Search

Free-living amoebae (FLA) are described as environmental reservoirs for some bacteria able to resist their phagocytosis. In the environment, the fate of Mycobacterium bovis (Mbo) and Mycobacterium avium subsp. paratuberculosis (Map) responsible for bovine tuberculosis and paratuberculosis, respectively, remains poorly understood and is considered potentially problematic in the eradication and control of these diseases. We hypothesize that FLA may play a role in the persistence of Mbo and Map in the environment. In this study, 90 samples were collected from herds affected by one or both diseases to investigate the diversity of amoeba and their associated bacteria. Metabarcoding analyses revealed that Acanthamoeba, Copromyxa, Naegleria, and Vermamoeba were the most represented genera of FLA, with Pseudomonadota being the bacteria most commonly found associated with FLA. Although no Mbo and Map DNA were identified by sequencing, traces were detected by ddPCR (digital droplet PCR), specifically targeting these bacteria. In conclusion, we described a wide diversity of FLA and associated bacteria in this environment. It also suggests that Map and Mbo could be associated, even weakly, with FLA in the environment. However, this needs to be confirmed by detecting a highest amount of DNA and, if possible, cultivable Map and/or Mbo associated with these environmental FLA., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

The complete genome sequence of the most ancestral type SI strain of Mycobacterium avium subspecies paratuberculosis 6756, isolated from a sheep, was determined. The genome was sequenced using PacBio technology, yielding a genome size of 4,830,294 nucleotides with no identified plasmids., Competing Interests: The authors declare no conflict of interest.

One of the obstacles to eradicating paratuberculosis or Johne's Disease (JD) seems to be the persistence of Mycobacterium avium subsp. paratuberculosis ( Map ) in the environment due to its ability to survive alone or vectorized. It has been shown that Map is widely distributed in soils and water. Previously, we isolated amoebae associated with Map strains in the environment of bovines from an infected herd. This work aims to verify our working hypothesis, which suggests that amoebae may play a role in the transmission of JD. In this study, we sampled water in the vicinity of herds infected with Map or Mycobacterium bovis ( M. bovis ) and searched for amoebae and mycobacteria. Live amoebae were recovered from all samples. Among these amoebae, four isolates associated with the presence of mycobacteria were identified and characterized. Map and other mycobacterial species were detected by qPCR and, in some cases, by culture. This study suggests that amoebae and Map may be found in the same environment and might represent a risk of exposure of animals to pathogenic mycobacteria. These data open up new perspectives on the control measures to be put in place to prevent contamination by Map .

Mammalian tuberculosis (TB) is a zoonotic disease mainly due to Mycobacterium bovis ( M. bovis ). A current challenge for its eradication is understanding its transmission within multi-host systems. Improvements in long-read sequencing technologies have made it possible to obtain complete bacterial genomes that provide a comprehensive view of species-specific genomic features. In the context of TB, new genomic references based on complete genomes genetically close to field strains are also essential to perform precise field molecular epidemiological studies. A total of 10 M. bovis strains representing each genetic lineage identified in France and in other countries were selected for performing complete assembly of their genomes. Pangenome analysis revealed a "closed" pangenome composed of 3900 core genes and only 96 accessory genes. Whole genomes-based alignment using progressive Mauve showed remarkable conservation of the genomic synteny except that the genomes have a variable number of copies of IS 6110 . Characteristic genomic traits of each lineage were identified through the discovery of specific indels. Altogether, these results provide new genetic features that improve the description of M. bovis lineages. The availability of new complete representative genomes of M. bovis will be useful to epidemiological studies and better understand the transmission of this clonal-evolving pathogen.

