The objective of this study was to use bioinformatics approaches to study IS900 distribution in the genomes of Map strains and to develop automated in silico IS900 restriction fragment length polymorphism (IS900-RFLP) analysis. Materials and methods: The complete genomes of the strains K-10, Telford and JII-1961 were chosen to cover the genetic diversity of Map divided into two strain types, S and C, according to the phylogeny of this species. Computer analysis included software located IS900 using BLAST and determined fragments from complete genome (FASTA) using BioPython. Digital representation was provided using matplotlib. Profile comparisons were carried out using Bionumerics software. Results, discussion and conclusion: The program developed in this study allowed automated location of IS900 sequences to identify their positions and their number. Between 17 to 22 copies of the IS900 sequence were found in the genomes studied. An in silico RFLP analysis was developed using complete genomes. This procedure utilised the BstEII restriction site conventionally used for Map to obtain the exact size of the DNA fragments carrying a copy of the IS900 sequence. IS900 RFLP profiles were compared using fragment sizes or by digital visualization of the restriction fragments separated according to their size by mimicking their migration in agarose gel. These digital profiles were compared to the many existing profiles in the literature. This study provided a program making it possible to automate IS900 distribution analysis in Map genomes to enrich our knowledge on the dynamics of distribution of this IS for epidemiological purposes, for understanding the evolution within the Map species and studying the biological implication of the presence of IS900.

In recent years the diversity of the French Mycobacterium bovis population responsible for bovine tuberculosis (bTB) outbreaks since 1970 has been described in detail. To further understand bTB evolution in France, we used single nucleotide polymorphisms (SNPs) based on whole genome sequence versus classical genotyping methods in order to identify accurate phylogenetic relationships between M. bovis strains. Whole genome sequencing was carried out on a selection of 87 strains which reflect the French M. bovis population’s genetic diversity. Sequences were compared to the M. bovis reference genome AF2122/97. Comparison among the 87 genomes revealed 9,170 sites where at least one strain shows a SNP with respect to the reference genome; 1,172 are intergenic and 7,998 in coding sequences, of which 2,880 are synonymous and 5,118 non-synonymous. SNP-based phylogenetic analysis using these 9,170 SNP is congruent with the cluster defined by spoligotyping and multilocus variable number of tandem repeat analysis typing. In addition, some SNPs were identified as specific to genotypic groups. These findings suggest new SNP targets that can be used for the development of high-resolving methods for genotyping as well as for studying M. bovis evolution and transmission patterns. The detection of non-synonymous SNPs on virulence genes enabled us to distinguish different clusters. Our results seem to indicate that genetically differentiated clusters could also display distinctive phenotypic traits.

National audience

National audience

National audience

National audience

National audience

National audience

National audience

International audience

National audience

International audience; Mycobacteria have a complex cell wall structure that includes many lipids which are often species specific. Besides giving a phenotypic signature these lipids are often involved in infectious processes of pathogenic mycobacteria by interfering with the host immune system. The biosynthesis pathway of the simplest glycopeptidolipids (GPLs) is relatively well understood and involves more than fifteen genes. Even though M. avium subsp. paratuberculosis (Map) produces a lipopeptide rather than GPL, its genome contains nevertheless a locus highly similar to the GPL biosynthetic pathway of M. avium subsp. avium (Mav). We showed that the module composition of the non-ribosomal protein synthase (Nrp) of Map, the enzyme involved in the synthesis of the peptidyl moiety, is dramatically different from that of other GPL producers such as M. smegmatis (Ms) and Mav. While Map isolates do not produce GPLs, they do produce lipopeptides without the carbohydrate moiety. However, the picture is not as clear regarding the diversity of lipopeptides produced among two lineages classified as type I/III or S-type (ovine) and type II or C-type (bovine) Map strains that have emerged from the common ancestor, M. avium subsp. hominissuis. The S-type isolates are readily distinguishable from C-type isolates based on genome studies and readily discriminated by genotyping methods. In addition to the genotypic distinctions between S- and C-type strains, phenotypic differences have been documented. To provide a genomic basis for the synthesis of the diversity of lipopeptides in Map, its recently published genome sequence was explored using in silico methods and completed by biochemical investigations. Interestingly we discovered a change in the chemical structure of the lipopeptide of the S strains. These findings add new phenotypic evidence that contribute to separate the S type to the C type. Furthermore deciphering the biosynthesis pathway of cell wall lipopeptides should contribute to better understand the determinants of the adaptation of a pathogen to a specific host but also the factors favoring transmission to a new host.