Paratuberculosis is a chronic infection of the intestine, mainly the ileum, caused by Mycobacterium avium subsp. paratuberculosis in cattle and other ruminants. This enzootic disease is present worldwide and has a negative impact on the dairy cattle industry. For this subspecies, the current genotyping tools do not provide the needed resolution to investigate the genetic diversity of closely related strains. These limitations can be overcome by the application of whole-genome sequencing (WGS), particularly for clonal populations such as M. avium subsp. paratuberculosis . The purpose of the present study was to undertake a WGS analysis with a panel of 200 animal field M. avium subsp. paratuberculosis strains selected based on a previous large-scale longitudinal study of Prim'Holstein and Normande dairy breeds naturally infected with M. avium subsp. paratuberculosis in the West of France. The pangenome analysis revealed that M. avium subsp. paratuberculosis has a closed pangenome. The phylogeny, based on alignment of 2,786 nonhomoplasic single nucleotide polymorphisms (SNPs), showed that the strain population is structured into three clades independently of the cattle breed or geographic distribution. The increased resolution of phylogeny obtained by WGS confirmed the homoplasic nature of the markers variable-number tandem repeat (VNTR) and short sequence repeat (SSR) used for M. avium subsp. paratuberculosis genotyping. These phylogenetic data also revealed independent introductions of the different genotypes in two main waves since at least 2003. WGS applied to this sampling demonstrated the presence of mixed infections in herds and at the individual animal level. Collectively, the phylogeny results inferred with French isolates compared to M. avium subsp. paratuberculosis isolates from around the world suggest introductions of M. avium subsp. paratuberculosis genotypes through the animal trade. Relationships between genetic traits and epidemiological data can now be investigated to better understand transmission dynamics of the disease. IMPORTANCE Mycobacterium avium subsp. paratuberculosis causes Johne's disease in ruminants, which is present worldwide and has significant negative impacts on the dairy cattle industry and animal welfare. Prevention and control of M. avium subsp. paratuberculosis infection are hampered by knowledge gaps in strain virulence, genotype distribution, and transmission dynamics. This work has revealed new insights into M. avium subsp. paratuberculosis strains currently circulating in western France and how they are related to strains circulating globally. We applied whole-genome sequencing (WGS) to obtain comprehensive information on genome evolution and discrimination of closely related strains. This approach revealed the history of M. avium subsp. paratuberculosis infection in France, refined the pangenomic characteristics of M. avium subsp. paratuberculosis , and demonstrated the existence of mixed infection in animals. Finally, this study identified predominant genotypes, which allow a better understanding of disease transmission dynamics. This information will facilitate tracking of this pathogen on farms and across agricultural regions, thus informing transmission pathways and disease control points.

Isoniazid is a cornerstone of modern tuberculosis (TB) therapy and targets the enoyl ACP reductase InhA, a key enzyme in mycolic acid biosynthesis. InhA is still a promising target for the development of new anti-TB drugs. Herein, we report the design, synthesis, and anti-tubercular activity of new isoniazid hybrids. Among these, 1H-1,2,3 triazole-tethered quinoline-isoniazid conjugates 16a to 16g exhibited high activity against Mycobacterium tuberculosis with minimal inhibitory concentrations in the 0.25-0.50 μg/mL range and were bactericidal in vitro. Importantly, these compounds were well tolerated at high doses on mammalian cells, leading to high selectivity indices. The hybrids were dependent on functional KatG production to inhibit mycolic acid biosynthesis. Moreover, overexpression of InhA in M. tuberculosis resulted in high resistance levels to 16a-16g and reduced mycolic acid biosynthesis inhibition, similar to isoniazid. Overall, these findings suggest that the synthesized quinoline-isoniazid hybrids are promising anti-tubercular molecules, which require further pre-clinical evaluation., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

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.

We explored the relevance of a Clustered regularly interspaced short palindromic repeats (CRISPR)-based genotyping tool for Streptococcus agalactiae typing and we compared this method to current molecular methods [multi locus sequence typing (MLST) and capsular typing]. To this effect, we developed two CRISPR marker schemes (using 94 or 25 markers, respectively). Among the 255 S. agalactiae isolates tested, 229 CRISPR profiles were obtained. The 94 and 25 markers made it possible to efficiently separate isolates with a high diversity index (0.9947 and 0.9267, respectively), highlighting a high discriminatory power, superior to that of both capsular typing and MLST (diversity index of 0.9017 for MLST). This method has the advantage of being correlated with MLST [through analysis of the terminal direct repeat (TDR) and ancestral spacers] and to possess a high discriminatory power (through analysis of the leader-end spacers recently acquired, which are the witnesses of genetic mobile elements encountered by the bacteria). Furthermore, this "one-shot" approach presents the benefit of much-reduced time and cost in comparison with MLST. On the basis of these data, we propose that this method could become a reference method for group B Streptococcus (GBS) typing., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Beauruelle, Treluyer, Pastuszka, Cochard, Lier, Mereghetti, Glaser, Poyart and Lanotte.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Conde, Price-Carter, Cochard, Branger, Stevenson, Whittington, Bannantine and Biet.)

Unlike other MAC members, Mycobacterium avium subsp. paratuberculosis (MAP) does not produce glycopeptidolipids (GPL) on the surface of the cell wall but a lipopentapeptide called L5P (also termed Lipopeptide-I or Para-LP-01) characterized in C-type (bovine) strains. This lipopeptide antigen contains a pentapeptide core, D-Phenylalanine-N-methyl-L-Valine-L-Isoleucine-L-Phenylalanine-L-Alanine, in which the N-terminal D-Phenylalanine is amido-linked with a fatty acid (C18-C20). The molecular and genetic characterization of this antigen demonstrated that L5P is unique to MAP. Knowledge of the structure of L5P enabled synthetic production of this lipopeptide in large quantities for immunological evaluation. Various studies described the immune response directed against L5P and confirmed its capability for detection of MAP infection. However, the hydrophobic nature of lipopeptide antigens make their handling and use in organic solvents unsuitable for industrial processes. The objectives of this study were to produce, by chemical synthesis, a water-soluble variant of L5P and to evaluate these compounds for the serological diagnosis of MAP using well-defined serum banks. The native L5P antigen and its hydrosoluble analog were synthesized on solid phase. The pure compounds were evaluated on collections of extensively characterized sera from infected and non-infected cattle. ROC analysis showed that L5P and also its water-soluble derivative are suitable for the development of a serological test for Johne's disease at a population level. However, these compounds used alone in ELISA have lower sensitivity (Se 82% for L5P and Se 62% for the water-soluble variant of L5P) compared to the Se 98% of a commercial test. Advantageously, these pure synthetic MAP specific antigens can be easily produced in non-limiting quantities at low cost and in standardized batches for robust studies. The fact that L5P has not been validated in the context of ovine paratuberculosis highlights the need to better characterize the antigens expressed from the different genetic lineages of MAP to discover new diagnostic antigens. 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bay, Begg, Ganneau, Branger, Cochard, Bannantine, Köhler, Moyen, Whittington and Biet.)

Mycobacterium bovis strain Mb3601 was isolated from the lymph node of an infected bovine in a bovine tuberculosis highly enzoonotic area of Burgundy, France. It was selected to obtain a complete genome for a new clonal complex, mainly constituted by SB0120-spoligotype strains that we propose to name "European 3". It was recently described as "clonal group I" based on whole-genome SNP analysis of 87 French strains. Here we describe the 4,365,068 bp complete genome obtained by the combination of PacBio and Illumina technologies. This genome of 65.64% G + C content includes 4024 predicted protein-coding genes, 52 tRNA, 3 rRNA and 11 copies of IS6110., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The Mycobacterium avium complex includes two closely related species, Mycobacterium avium and Mycobacterium intracellulare . They are opportunistic pathogens in humans and responsible for severe disease in a wide variety of animals. Yet, little is known about factors involved in their pathogenicity. Here, we identified, purified and characterized adhesins belonging to the heparin-binding hemagglutinin (HBHA) and laminin-binding protein (LBP) family from M. intracellulare ATCC13950 and examined clinical isolates from patients with different pathologies associated with M. intracellulare infection for the presence and conservation of HBHA and LBP. Using a recombinant derivative strain of M. intracellulare ATCC13950 producing green fluorescent protein and luciferase, we found that the addition of heparin inhibited mycobacterial adherence to A549 cells, whereas the addition of laminin enhanced adherence. Both HBHA and LBP were purified by heparin-Sepharose chromatography and their methylation profiles were determined by mass spectrometry. Patients with M. intracellulare infection mounted strong antibody responses to both proteins. By using PCR and immunoblot analyses, we found that both proteins were highly conserved among all 17 examined clinical M. intracellulare isolates from patients with diverse disease manifestations, suggesting a conserved role of these adhesins in M. intracellulare virulence in humans and their potential use as a diagnostic tool.