National audience

National audience

International audience; Mycobacterium bovis is the etiologic agent of bovine tuberculosis, a chronic infectious disease affecting livestock, wild animals, and sometimes humans. We report here three draft genome sequences of Mycobacterium bovis strains of spoligotypes SB0821 and SB0134, isolated from wildlife but circulating in wildlife-livestock multihost systems, and SB0121, circulating exclusively in cattle.

Caracteristiques des adhesines hbha et lbp exprimees chez [i]mycobacterium intracellulare[/i]. 14. Journées de Mycobactériologie de Langue Française

National audience

Session Poster : Diagnostic and Detection; National audience; Background: Unlike other MAC members, Mycobacterium avium subsp. paratuberculosis (MAP) does not produce GPL on the surface of the cell wall but a lipopentapeptide called L5P or ParaLP01. The molecular and genetic characterization of this antigen demonstrated that L5P is specific to MAP. Currently available diagnostic tests are based on the use of whole cell antigens that are mainly not MAP specific and that require a pre-absorption step with antigens of M. phlei. This diagnosis detect animals with a late stage infection but are not sensitive enough to detect early stage infection. We hypothesize that the pre-absorption step prevents detection of informative populations of immunoglobulins, especially in animals with subclinical faecal shedding of MAP that are not detected by the current serology-based diagnostics. The L5P antigen has now been used in various studies to evaluate a MAP specific sero-diagnostic. This synthetic molecule was used as a pure product, not requiring pre-absorption step. However a number of parameters must be assessed including the formulation of L5P and with reference to a panel of well defined serum samples.Objective: To assess the potential of L5P and its hydrosoluble variants for the serological diagnosis of MAP infection using collections of sera from different contexts.Method: In order to find the best compound for use in serology we chemically synthesized L5P and derivatives of L5P including water-soluble variants. These pure compounds were evaluated on collections of extensively characterized sera from infected and non-infected cattle, goats and sheep. In addition, the panels of sera included animals infected with M. bovis and M. avium subsp. hominissuis.Results: ROC analysis showed that L5P and also its water-soluble derivatives are suitable for the development of serological diagnosis. Advantageously, these pure synthetic MAP specific antigens can be produced at low cost. The use of L5P has not been validated in the contexts of ovine paratuberculosis. In the context of infections due to other mycobacteria such as M. bovis or the more closely related species M. avium subsp. hominissuis, the L5P did not cross react and therefore may be a valuable antigen to solve ambiguous results in other tests.

Session Poster : Maps Genomics and Diversity; National audience; Background: Genotyping applied to strains of Mycobacterium avium subsp. paratuberculosis (MAP) has become an indispensable tool for epidemiological surveillance of this significant veterinary pathogen. For MAP, multi-locus variable number tandem repeat analysis (MLVA) targeting mycobacterial interspersed repetitive units (MIRUs) and other variable-number tandem repeats (VNTRs) was established using 8 markers. Within the past years this standard, portable, reproducible and discriminatory typing method has been frequently applied alone or in combinations with short-sequence-repeat (SSR) sequencing. As such, a number of genotypes of strains of diverse origins have been identified in laboratories worldwide. With the large diffusion of these genotyping methods standardization between laboratories and knowledge of existing INMV profiles and archiving of newly defined genotypes need to be managed. Objective: To develop a web application called “Mac INMV plus database” including MLVA and SSR. This freely accessible service allows users to compare VNTR and SSR subtype data of their strains analyzed by MLVA and with those of existing reference strains.Results: A freely accessibledatabase was created with genotyping data of strains of MAP and of other M. avium complex (MAC) members. This database gathers all profiles obtained in the laboratory and those published in the literature. The database information include incrementing species and subspecies status, INMV and SSR profiles with corresponding alleles/repeat numbers at each locus, and combined numerical genotypes. The user can consult and query all the existing profiles in order to see if profiles identified in their lab already exist. If profiles are not known, the user can request new attribution numbers online. All information can be exported under EXCEL or PDF format. An extensive documentation regarding the genotyping method, protocols, primer sequences, marker information, and associated isolate specific metadata is available to the users.