Streptococcus agalactiae is a major pathogen and is the leading cause of neonatal infections in industrialized countries. The diversity of strains isolated from two pregnant women was investigated. Here, we present the draft genome sequences of strains W8A2, W8A6, W10E2, and W10F3, obtained in order to ascertain their phylogenetic affiliation., (Copyright © 2020 Beauruelle et al.)

Genotyping of Mycobacterium avium subsp. paratuberculosis (Map) is an indispensable tool for 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 variable-number tandem repeats (VNTRs) was established using 8 markers. In the recent past this standard, portable, reproducible and discriminatory typing method has been frequently applied alone or in combinations with multi-locus short-sequence-repeat (MLSSR) sequencing. With the widespread use of these genotyping methods, standardization between laboratories needs to be managed, and knowledge of existing profiles and newly defined genotypes should be indexed and shared. To meet this need, a web application called "MAC-INMV-SSR database" was developed. This freely accessible service allows users to compare MLVA and MLSSR subtype data of their strains with those of existing reference strains analyzed with the same genotyping methods., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bovine tuberculosis (TB) is a zoonotic disease, mainly caused by Mycobacterium bovis . France was declared officially TB free in 2001, however, the disease persists in livestock and wildlife. Among wild animals, deer are particularly susceptible to bovine TB. Here, a whole genome sequence (WGS) analysis was performed on strains with the same genetic profile-spoligotype SB0121, Multiple Loci VNTR Analysis (MLVA) 6 4 5 3 11 2 5 7-isolated from different types of outbreaks, including from deer or cattle herds, or zoological or hunting parks where the presence of infected deer was a common trait in most of them. The results of the phylogeny based on the SNP calling shows that two sub-clusters co-exist in France, one related to deer bred to be raised as livestock, and the other to hunting parks and zoos. The persistence over almost 30 years of sporadic cases due to strains belonging to these clusters highlights the deficiency in the surveillance of captive wildlife and the need for better monitoring of animals, especially before movement between parks or herds.

Lung inflammation induced by silica impairs host control of tuberculosis, yet the underlying mechanism remains unclear. Here, we show that silica-driven exacerbation of M. tuberculosis infection associates with raised type 2 immunity. Silica increases pulmonary Th2 cell and M2 macrophage responses, while reducing type 1 immunity after M. tuberculosis infection. Silica induces lung damage that prompts extracellular self-DNA release and activates STING. This STING priming potentiates M. tuberculosis DNA sensing by and activation of cGAS/STING, which triggers enhanced type I interferon (IFNI) response and type 2 immunity. cGAS-, STING-, and IFNAR-deficient mice are resistant to silica-induced exacerbation of M. tuberculosis infection. Thus, silica-induced self-DNA primes the host response to M. tuberculosis-derived nucleic acids, which increases type 2 immunity while reducing type 1 immunity, crucial for controlling M. tuberculosis infection. These data show how cGAS/STING pathway activation, at the crossroads of sterile inflammation and infection, may affect the host response to pathogens such as M. tuberculosis., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

Mycobacterium avium subsp. paratuberculosis is responsible for paratuberculosis in animals. This disease, leading to an inflammation of the gastrointestinal tract, has a high impact on animal health and an important economic burden. The environmental life cycle of M. avium subsp. paratuberculosis is poorly understood and several studies suggest that free-living amoebae (FLA) might be a potential environmental host. FLA are protozoa found in water and soil that are described as reservoirs of pathogenic and non-pathogenic bacteria in the environment. Indeed, bacteria able to survive within these amoebae would survive phagocytosis from immune cells. In this study, we assessed the in vitro interactions between several strains of M. avium subsp. paratuberculosis and Acanthamoeba castellanii . The results indicate that the bacteria were able to grow within the amoeba and that they can survive for several days within their host. To explore the presence of M. avium subsp. paratuberculosis in environmental amoebae, we sampled water from farms positive for paratuberculosis. A M. avium subsp. paratuberculosis strain was detected within an environmental amoeba identified as related to the poorly described Rosculus genus. The bacterial strain was genotyped, showing that it was similar to previous infectious strains isolated from cattle. In conclusion, we described that various M. avium subsp. paratuberculosis strains were able to grow within amoebae and that these bacteria could be found on farm within amoebae isolated from the cattle environment. It validates that infected amoebae might be a reservoir and vector for the transmission of M. avium subsp. paratuberculosis .

Mycobacteria have a complex cell wall structure that includes many lipids; however, even within a single subspecies of Mycobacterium avium these lipids can differ. Total lipids from an M. avium subsp. paratuberculosis (Map) ovine strain (S-type) contained no identifiable glycopeptidolipids or lipopentapeptide (L5P), yet both lipids are present in other M. avium subspecies. We determined the genetic and phenotypic basis for this difference using sequence analysis as well as biochemical and physico-chemical approaches. This strategy showed that a nonribosomal peptide synthase, encoded by mps1, contains three amino acid specifying modules in ovine strains, compared to five modules in bovine strains (C-type). Sequence analysis predicted these modules would produce the tripeptide Phe-N-Methyl-Val-Ala with a lipid moiety, termed lipotripeptide (L3P). Comprehensive physico-chemical analysis of Map S397 extracts confirmed the structural formula of the native L3P as D-Phe-N-Methyl-L-Val-L-Ala-OMe attached in N-ter to a 20-carbon fatty acid chain. These data demonstrate that S-type strains, which are more adapted in sheep, produce a unique lipid. There is a dose-dependent effect observed for L3P on upregulation of CD25+ CD8 T cells from infected cows, while L5P effects were static. In contrast, L5P demonstrated a significantly stronger induction of CD25+ B cells from infected animals compared to L3P., (© 2017 John Wiley & Sons Ltd.)

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., (Copyright © 2017 Branger et al.)

Mycobacterium bovis is the etiologic agent of bovine tuberculosis, a chronic infectious disease, affecting livestock, wild animals, and sometimes humans. We report the draft genome sequence of a Mycobacterium bovis strain isolated from wild boar of spoligotype SB0120 (or BCG-like) also present in wildlife-livestock multi-host systems., (Copyright © 2016 Branger et al.)

The description of the population of M. bovis strains circulating in France from 1978 to 2013 has highlighted the discriminating power of the MLVA among predominant spoligotype groups. In the present study we aimed to characterize clonal groups via MLVA and to better understand the strain's population structure. MLVA was performed with eight MIRU-VNTR loci, most of them defined by the Venomyc European consortium. The discriminatory index of each MLVA loci was calculated for SB0120, SB0134, SB0121 and the "F4-family", the main spoligotype groups in France. Differences in global DI per spoligotype, but also by locus within each spoligotype, were observed, which strongly suggest the clonal complex nature of these major groups. These MLVA results were compared to those of other European countries where strain collections had been characterized (Spain, Portugal, Italy, Northern Ireland and Belgium). Overall, QUB 3232 and ETR D are respectively the most and the least discriminative loci, regardless of the strains geographical origin. However, marked DI differences are observed in the rest of the MIRU-VNTR loci, again highlighting that strain genetic variability in a country depends on the dominant existing clonal complexes. A web application for M. bovis, including spoligotyping and MIRU-VNTR typing data, was developed to allow inter-laboratory comparison of field isolates. In conclusion, combination of typing methods is required for M. bovis optimum discrimination and differentiation of groups of strains. Thus, the loci employed for MLVA in a country should be those which are the most discriminative for the clonal complexes which characterize their M. bovis population., (Copyright © 2016 Elsevier B.V. All rights reserved.)