National audience; Peptidyl moiety of cell wall lipopentapeptide (L5P) specific of Mycobacterium avium subsp paratuberculosis (Map) is immunogenic and a target for specific humoral response in Map infected animals. A chemically synthesized L5P is able to induce specific cell mediated immune response (CMIR) in IFN-γ release assay (IGRA) in selected cows from Map infected herds comparatively to non-infected or M. bovis infected. Following these observations, the aim of this study was to evaluate if L5P was an antigen of early specific immune response and potentially a predictive tool of Map infection. 113 heifers of 6 herds were included in a two years’ longitudinal study: 71 animals from three Map culture-confirmed herds, 11 animals from a Map infected herd Silirum® vaccinated during the study and 31 animals from two certified Map free herds. The analysis of the CMIR was investigated by IGRA following whole blood stimulation with synthetic L5P or mycobacterium purified protein derivative (PPD) from M. avium (PPDa), M. bovis (PPDb), Map (PPDj) and M. phlei (PPDp). Humoral immune response was quantified by L5P-based ELISA using an internal procedure and two commercial Map kits. Moreover, bacilli excretion was estimated by isolation and culture from faecal sample. PPDs’ CMIR was more or less high depending of infected herds context, became high over 2 S/P ratio for 10/11 animals just after Silirum® vaccination and was low, less than 0.1 S/P ratio, in certified Map free herds. L5P CMIR was observed in 9 of 71 animals from Map culture-confirmed herds. These 9 animals with a L5P CMIR positive between 0.05 and 0.6 S/P ratio were from the same herd, knowing that L5P CMIR was previously detected in all included Map culture-confirmed herds. L5P CMIR was fluctuant as already described for PPD but was significantly correlated with PPDj CMIR. For 2 of the 9 animals, the L5P CMIR was predictive of the Map positive serology, whereas it was concomitant with seropositivity for 2 others and that for 5 animals was several times observed without seroconversion. And no seroconversion was observed in other herds. The continuation of this study would assess the predictive potential of L5P CMIR for paratuberculosis diagnosis.

Session : Maps Genomics and Diversity; National audience; Mycobacteria have a complex cell wall structure that includes many lipids which are often species specific. Besides giving a phenotypic signature these lipids are often involved in infectious processes of pathogenic mycobacteria by interfering with the host immune system. The biosynthesis pathway of the simplest glycopeptidolipids (GPLs) is relatively well understood and involves more than fifteen genes. Even though M. avium subsp. paratuberculosis (Map) produces a lipopeptide rather than GPL, its genome contains nevertheless a locus highly similar to the GPL biosynthetic pathway of M. avium subsp. avium (Mav). We showed that the module composition of the non-ribosomal protein synthase (Nrp) of Map, the enzyme involved in the synthesis of the peptidyl moiety, is dramatically different from that of other GPL producers such as M. smegmatis (Ms) and Mav. While Map isolates do not produce GPLs, they do produce lipopeptides without the carbohydrate moiety. However, the picture is not as clear regarding the diversity of lipopeptides produced among two lineages classified as type I/III or S-type (ovine) and type II or C-type (bovine) Map strains that have emerged from the common ancestor, M. avium subsp. hominissuis. The S-type isolates are readily distinguishable from C-type isolates based on genome studies and readily discriminated by genotyping methods. In addition to the genotypic distinctions between S- and C-type strains, phenotypic differences have been documented. To provide a genomic basis for the synthesis of the diversity of lipopeptides in Map, its recently published genome sequence was explored using in silico methods and completed by biochemical investigations. Interestingly we discovered a change in the chemical structure of the lipopeptide of the S strains. These findings add new phenotypic evidence that contribute to separate the S type to the C type. Furthermore deciphering the biosynthesis pathway of cell wall lipopeptides should contribute to better understand the determinants of the adaptation of a pathogen to a specific host but also the factors favoring transmission to a new host.