After Mycobacterium avium subsp. paratuberculosis (Map) infection the cell-mediated immune (CMI) response indicative of early Th1 activation may be detected using interferon-gamma release assay (IGRA). Currently, the purified protein derivatives (PPDs), i.e., the total extract of mycobacteria antigens are used to recall CMI responses against Map. This study aimed to assess the ability of the chemically synthesized Map specific cell wall lipopentapeptide L5P to induce CMI response in cows infected by Map compared to PPD. L5P and PPD elicited an IFN-γ response in 12 and 35 animals from two Map infected herds respectively, but IFN-γ was not detected in the 13 cows recruited from a non-infected herd. Levels of IFN-γ detected were higher with PPD than with L5P. There was no correlation between the IFN-γ response and the humoral response to Map or faecal culture., (Copyright © 2015. Published by Elsevier Ltd.)

To study the dynamics of bovine tuberculosis (bTB) in France, 4,654 M. bovis strains isolated mainly from livestock and wildlife since 1978 were characterized by spoligotyping and MLVA based on MIRU-VNTR. In our study spoligotyping allowed the discrimination of 176 types although 3 spoligotypes are predominant and account for more than half of the total strain population: SB0120 (26%), SB0134 (11%) and SB0121 (6%). In addition, 11% of the isolates, principally from Southern France, showing close spoligotypes and MIRU-VNTR types have been gathered in a family designated as the "F4-family". MLVA typing allowed extensive discrimination, particularly for strains with predominant spoligotypes, with a total of 498 genotypes, several of which were highly regionalized. The similarity of the strains' genetic relationships based on spoligotyping and MIRU-VNTR markers supports the co-existence of different clonal populations within the French M. bovis population. A genetic evolution of the strains was observed both geographically and in time. Indeed, as a result of the reduction of bTB due to the national control campaigns, a large reduction of the strains' genetic variability took place in the last ten years. However, in the regions were bTB is highly prevalent at present, cases in both livestock and in wildlife are due to the spread of unique local genotype profiles. Our results show that the highly discriminating genotyping tools used in this study for molecular studies of bTB are useful for addressing pending questions, which would lead to a better insight into the epidemiology of the disease, and for finding proper solutions for its sustainable control in France.

Mycobacterium avium subsp. paratuberculosis comprises two genotypically defined groups, known as the cattle (C) and sheep (S) groups. Recent studies have reported phenotypic differences between M. avium subsp. paratuberculosis groups C and S, including growth rates, infectivity for macrophages, and iron metabolism. In this study, we investigated the genotypes and biological properties of the virulence factor heparin-binding hemagglutinin adhesin (HBHA) for both groups. In Mycobacterium tuberculosis, HBHA is a major adhesin involved in mycobacterium-host interactions and extrapulmonary dissemination of infection. To investigate HBHA in M. avium subsp. paratuberculosis, we studied hbhA polymorphisms by fragment analysis using the GeneMapper technology across a large collection of isolates genotyped by mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) and IS900 restriction fragment length polymorphism (RFLP-IS900) analyses. Furthermore, we analyzed the structure-function relationships of recombinant HBHA proteins of types C and S by heparin-Sepharose chromatography and surface plasmon resonance (SPR) analyses. In silico analysis revealed two forms of HBHA, corresponding to the prototype genomes for the C and S types of M. avium subsp. paratuberculosis. This observation was confirmed using GeneMapper on 85 M. avium subsp. paratuberculosis strains, including 67 strains of type C and 18 strains of type S. We found that HBHAs from all type C strains contain a short C-terminal domain, while those of type S present a long C-terminal domain, similar to that produced by Mycobacterium avium subsp. avium. The purification of recombinant HBHA from M. avium subsp. paratuberculosis of both types by heparin-Sepharose chromatography highlighted a correlation between their affinities for heparin and the lengths of their C-terminal domains, which was confirmed by SPR analysis. Thus, types C and S of M. avium subsp. paratuberculosis may be distinguished by the types of HBHA they produce, which differ in size and adherence properties, thereby providing new evidence that strengthens the genotypic differences between the C and S types of M. avium subsp. paratuberculosis.

Background: Paratuberculosis remains today a major global problem in animal health, especially for dairy cattle. However, the diagnosis of its etiologic agent, Mycobacterium avium subsp. paratuberculosis (Map), still lacks sensitivity because of the lack of available antigens. Little is known about the virulence factors for this pathogen. In this study we have developed a method to produce and purify the heparin-binding hemagglutinin (HBHA), a major adhesin of Mycobacteria, from a culture of Map., Findings: For this extremely slow-growing Mycobacterium, a culture was established in a 3-liter bioreactor. Using the bioreactor the amount of the Map biomass was increased 5-fold compared to a classical culture in flasks. The map-HBHA was purified from a Map lysate by heparin-Sepharose chromatography on HiTrap columns. Binding of map-HBHA onto heparin-Sepharose can be reduced in the presence of salt. Consequently, all steps of sample preparation and column equilibration were carried out in 20 mM Tris-HCl (pH 7.2). The map-HBHA was eluted by a linear NaCl gradient. High resolution mass spectrometry analyses revealed that the native form of map-HBHA has posttranslational modifications, including the removal of the initiation methionine, acetylation of the alanine residue at the N-terminal extremity and the presence of methylated lysines in the C-terminal domain of the protein., Conclusions: An optimized culture of Map in a bioreactor was established to purify the native map-HBHA from a Map lysate by heparin-Sepharose chromatography. The availability of this antigen offers the possibility to study the structure of the protein and to examine its role in pathogenicity, in particular to better understand the specific interactions of Map with the intestinal tissue. The map-HBHA obtained in its native immunogenic form may also be useful to improve the diagnostic test, especially for the development of a new T-cell-based interferon gamma release assays.

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.

Members of the Mycobacterium avium complex (MAC) are ubiquitous bacteria that can be found in water, food, and other environmental samples and are considered opportunistic pathogens for numerous animal species, mainly birds and pigs, as well as for humans. We have recently demonstrated the usefulness of a PCR-based mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing for the molecular characterization of M. avium subsp. paratuberculosis and M. avium strains exclusively isolated from AIDS patients. In the present study we extended our analysis, based on eight MIRU-VNTR markers, to a strain collection comprehensively comprising the other M. avium subspecies, including M. avium subsp. avium, M. avium subsp. hominissuis, and M. avium subsp. silvaticum, isolated from numerous animal species, HIV-positive and HIV-negative humans, and environmental sources. All strains were fully typeable, with the discriminatory index being 0.885, which is almost equal to that obtained by IS1311 restriction fragment length polymorphism (RFLP) typing as a reference. In contrast to IS1311 RFLP typing, MIRU-VNTR typing was able to further discriminate M. avium subsp. avium strains. MIRU-VNTR alleles strongly associated with or specific for M. avium subspecies were detected in several markers. Moreover, the MIRU-VNTR typing-based results were consistent with a scenario of the independent evolution of M. avium subsp. avium/M. avium subsp. silvaticum and M. avium subsp. paratuberculosis from M. avium subsp. hominissuis, previously proposed on the basis of multilocus sequence analysis. MIRU-VNTR typing therefore appears to be a convenient typing method capable of distinguishing the three main subspecies and strains of the complex and providing new epidemiological knowledge on MAC.

Staphylococcus aureus is responsible for a large panel of infections in humans and animals. In cows, S. aureus provokes chronic intramammary infections. Little information is available about the regulation of virulence factors in bovine isolates. Moreover, oxygenation, which is low in an inflamed mammary gland, could play an important role during the infectious process. We investigated the impact of oxygen on regulatory and virulence factors transcription for three S. aureus bovine isolates cultivated in CYPG medium into a fermentor under moderate oxygenation or low oxygenation. A selective panel of regulatory factors and virulence factors was studied through their mRNA profiles by real-time PCR according to growth phases and oxygenation. RNAIII, rot and sarR genes, for the regulatory factors, and asp23 and cflA genes, for the virulence factors, were strongly expressed, whatever the oxygenation and the strains. Under low oxygenation, whatever the strain, an enhanced expression of srr, clfA and spa genes was detected. Some regulators such as sae, sarA and sigB were differentially transcribed according to the strain and the oxygenation condition. This study sustains the complexity of S. aureus genes global regulation and suggests the coexistence of different pathways that can be activated depending on the strain and the oxygen availability.

Among the toxins that Staphylococcus aureus is able to secrete, bi-component toxins named leukotoxins target specifically leukocytes, mainly phagocytic cells. Isolates from cows, goats and ewes with mastitis were selected on the basis of the presence or not of the genes encoding the recently described LukM/LukF'-PV leukotoxin. Of the 128 isolates tested, 126 had moderate to high leukotoxic activity to bovine polymorphonuclear cells (PMN). The supernatants of lukM-positive isolates were much more leukotoxic than the supernatants of lukM-negative isolates: mean leukotoxic titers were 122 versus 20 and 581 versus 26 for isolates of bovine and caprine origin, respectively. Among lukM/lukF'-PV positive isolates, those of caprine and ovine origins were more leukotoxic than were isolates of bovine origin (P < 0.01). The two most abundant proteins in the culture supernatant of a highly toxic isolate were purified and identified as the two components of LukM (LukM and LukF'-PV) on the basis of their molecular mass, N-terminal amino acid sequence, and high synergistic activity. LukM/LukF'-PV induced the flattening of bovine PMN at a concentration as low as 3.6 ng/ml (0.1 nM). A higher concentration (18 ng/ml) was necessary to produce the same effect on caprine or ovine PMN. Affinity-purified antibodies to LukM or to LukF'-PV neutralized the leukotoxic effect of all the culture supernatants. They neutralized with the same efficiency the toxic activity of supernatants from lukM/lukF'-PV positive or negative isolates. These results establish that LukM/LukF'-PV is very active on PMN of ruminants and suggest that this leukotoxin could be the most active leukotoxin produced by mastitis isolates. They prompt further studies to delineate the contribution of LukM/LukF'-PV to the pathogenesis of mastitis in ruminants and the protective effect of antibodies to this leukotoxin.

The expression of Staphylococcus aureus virulence proteins is under the control of RNA III, a central pleiotropic regulator transcribed from the agr locus. RNA III is activated by at least two two-component systems, one encoded by the agr locus (AgrC-AgrA) and another encoded outside of this locus (TRAP-RAP). In this work, we developed new typing methods based on genes encoding these two systems, which we used to characterize a nonclonal population of S. aureus bovine mastitis isolates. Twelve agr restriction types were identified in this population, but the majority of strains (56.3%) were grouped in the R III-A1 type. No strain isolated from humans, whose agr sequence is available from GenBank, was found to belong to this major type. Restriction maps constructed for all of those agr variants allowed the linking of all types in an evolution scheme and their grouping in one of the four agr interference groups. This analysis indicates that groups 2, 3, and 4 probably evolved from the more frequently encountered type, which belongs to group 1. agr group 1 was also found to be the most prevalent (69.0% of the strains) and the most polymorphic interference group. By developing an agr group-specific multiplex PCR, we confirmed the above classification of strains in the agr interference groups. Four allelic variants of trap were also identified, indicating that this two-component system is also polymorphic. The majority of strains was grouped in the trap 1 type (71.8%). Whereas no relationships between agr group and trap types were found, strains of similar agr restriction type were also of similar trap type (with the exception of strains belonging to the agr R IV-A5 and R VI-A8 types). Our analysis indicates that S. aureus isolated from cows has predominantly a clonal structure and that the highly prevalent agr R III-A1, trap 1 type (56.3% of the strains) probably possesses a genetic background which endows it with superior ability to infect the bovine mammary gland.